Research Article Urea Unfolding Study of E. coli Alanyl

Research ArticleUrea Unfolding Study of E coli Alanyl-tRNA Synthetaseand Its Monomeric Variants Proves the Role of C-TerminalDomain in Stability

Baisakhi Banerjee and Rajat Banerjee

Department of Biotechnology and Dr B C Guha Centre for Genetic Engineering and Biotechnology University of Calcutta 35Ballygunge Circular Road Kolkata 700 019 India

Correspondence should be addressed to Rajat Banerjee rbbcgcgmailcom

Received 10 August 2015 Accepted 20 September 2015

Academic Editor Hieronim Jakubowski

Copyright copy 2015 B Banerjee and R Banerjee This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

E coli alanyl-tRNA exists as a dimer in its native form and the C-terminal coiled-coil part plays an important role in thedimerization processThe truncatedN-terminal containing the first 700 amino acids (1ndash700) forms amonomeric variant possessingsimilar aminoacylation activity like wild type A point mutation in the C-terminal domain (G674D) also produces a monomericvariant with a fivefold reduced aminoacylation activity compared to the wild type enzyme Urea induced denaturation of thesemonomeric mutants along with another alaRS variant (N461 alaRS) was studied together with the full-length enzyme using variousspectroscopic techniques such as intrinsic tryptophan fluorescence 1-anilino-8-naphthalene-sulfonic acid binding near- and far-UV circular dichroism and analytical ultracentrifugation Aminoacylation activity assay after refolding from denatured staterevealed that the monomeric mutants studied here were unable to regain their activity whereas the dimeric full-length alaRS getsback similar activity as the native enzymeThis study indicates that dimerization is one of the key regulatory factors that is importantin the proper folding and stability of E coli alaRS

1 Introduction

Aminoacyl-tRNA synthetases (aaRSs) belong to a groupof enzymes that are important in the protein biosynthesisprocess They covalently link an amino acid to its cognatetRNA molecule containing the triplet anticodon specificfor that amino acid Despite catalyzing the similar kind ofaminoacylation reaction depending on the distinct activesite topologies aaRSs have been classified into two unrelatedclasses class I and class II [1ndash4] In class I enzymes the activesite is formed by the Rossmann dinucleotide binding domainwhereas in class II synthetases a seven-stranded antiparallel120573-fold is responsible for the active site formation [5]

E coli alanyl-tRNA synthetase class IIa enzyme has beenpreviously described either as tetramer [6] or as dimer [7 8]Recently based on analytical ultracentrifugation experimentDignam et al have shown that this enzyme is a homodimerof 875 residues with molecular weight of 96000Da persubunitsmonomer [9] There are four functional domains

that constitute the structure of this particular enzyme Theclass IIa aminoacylation and tRNA recognition domain (N-terminal 1ndash461 amino acids) is responsible for the aminoa-cylation of tRNA and also activation of alanine The edit-ing domain (amino acid 553ndash705) hydrolyzes misactivatedglytRNAAla and sertRNAAla The C-terminal oligomerisationdomain (amino acid 705ndash875) which is responsible for thedimer formation plays a role in the tRNA recognition [10]Interestingly this enzyme also has a unique transcriptionalregulation mechanism [11] It was demonstrated that alaRSbinds the palindromic DNA sequence flanking its own genersquostranscriptional start site and repress its own gene transcrip-tion [11] It was also reported that the amount of repressionis increased in the presence of elevated concentration of itscognate amino acid substrate L-alanine [11]

Recently crystal structure of the full-length enzymefrom Archaeoglobus fulgidus in the presence of tRNA wasreported [12] Moreover the structure of the N-terminalcatalytic fragment andC-terminal oligomerization domain of

Hindawi Publishing CorporationJournal of Amino AcidsVolume 2015 Article ID 805681 12 pageshttpdxdoiorg1011552015805681

2 Journal of Amino Acids

the same enzyme from E coli Aquifex aeolicus Pyrococcushorikoshii and Archaeoglobus fulgidus was also reportedeither in complex with its ligands or in absence of any [10 13ndash17] Previous reports showed that the N-terminal 461 aminoacid residues are sufficient for the aminoacylation of tRNAAla

[18] Moreover alaRS monomer containing the N-terminal700 amino acids has almost the same 119896cat value for tRNA

Ala

aminoacylation when compared to the full-length dimeric(875 amino acidsmonomer) protein [18] In another study itwas shown that point mutation in the amino acid residue 674(where the glycine has been changed to aspartic acid) resultsin a full-length monomer that has 20-fold reduction in the119896cat value for tRNA aminoacylation [19]

Keeping inmind the unusual features of this enzymewithlarge multidomain dimeric structure in this study we haveinvestigated how the C-terminal domain affects the stabilityof this enzyme In this report we have constructed threemutant proteins The first mutant is a full-length monomerhaving a mutation in the 674 position (G674D previouslydemonstrated to form full-length monomer vide supra)The second mutant contains the N-terminal aminoacylationdomain (1ndash700 N700) The third mutant has N-terminal 461amino acids (1ndash461 N461) aminimum fragment required forthe proper aminoacylation of tRNAAlaThesemutant proteinsand the wild type enzyme were subjected to denaturationin the presence of urea to decipher the unfolding pathwayof these proteins under equilibrium condition This studyreveals that WT-alaRS is more stable than the mutant alaRSsand also have the capacity to refold back from the fullydenatured state unlike the other mutants studied here

2 Materials and Methods

21 Materials The clone of full-length E coli alaRS (WT-alaRS) N700 alaRS and plasmid containing tRNAAla tran-script was a kind gift from Professor Paul Schimmel (SkaggsInstitute for Chemical Biology The Scripps Research Insti-tute California) All the chemicals that are used in thisstudy are of analytical grade and were purchased from Sigma(St Louis MO) SRL (India) E-Merck (Germany) and HI-MEDIA (India)

22 Construction of E coli N461 and G674D alaRS MutantFor construction of N461 truncated enzyme and G674D vari-ant the primer 51015840ATT TAAAGGCTA TGA CTAACTGGAACT GAA 31015840 and 51015840 AGCCGCA CTGATGATAT TGGT 31015840were used respectively Mutagenesis was carried out by usingin vitro site-directed mutagenesis protocol by Stratagene andthe mutation was confirmed by DNA sequencing

23 Purification of E coli Alanyl-tRNA Synthetases Wild typealaRS N461 and G674D alaRS with a N-terminal histidinetag were expressed in BL21 (DE3) after transformation ofplasmid pET28a containing the desired constructs The plas-mid pET20a encoding N-terminal histidine tag N700 alaRSwas also transformed in BL21 (DE3) Single colonies werepicked in all cases inoculated into 10mL of Luria-Bertani(LB) medium containing 50 120583gmL Kanamycin except N700where 100 120583gmL of ampicillin was added and grown

overnight at 37∘C with constant shaking at 250 rpm On thefollowingmorning 1 of overnight culturewas transferred to1 L of LB medium and grown at 37∘C until OD

600= 06 cells

were then induced with 1mM of IPTG and further grownat the same temperature for 4 hrs The cells were pelleted bycentrifugation at 5000 rpm for 15min at 4∘C Except N700alaRS the pelleted cells were lysedwith bufferA (50mMTris-HCl pH 80 containing 500mMNaCl 10mM imidazole 10glycerol and 1mM 120573-mercaptoethanol) For N700 alaRS thebuffer composition was 100mMTris-HCl pH 80 containing10 glycerol and 1mM 120573-mercaptoethanol The suspendedcells were sonicated and centrifuged at 25000 rpm for 1 hThe supernatant was loaded on an Ni-NTA agarose column(preequilibrated with buffer A)The columnwas thenwashedwith 2 column volumes of buffer A finally E coli alaRSand its mutants were eluted with a gradient of imidazoleconcentration ranging from 20 to 200mM in buffer A All theeluted fractions were checked by 12 sodiumdodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis The majoreluted fractions containing desired proteins were pooledand dialyzed against buffer C (100mM Tris-HCl pH 80containing 100mMNaCl and 20 glycerol) at 4∘C After 36ndash40 hrs of dialysis samples were collected from the dialysisbag and protein concentration was determined by measuringabsorbance at 280 nm using the molar extinction coefficientof the protein (71195Mminus1 cmminus1 for WT-alaRS as a monomerand G674D 65320Mminus1 cmminus1 for N700 and 60580Mminus1 cmminus1for N461)

24 Fluorescence Spectroscopy Fluorescence measurementswere performed in a Hitachi F-7000 spectrofluorimeter with1mm quartz cuvette at 25∘C All the experiments wererepeated at least 3 times The fluorescence emission wasrecorded in the wavelength region of 310ndash450 nm after excit-ing the protein solution at 295 nm for selective observationof tryptophan residues The bandpasses were 5 nm for bothexcitation and emission The protein concentrations were4 120583M Proteins were incubated in the presence or absenceof increasing concentration of urea (0ndash8M) for 16ndash18 h at25∘C in 100mM Tris-HCl pH 80 buffer at room tempera-ture before the spectra were recorded Controls containingappropriate concentration of denaturants were prepared andwere subtracted from the corresponding samples spectra Forthe experiments where ANS (30 120583M) had been used theexcitation wavelength was 420 nm to avoid inner filter effectand emission was recorded at 482 nm Appropriate blankswere also subtracted from each experiment

25 Circular Dichroism (CD) Spectroscopy The CD spectrameasurements were carried out in a JASCO J-815 spec-tropolarimeter using 100mM Tris-HCl (pH 80) bufferThe spectra were recorded at 25∘C using 1mm and 10mmpath length cuvettes for far-UV (210ndash260 nm) and near-UV(260ndash320 nm) CD measurement respectively with a scanspeed 50 nmmin The final spectrum was average of fiveindependent measurements For far-UV and near-UV CDexperiments the protein concentrations were kept at 4120583Mand 15 120583M respectively The sample spectra were correctedby subtracting from the buffer spectra containing the same

Journal of Amino Acids 3

concentration of urea without the protein The followingequation was used to calculate the mean residue ellipticity(MRE) of the proteinMRE= (100times observed 120579 times MW)(10 times119897 times 119862 times 119899) in degsdotcm2sdotdmolminus1 where 120579 is the observedellipticity in millidegrees MW the molecular weight inkilodalton (kDa) 119862 the concentration in mgmL 119899 thenumber of amino acids and 119897 the path length in cm [20 21]

26 Curve Fitting Nonlinear least square fitting of the equi-librium denaturation data (both fluorescence intensity ratioand CD) was done according to the two following equationscorresponding to two-state and three-state models respec-tively [22]

119878obs =119878119873+ 119878119880119890minus(Δ1198661minus1198981[119863])RT

1 + 119890minus(Δ1198661minus1198981[119863])RT (1)

where 119878obs denotes observed intensity of spectroscopic signal119878119873

and 119878119880

denote the intensity of signals for the nativeand unfolded species respectively Δ119866 is the free energy ofunfoldingwithout denaturant119898 is the slope of the transitionand concentration of the denaturant is 119889 Consider119878obs

=119878119873119878119868119890minus(Δ1198661minus1198981[119863])RT + 119878

119880119890minus(Δ1198661minus1198981[119863])RT119890minus(Δ1198662minus1198982[119863])RT

1 + 119890minus(Δ1198661minus1198981[119863])RT + 119890minus(Δ1198661minus1198981[119863])RT119890minus(Δ1198662minus1198982[119863])RT

(2)

In the above equation 119878obs denotes intensity of the observedsignal for spectroscopic data 119878

119873 119878119868 and 119878

119880denote the

intensities of signal for the native intermediate and unfoldedstates respectively 119877 represents the universal gas constantand 119879 represents the temperature in absolute scale Existenceof one intermediate state is assumed while deriving theequation It was also considered that Δ119866 varies linearly withdenaturant concentration Δ119866

1= Δ119866

119874+ 119898119897[119863] There are

five independent parameters in this equation such asΔ1198661 the

change in free energy between the native and the intermediatestates Δ119866

2 the change in free energy between the native

and the unfolded states 1198981 the slope of the first transition

1198982 the slope of the second transition and 119878

119868 the value of

the spectroscopic parameter for the pure intermediate stateThese parameters were floated globally and the fitting of datawas done using software KyPlot (version 20 beta 15 (32 bits)Koichi Yoshioka 1997ndash2001) Further the chi-square values(1205942) were determined from the fitted data The set of valuesthat resulted in minimum (1205942) was taken as the best fit Dataobtained from at least three independent experiments wereused to calculate the average value

27 Analytical Ultracentrifugation Beckman Optima XL-Ianalytical ultracentrifuge containing absorbance optics wasused to perform analytical ultracentrifugation experimentsThe rotor used was An50Ti Experiments were done at20∘C Sedimentation velocity experiments were performedat 28000 rpm in a charcoal-filled Epon double-sector cen-terpiece Protein samples and the reference buffer solutionswere centrifuged at 5000 rpm for 4min at room temper-ature in a tabletop centrifuge to remove any suspendedcontaminants Protein samples were maintained in 100mMTris-HCl pH 80 buffer with or without the denaturant

Parameters like density and viscosity of the solvents werecalculated using the program SEDNTERP [23] The valueof partial specific volume was found to be 07321mLgminus1 ascalculated at experimental temperature that is 20∘C fromthe sequence of amino acid using SEDNTERP Before thecollection of data all the samples were allowed to equilibratefor 1 h under vacuum and temperature was allowed to getstabilised Each centrifuged cell was scanned in a sequentialmanner by setting zero time delay between successive scansVelocity data were collected at 280 nm at 0003 cm intervalswith one average in a continuous scan mode Sedimentationvelocity data were transferred to the program SEDFIT All thedata were analyzed with SEDFIT software (version 141 2013)using the following specifications A continuous 119888(119904) modelwas used with a range of 05minus12 S that had confidence levelof 068 (1 standard deviation) [24] Fitting was performedusing alternating rounds of the simplex and Marquardt-Levenberg algorithms were employed for the fitting of datauntil no further decrease in root mean square deviation(RMSD) was observed Samples were prepared 12 h priorto the experiment protein concentrations were adjustedto 4 120583M For sedimentation equilibrium experiments six-channel charcoal-filled epon centerpieces were used Sedi-mentation equilibrium was attained at a rotor temperature of20∘C at different rotor speeds for different proteins for com-plete attainment of equilibrium Absorbance profiles wereobtained at a wavelength of 280 nm Extinction coefficientratios were estimated using spectrophotometry at differentwavelengths Further 3ndash6 sedimentation equilibrium datasets that are obtained at different loading concentrationswere analyzed by SEDPHAT (version 104 2012) using globalmodelling [25] During the fitting of data sets molecularweight values were allowed to float Molecular weights (Mr)were obtained with least chi-square and least RMSD value byamodel-dependentmethod Loading concentrations of alaRSproteins were kept at 5120583M 10 120583M and 20120583MAll the proteinconcentrations are that of the monomer

28 Aminoacylation Assay E coli tRNAAla was purified asdescribed in a previously published protocol [7] All thealaRSs (protein concentration 4120583M) were first unfolded byusing different concentrations of denaturant as describedin spectroscopic experiments The assay were then startedby rapidly diluting these denatured samples ten timeswith aminoacylation buffer (20mM Hepes-NaOH (pH 75)100mM KCl 02mM EDTA 02mM L-alanine 2mM ATPand 5mM MgCl2) containing trace amount of L-[3-3H]Alanine (250 120583Ci Perkin Elmer Lifesciences) 4 120583M E colitRNAAla and desired amount of denaturant Aminoacyla-tion was performed in 37∘C in aminoacylation buffer Tenmicrolitres of aliquots was removed at different time inter-vals These aliquots were spotted on 3MM filter paper disks(Whatman) Thereafter they were washed three times in 10trichloroacetic acid and dried The amount of radioactivityretained was determined by liquid scintillation counting wasemployed to determine the amount of radioactivity retainedin the disks Acceptor activity was taken to be 50 nmol permgof RNA


3 Result

31 G674DN700 andN461 alaRSAll Forms FoldedMonomerPrevious report indicated that thewild typeE coli alaRSwas adimeric enzyme of identical subunits [9] It was also reportedthat the Gly to Asp mutation in the 674 position of E colialaRS results in a protein which forms a full-lengthmonomer[19] To reestablish the oligomeric nature of wild type alaRS(WT-alaRS) G674D alaRS along with N700 alaRS and N461alaRS sedimentation equilibrium experiments had been done(Figure 1(a)) The N461 mutant protein formed aggregates atconcentrations higher than 10 120583M Fitting the data in SED-PHAT using a single species model yielded a good fit with amolecular weight ranging from 186280 to 197770Da forWT-alaRS (theoretical molecular weight calculated from aminoacid composition 192500Da) 92932 to 95331Da for G674D(theoretical molecular weight 96250Da) 65971 to 72843DaforN700 alaRS (theoreticalmolecular weight 77000Da) and44601 to 50000Da for N461 alaRS (theoretical molecularweight 50710Da) The theoretical molecular weight wascalculated from amino acid composition Thus under ourexperimental conditions the proteins exist as monomeric ordimeric states as reported earlier [9 18 19]

Circular dichroism proved to be a very useful indicatorof the overall secondary and tertiary structure of a particularprotein [21 26] The far-UV CD spectrum (Figure 1(b))showed a well-folded secondary structure for WT-alaRSG674D N700 and N461 mutant proteins The near-UVCD spectrum showed that N700 and G674D have almostsimilar kind of tertiary interaction that differs from the full-length enzymeThese two mutant proteins have an increasedintensity in the region of 285ndash305 nm (that arises due to thetryptophan residue in a protein) than WT-alaRS This couldbe due to the different folding pattern ofmonomericmutantsIn contrast N461 showed a near-UV CD spectrum that isdifferent from full-length as well as N700 and G674D alaRS(Figure 1(c)) This suggests that the tertiary interactions inthese three mutant variants might be different from the full-length enzyme

32 Unfolding of Wild Type alaRS and Its Mutants Is aMultistep Process Many studies used intrinsic tryptophanfluorescence to investigate conformational changes in pro-teins [27 28] E coli alaRS has a total of eight tryptophanresidues Seven of them are present in the N-terminal halfof the protein (positions 112 120 129 143 171 194 and212) The remaining tryptophan residue is present in the C-terminal domain (position 870) We examined the fluores-cence emission spectrum of the wild type and three mutantalaRSs at different urea concentrations At 0M urea WT-alaRS showed an emission maximum at 336 nm which isconsistent with our previous study [29]With increasing ureaconcentration the emission maximum was red-shifted andfinally levelled off around 350 nm at 8M urea In contrastN700 alaRS showed an emission maximum closer to 339 nmwhereas the other two mutants N461 (335 nm) and G674D(336 nm) showed an emission maximum similar to the wildtype full-length enzyme At 8M urea red shifted emissionmaximum indicated a complete exposure of the buried

Table 1 The thermodynamic parameters derived from the ureainduced denaturation of WT-alaRS G674D N700 and N461 alaRSusing the data of fluorescence emission maximum A three-stateequation was used to fit the data (equation (2)) as described inSection 2 using the graph plotting software KyPlot (version 20 beta15 (32 bits) Koichi Yoshioka 1997ndash2001) The excitation wavelengthwas kept at 295 nm and emission spectra were recorded from 310to 450 nm The emission maximum value was calculated by takingthe first derivative of the spectra Bandpasses for both excitation andemission were kept at 5 nm

Fluorescence Δ1198661(Kcalmol)Δ1198662

(Kcalmol)1198981

(KcalmolM)1198982

(KcalmolM)WT 69 plusmn 02lowast 117 plusmn 03lowast 13 plusmn 01lowast 18 plusmn 01lowast

G674D 47 plusmn 01lowast 89 plusmn 11lowast 09 plusmn 01lowast 12 plusmn 02lowast

N700 59 plusmn 01lowast 87 plusmn 02lowast 11 plusmn 01lowast 13 plusmn 02lowast

N461 35 plusmn 02lowast 132 plusmn 03lowast 08 plusmn 01lowast 16 plusmn 01lowastlowastValues are the means of at least three independent experiments withstandard errors indicated

tryptophan residues (Figure 2) The denaturation curve forall the alaRSs including wild type alaRS when plotted againstthe denaturation concentration showed two transitions withan indication of probable intermediate formation Thermo-dynamic parameters (Table 1) were calculated for all the fouralaRS proteins using (2) (for three-state model) as describedin Section 2

33 The Intermediate in the Urea Induced Unfolding Path-way Contains Exposed Hydrophobic Surfaces ANS has beenwidely used as a marker to locate exposed hydrophobicpatches on protein surface [30] Intrinsic tryptophan fluores-cencemeasurement indicated the presence of an intermediatein the urea unfolding pathway of all the alaRS proteins Tounderstand this intermediate forming phenomenon in detailANS binding experiments were carried out To understandthe ANS saturation of all the alaRS proteins a separatetitration experiment was done The result indicates that thedissociation constant for ANS binding for all the proteins fallsunder the 1ndash5120583M range (data not shown) Depending on thisresult 30 120583M ANS (as a final concentration) was added toalaRS proteins with increasing urea concentration (Figure 3)For theWT-alaRS ANS fluorescence gradually increased andreached a maximum at around 3M urea and then decreasedslowly and levelled off at 8M of urea concentration A similarincrease of ANS fluorescence was also observed for N700 andinN461 alaRS but the change was negligible for G674D alaRS(Figure 3) Taken together these results suggested that theremight be accumulation of an intermediate species duringdenaturation of WT and N700 and N461 alaRS that couldbind ANS strongly The increase in the ANS fluorescenceintensity might result from either increased quantum yieldor increased binding stoichiometry As the emission maximadid not alter much after ANS binding in WT-alaRS N700and N461 alaRS the observed enhanced fluorescence inten-sity was primarily due to increased binding sites (data notshown)

34 Far-UV CD Reveals Disrupted Secondary Structure of theIntermediate To observe the effect of urea on the secondary


Abso

rban

ce (O

D)

Radius (cm)

2

18

16

14

12

1

08585 59 595 6 605 61 615

002

001

0

Resid

uals

Radius (cm)585 59 595 6 605 61 615

WT-alaRSG674D

N700 alaRSN461 alaRS

minus002

minus001

06

055

05

045

04

035

03

025

02685 69 695 7 705 71 715

Abso

rban

ce (O

D)

Radius (cm)

685 69 695 7 705 71 715

Radius (cm)

006

004

002

0

Resid

uals

minus002

minus004

minus006

Abso

rban

ce (O

D)

Radius (cm)

12

11

1

09

08

07

06

05635 64 645 65 655 66 665

Radius (cm)635 64 645 65 655 66 665

006

004

002

0Re

sidua

ls

minus002

minus004

minus006

(a)

Figure 1 Continued


0

minus200

minus400

minus600

minus800

minus1000

minus1200

minus1400210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

WT-alaRSG674D


(b)

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

WT-alaRSG674D


10

8

6

4

2

0

260 270 280 290 300 310 320

(c)

Figure 1 Biophysical characterization of mutant alaRSs along with WT protein (a) Molecular weight determination of WT-alaRS G674DN700 alaRS and N461 alaRS by analytical ultracentrifugation (AUC) Sedimentation equilibrium profiles of WT-alaRS and its mutants atconcentrations 5120583M (upper left panel) 10 120583M (upper right panel) and 20120583M (lower panel) at 20∘C The buffer was 100mM Tris-HCl pH80 Fitting of the data was performed using SEDPHAT software (b) Far-UV CD and (c) near-UV CD spectra of WT-alaRS G674D N700alaRS and N461 alaRS The result has been represented as mean residue ellipticity (MRE) at 25∘C Protein concentrations were kept at 4120583Mfor far-UV CD and 15 120583M for near-UV CD with the same buffer as the analytical ultracentrifugation experiment Path length of the cuvetteswas 1mm and 10mm for far-UV CD and near-UV CD respectively Scan speed was 50 nmmin and five independent scans were performedand from that average spectra were taken

12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(a)

12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(b)

120582m

ax(n

m)

352

350

348

346

344

342

340

338

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

(c)

12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(d)

Figure 2 Unfolding profile of WT-alaRS (a) G674D (b) N700 alaRS (c) and N461 (d) The graph was made by plotting the fluorescenceintensity ratio change (119865

340119865350

) and alterations in emission maximum with increasing urea concentration All the protein concentrationswere kept at 4 120583M Samples were excited at 295 nm where excitation and emission bandpasses were kept as 5 nm for both A circulating waterbath was used to maintain the temperature at 25∘CThe buffer was 100mM Tris-HCl pH 80


WT-alaRSG674D


3

25

2

15

1

05

00 2 4 6 8

F482

Urea (M)

Figure 3 Change in normalized ANS fluorescence intensity withincreased urea concentration ANS and alaRS concentrations were30 120583M and 4 120583M respectively Excitation wavelength was 420 nmand the bandpasses for both the excitation and emission were keptat 5 nm The spectra were measured at 25∘C The samples were keptat dark for 1 h after the ANS addition The values at 482 nm wereplotted after they were normalized considering the 0M values as 1

structure far-UV CD experiments of all the alaRS proteinswere performed The CD spectrum profile of wild typeenzyme was stable up to 1M urea and then showed contin-uous denaturation from 2 to 8M urea (Figure 4(a)) whereastheN700 andG674Dmutantswere stable up to 2Murea thenit got denatured from 3 to 8M urea (Figures 4(c) and 4(b)resp) However the N461 mutant is stable up to 1M ureashows an intermediate level of denaturation from 2 to 5Murea and then completely denatures above 5M (Figure 4(d))From the far-UV CD data it was quite clear that WT-alaRSloses its secondary structure with increasing urea concentra-tion whereas N700 and G674D alaRS retain their secondarystructure till 3M of urea concentration This feature oftensuggests the presence of a folding intermediate Interestinglywhen mean residual ellipticity at 222 nm (MRE

222) was

plotted against the denaturant concentration to understandthe changes in the 120572-helix content of the proteins WT-alaRS data fitted well with a three-state equation whereas theG674D N700 and N461 alaRSs followed a two-state modelequation (1) The calculated thermodynamic parameters arerepresented in Table 2

35 Only WT-alaRS Has the Capability to Refold Backto Its Native-Like Architecture and Retained Activity Theaminoacylation assay was performed by rapidly diluting theovernight denatured protein samples with buffer A completereversibility of the enzyme activity was observed in case ofwild type alaRS whereas all the other three mutants wereincapable of regaining back their activity when the sampleswere diluted from 8M to 08M or lower urea concentration(Figure 5)

36 Wild Type alaRS Intermediate Had Diminished Ter-tiary Interaction A large change in the ANS fluorescence

Table 2 Thermodynamic parameters as obtained by fitting the far-UV CD data of WT-alaRS and its variants The data was fitted intwo-state (equation (1)) and three-state equation (equation (2)) formonomeric variants andWT-alaRS respectively using the softwareKyPlot (version 20 beta 15 (32 bits) Koichi Yoshioka 1997ndash2001)The spectra were recorded in a 1mm path length cuvette and withina region of 210ndash260 nm keeping the speed of the scan at 50 nmminFor averaging data from five independent scans were taken

CD Δ1198661

(Kcalmol)Δ1198662

(Kcalmol)1198981

(KcalmolM)1198982


G674D 27 plusmn 02lowast mdash 07 plusmn 02lowast mdashN700 26 plusmn 03lowast mdash 06 plusmn 02lowast mdashN461 438 plusmn 03lowast mdash 08 plusmn 01lowast mdashlowastValues are the means of at least three independent experiments withstandard errors indicated

intensity and retention of secondary structure were oftenobserved in case of intermediates like molten globule [31]Molten globule state of a protein was reported to havethe following characteristics wild-type-like secondary struc-ture (invariant or little changed in far-UV CD spectrum200ndash250 nm) partially or completely diminished tertiaryinteractions (large change in near-UV CD spectrum 260ndash320 nm) and increased number of exposed hydrophobicpatches (increased ANS binding) Though WT and N700alaRS had shown increased ANS binding the secondarystructure was lost as observed from the far-UV CD spectra(Figure 6) so the intermediate formed in this pathway mightnot be a molten globule-like intermediate Changes in thetertiary structure can be deduced from CD study in the near-UV region (260ndash320 nm) where signal largely contributedto aromatic amino acids and cysteine [26] To characterizethe nature of the intermediate formed around 3M of ureaconcentration near-UV CD experiments were carried outThe near-UV CD spectrum of WT and mutant alaRSs isshown in the presence of 0M 3M and 8Mof urea (Figure 6)In all the cases the native protein (0M urea) displayed adistinct positive side chain CD spectrumwhereas in presenceof 3M as well as 8M urea signal has completely disappearedindicating a complete loss of tertiary interaction

37 Urea Induced Intermediate of Wild Type alaRS Is anEnsemble of Dimer and Monomer When an oligomericprotein unfolds in the presence of denaturants formationof lower oligomers due to loss of interfacial interactions ispossible It is also interesting to identify the exact nature ofthis particular state that forms in the denaturation pathwayof oligomeric proteins To understand this in more detailwe had determined the sedimentation coefficients at differenturea concentartions E coli alaRS exists as a dimer in theabsence of any denatutants with a sedimentation coefficientof sim61 S consistent with the previous report as dimer [9 29]In the presence of 8M urea sedimentation value of sim3 Swas obtained which corresposponds to the presesnce of onlymonomeric species Remerkably at 3M urea concentrationthe sedimentation data indicated existence of two species


200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

minus1000210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(a)

200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus1000

minus800

minus1200

minus1000

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(b)200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(c)

1000

500

0

minus500

minus1000

minus1500

0

minus200

minus400

minus600

minus1000

minus1200

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(d)

Figure 4 Far-UV CD spectra of WT-alaRS (a) G674D (b) N700 alaRS (c) and N461 alaRS (d) (4 120583M) using native protein (black line) anddifferent concentrations of urea such as 1M (blue line) 2M (green line) 3M (red line) 5M (purple line) and 8M urea (violet line) Thetemperature was at 25∘C and the buffer composition was 100mM Tris-HCl pH 80 Inset shows the changes in the mean residue ellipticity(MRE

222) as a function of increasing concentration of urea The solid line represents the global fit The cuvette used has 1mm path length

and the scan speed was 50 nmminThe final spectra were an average of five independent scanned spectra All of the spectra were subtractedfrom blanks containing appropriate concentration of denaturant

one with a value of sim6 S and the other one with sim39 Sconfirming the presence of both dimeric and monomericspecies However surprisingly at 25M and 35M of ureaWT-alaRS exists exclusively as a dimer and a monomerrespectively as depicted in Figure 7

4 Discussion

E coli alaRS is a large multidomain homodimeric pro-tein whose structure-function relationships are not fullyunderstood Available biochemical and genetic mutationalinvestigations [18 32 33] are insufficient to provide satis-factory information about interactions among its subunitsas well as the intrasegmental and domain interactions inits native conformation Denaturation studies provide valu-able information about the nature of forces contributing toproper folding of a protein as well as to its proper func-tioning Previously many folding studies were conductedon oligomeric proteins to understand their conformationalstability [34ndash38] However not much information is available

on the stability of E coli alaRS It was reported recentlyfrom our group that the full-length alaRS forms an inactivedimeric molten globule-like intermediate in the presenceof low concentration of guanidinium hydrochloride [26]Though both urea and guanidinium hydrochloride have adenaturing effect on proteins urea has only chaotropic effectwhereas guanidinium hydrochloride has both the ionic andchaotropic effects [39] Owing to this difference in theirmechanism of action different proteins behave differentlyin the presence of these two denaturants as evident fromseveral earlier studies [40ndash44] It was established that theoligomerization of alaRS depends on the interaction betweenthe C-terminal coiled-coil domains and a deletion of theC-terminal 175 amino acid produces a mutant monomerictruncated protein (amino acid 1ndash700) that has the sameaminoacylation activity as the wild type protein [32] Theseresults clearly indicated that alaRS does not need its dimericstructure for aminoacylation N-terminal part (N-terminal700 amino acid) of this protein is capable of doing thataccurately To understand the significance of the C-terminal


50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(a)

12 14 16

50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(b)35

30

25

20

15

10

5

012 14 160 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(c)

30

25

20

15

10

5

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(d)

Figure 5 Aminoacylation assays of WT-alaRS (a) G674D (b) N700 alaRS (c) and N461 alaRS (d) were followed at 37∘C for 10min usingnative alaRS (open circle) overnight incubated denatured protein containing 03M urea (filled square) and 08M urea (open triangle) andten times diluted protein samples from 3M (open square) and 8M (filled circle) of urea Liquid scintillation counting was used to calculatethe amount of radioactivity preserved The concentrations of alaRS and tRNA were 400 nM and 4 120583M respectively

domain in folding and stability if any we studied ureainduced unfolding pathway of WT-alaRS and its monomericmutants The first mutant (G674D) was constructed to pro-vide the information about the contribution of dimerizationon the stability of the full-length alaRS The second mutantwas constructed to highlight the role of C-terminal residues(701ndash875) on the stability of full-length monomeric alaRSThe N461 mutant provides information about the stability ofthe minimum fragment of the alaRS protein that is neededfor the correct aminoacylation of tRNAAla

Intrinsic tryptophan fluorescence (tryptophan emissionmaximum shift and the 119865

340119865350

ratio) has indicated thepresence of an intermediate in the denaturation pathway ofwild type alaRS and all its monomeric mutants The datawhen fitted into a three-state equation yielded the thermody-namic parameters as shown inTable 1TheΔ119866total(Δ1198661+Δ1198662)of WT-alaRS being 186 Kcalmol is higher than the rest ofthe mutant proteins that shows similar Δ119866 values (G674Dmdash136 Kcalmol N700mdash146 and N461mdash167 Kcalmol) ANSbinding studies also indicated the probability of formation ofan intermediate in all the alaRS proteins except G674D alaRSHowever far-UV CD experiment ruled out the possibilityof formation of an intermediate in the unfolding pathway

of all investigated mutant alaRS proteins When the far-UVCD data (mean residue ellipticity at 222 nm) of monomericalaRSs (G674D N700 and N461) were plotted with increas-ing denaturant concentration the data fitted well in a two-state equation whereas the WT-alaRS still produces a goodfit with a three-state equation This finding was not unusualconsidering the fact that far-UV CD signals provide theinformation of the secondary structures of the entire proteinmolecule In contrast the fluorescence data corresponds tothe changes in themicroenvironment of tryptophan residuesthat is it provides information about the local or tertiaryenvironment [45] In WT-alaRS all the tryptophans areclustered in the N-terminal part of the protein (within thefirst 461 amino acids) except one thus the possibility ofobtaining similar pattern of fluorescence transitions for all thealaRS proteins is not surprising

Sedimentation velocity experiments proved that at 3Murea E coli alaRS exists as a mixture of monomericand dimeric species Surprisingly tertiary interactions werealmost completely lost as indicated by the near-UV CDspectrum at 3M urea concentration for all the alaRS proteinsincluding WT-alaRS In a previous report our group hasdemonstrated thatWT-alaRS lost its tertiary structure before


6

4

2

0

minus2

MRE

(deg

middotcm2middotd

molminus1)

260 270 280 290 300 310 320

Wavelength (nm)

(a)

MRE

(deg

middotcm2middotd

molminus1)

7

6

5

4

3

2

1

0260 270 280 290 300 310 320

Wavelength (nm)

(b)

MRE

(deg

middotcm2middotd

molminus1)

6

4

2

0

260 270 280 290 300 310 320

Wavelength (nm)

(c)

MRE

(deg

middotcm2middotd

molminus1)

10

8

6

4

2

0

minus2

minus4

260 270 280 290 300 310 320

Wavelength (nm)

(d)

Figure 6 Near-UV CD spectra of WT-alaRS (a) G674D (b) N700 alaRS (c) and N461 alaRS (d) (15 120583M) without denaturant (open circle)in the presence of 3M urea (filled circle) and 8M urea (open triangle) The buffer composition was the same as the far-UV CD and thetemperature was kept at 25∘C The experiment was recorded with a cuvette having a path length of 10mm and 50 nmmin scan speed wasmaintained Five independent spectra were averaged

the dissolution of the quaternary structure [27] Similarlyin this study it was clearly evident from the sedimentationvelocity and near-UV CD that at 3M ureaWT-alaRS has lostits tertiary interaction whereas at this denaturant concentra-tion some of its quaternary interaction was still present

When the proteinwas refolded from the 8M to 08Mureaor lower denaturant concentration and the aminoacylationassay was carried out the mutant proteins were unable toregain its native conformation as evident from their loss ofactivity In contrast WT-alaRS was able to regain its native-like activity after refolding The finding that oligomerizationprovides stability to a protein is not very uncommon Forexample a study conducted on soybean agglutinin has shownthat oligomerization imparts a greater stability to the nativestructure of this legume lectins [46] Comparison of thefolding mechanism of these mutant proteins with the WT-alaRS revealed the role of dimerization in the stability ofthe native dimeric structure of the protein This stability tothe protein may be conferred by a specific recognition atthe dimeric interface essential for folding or due to probablelong-range or quaternary interactions From this study it wasevident that dimerization of alaRS endows the protein with agreat deal of conformational stability

5 Conclusion

The present study indicates that the urea induced denat-uration of WT-alaRS is a two-state process with no trueintermediate being formed Also the higher stability of thefull-length dimeric protein indicates that the C-terminalpart plays an important role in the stability of the entireprotein

Abbreviations

aaRS Aminoacyl-tRNA synthetasealaRS Alanyl-tRNA synthetaseANS 1-Anilino-8-naphthalene-sulfonateaa Amino acidAUC Analytical ultracentrifugationCD Circular dichroismSDS Sodium dodecyl sulphate

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


04

03

02

01

02 4 6 8 10 12

Sedimentation coefficient (S)

c(s)

0M05M15M2M

25M3M35M8M

Figure 7 Sedimentation coefficient distribution of WT-alaRS(4 120583M) in native form and in the presence of different concentrationsof urea SEDFITprogramwas used to fit sedimentation velocity dataConsidering continuous 119888(119904) distribution model was considered forthe fitting of data The experiment was performed at 28000 rpmcentrifugation speed at 20∘C

Acknowledgments

Baisakhi Banerjee is supported by University with Potentialfor Excellence UGC (UPE UGC) Fellowship The authorsgratefully acknowledge Professor Paul Schimmel (SkaggsInstitute for Chemical Biology The Scripps Research Insti-tute California) for his gift of full-length E coli alaRS andN700 alaRS plasmid and tRNAAla transcript Professor Gau-tam Basu and Dr Saumya Dasgupta Bose Institute Kolkataare acknowledged for their help during CD measurementsThe authors are thankful to Nanocentre Facility University ofCalcutta Salt Lake for AUC facilityThis workwas supportedby funds from the Department of Science and Technology(India) to Rajat Banerjee (SRSOBB-00182012)

References

[1] S Cusack ldquoEleven down and nine to gordquo Nature StructuralBiology vol 2 no 10 pp 824ndash831 1995

[2] S Cusack ldquoAminoacyl-tRNA synthetasesrdquo Current Opinion inStructural Biology vol 7 no 6 pp 881ndash889 1997

[3] S Cusack C Berthet-Colominas M Hartlein N Nassar andR Leberman ldquoA second class of synthetase structure revealedby X-ray analysis of Escherichia coli seryl-tRNA synthetase at25 Ardquo Nature vol 347 no 6290 pp 249ndash255 1990

[4] G Eriani M Delarue O Poch J Gangloff and D MorasldquoPartition of tRNA synthetases into two classes based onmutually exclusive sets of sequence motifsrdquoNature vol 347 no6289 pp 203ndash206 1990

[5] M G Rossmann D Moras and K W Olsen ldquoChemical andbiological evolution of a nucleotide-binding proteinrdquo Naturevol 250 no 5463 pp 194ndash199 1974

[6] S D Putney R T Sauer and P R Schimmel ldquoPurification andproperties of alanine tRNA synthetase from Escherichia coli Atetramer of identical subunitsrdquo Journal of Biological Chemistryvol 256 no 1 pp 198ndash204 1981

[7] S M Sood K A W Hill and C W Slattery ldquoStability ofEscherichia coli alanyl-tRNA synthetase quaternary structureunder increased pressurerdquo Archives of Biochemistry and Bio-physics vol 346 no 2 pp 322ndash323 1997

[8] S M Sood C W Slattery S J Filley M-X Wu and K A WHill ldquoFurther characterization of Escherichia coli alanyl-tRNAsynthetaserdquoArchives of Biochemistry andBiophysics vol 328 no2 pp 295ndash301 1996

[9] J D Dignam J Guo W P Griffith N C Garbett A Hol-loway and T Mueser ldquoAllosteric interaction of nucleotides andtRNAala with E coli alanyl-tRNA synthetaserdquo Biochemistry vol50 no 45 pp 9886ndash9900 2011

[10] M Naganuma S-I Sekine R Fukunaga and S YokoyamaldquoUnique protein architecture of alanyl-tRNA synthetase foraminoacylation editing and dimerizationrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 21 pp 8489ndash8494 2009

[11] S D Putney and P Schimmel ldquoAn aminoacyl tRNA synthetasebinds to a specific DNA sequence and regulates its genetranscriptionrdquo Nature vol 291 no 5817 pp 632ndash635 1981

[12] M Naganuma S-I Sekine Y E Chong et al ldquoThe selectivetRNA aminoacylation mechanism based on a single GsdotU pairrdquoNature vol 510 no 7506 pp 507ndash511 2014

[13] M Guo Y E Chong R Shapiro K Beebe X-L Yang andP Schimmel ldquoParadox of mistranslation of serine for alaninecaused by AlaRS recognition dilemmardquo Nature vol 462 no7274 pp 808ndash812 2009

[14] M A Swairjo and P R Schimmel ldquoBreaking sieve for stericexclusion of a noncognate amino acid from active site of a tRNAsynthetaserdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 102 no 4 pp 988ndash993 2005

[15] M Sokabe T Ose A Nakamura et al ldquoThe structure ofalanyl-tRNA synthetasewith editing domainrdquoProceedings of theNational Academy of Sciences of theUnited States of America vol106 no 27 pp 11028ndash11033 2009

[16] M Guo Y E Chong K Beebe R Shapiro X-L Yang andP Schimmel ldquoThe C-Ala domain brings together editing andaminoacylation functions on one tRNArdquo Science vol 325 no5941 pp 744ndash747 2009

[17] M A Swairjo F J Otero X-L Yang et al ldquoAlanyl-tRNAsynthetase crystal structure and design for acceptor-stem recog-nitionrdquoMolecular Cell vol 13 no 6 pp 829ndash841 2004

[18] S D Putney N J Royal H Neuman de Vegvar W C HerlihyK Biemann and P Schimmel ldquoPrimary structure of a largeaminoacyl-tRNA synthetaserdquo Science vol 213 no 4515 pp1497ndash1501 1981

[19] M Jasin L Regan and P Schimmel ldquoTwo mutations in thedispensable part of alanine tRNA synthetase which affect thecatalytic activityrdquo The Journal of Biological Chemistry vol 260no 4 pp 2226ndash2230 1985

[20] N Gull P Sen R H Khan and Kabir-Ud-Din ldquoSpectroscopicstudies on the comparative interaction of cationic single-chainand gemini surfactants with human serum albuminrdquo Journal ofBiochemistry vol 145 no 1 pp 67ndash77 2009

[21] Y-H Chen J T Yang and H M Martinez ldquoDeterminationof the secondary structures of proteins by circular dichroismand optical rotatory dispersionrdquo Biochemistry vol 11 no 22 pp4120ndash4131 1972


[22] J D Dignam X Qu and J B Chaires ldquoEquilibrium unfoldingof Bombyx mori glycyl-tRNA synthetaserdquo Journal of BiologicalChemistry vol 276 no 6 pp 4028ndash4037 2001

[23] M C Moncrieffe J G Grossmann and N J Gay ldquoAssemblyof oligomeric death domain complexes during Toll receptorsignalingrdquoThe Journal of Biological Chemistry vol 283 no 48pp 33447ndash33454 2008

[24] P Schuck ldquoSize-distribution analysis of macromolecules bysedimentation velocity ultracentrifugation and Lamm equationmodelingrdquo Biophysical Journal vol 78 no 3 pp 1606ndash16192000

[25] J Vistica J Dam A Balbo et al ldquoSedimentation equilib-rium analysis of protein interactions with global implicit massconservation constraints and systematic noise decompositionrdquoAnalytical Biochemistry vol 326 no 2 pp 234ndash256 2004

[26] SM Kelly andN C Price ldquoThe use of circular dichroism in theinvestigation of protein structure and functionrdquoCurrent Proteinamp Peptide Science vol 1 no 4 pp 349ndash384 2000

[27] M R Eftink ldquoIntrinsic fluorescence of proteinsrdquo in Topicsin Fluorescence Spectroscopy vol 6 of Topics in FluorescenceSpectroscopy pp 1ndash15 Springer New York NY USA 2000

[28] C N Pace ldquoDetermination and analysis of urea and guanidinehydrochloride denaturation curvesrdquo inMethods in Enzymologyvol 131 pp 266ndash280 Elsevier 1986

[29] B Banerjee and R Banerjee ldquoGuanidine hydrochloride medi-ated denaturation of E coli alanyl-tRNA synthetase identifica-tion of an inactive dimeric intermediaterdquo The Protein Journalvol 33 no 2 pp 119ndash127 2014

[30] G V Semisotnov N A Rodionova O I Razgulyaev V NUversky A F Gripasrsquo and R I Gilmanshin ldquoStudy of thelsquomolten globulersquo intermediate state in protein folding by ahydrophobic fluorescent proberdquo Biopolymers vol 31 no 1 pp119ndash128 1991

[31] H Christensen and R H Pain ldquoMolten globule intermediatesand protein foldingrdquo European Biophysics Journal vol 19 no 5pp 221ndash229 1991

[32] M Jasin L Regan and P Schimmel ldquoModular arrangement offunctional domains along the sequence of an aminoacyl tRNAsynthetaserdquo Nature vol 306 no 5942 pp 441ndash447 1983

[33] M Jasin L Regan and P Schimmel ldquoDispensable pieces of anaminoacyl tRNA synthetase which activate the catalytic siterdquoCell vol 36 no 4 pp 1089ndash1095 1984

[34] S Bjelic A Karshikoff and I Jelesarov ldquoStability and fold-ingunfolding kinetics of the homotrimeric coiled coil Lpp-56rdquoBiochemistry vol 45 no 29 pp 8931ndash8939 2006

[35] M S Gittelman and C R Matthews ldquoFolding and stability oftrp aporepressor fromEscherichia colirdquoBiochemistry vol 29 no30 pp 7011ndash7020 1990

[36] S Blond and M E Goldberg ldquoKinetics and importance of thedimerization step in the folding pathway of the 1205732 subunitof Escherichia coli tryptophan synthaserdquo Journal of MolecularBiology vol 182 no 4 pp 597ndash606 1985

[37] M G Mateu ldquoConformational stability of dimeric andmonomeric forms of the C-terminal domain of human immun-odeficiency virus-1 capsid proteinrdquo Journal ofMolecular Biologyvol 318 no 2 pp 519ndash531 2002

[38] M E Milla and R T Sauer ldquoP22 Arc repressor folding kineticsof a single-domain dimeric proteinrdquo Biochemistry vol 33 no5 pp 1125ndash1133 1994

[39] O D Monera C M Kay and R S Hodges ldquoProtein denatura-tion with guanidine hydrochloride or urea provides a different

estimate of stability depending on the contributions of electro-static interactionsrdquo Protein Science vol 3 no 11 pp 1984ndash19911994

[40] M S Akhtar A Ahmad and V Bhakuni ldquoGuanidiniumchloride- and urea-induced unfolding of the dimeric enzymeglucose oxidaserdquo Biochemistry vol 41 no 11 pp 3819ndash38272002

[41] A C Clark J F Sinclair andTO Baldwin ldquoFolding of bacterialluciferase involves a non-native heterodimeric intermediate inequilibriumwith the native enzyme and the unfolded subunitsrdquoThe Journal of Biological Chemistry vol 268 no 15 pp 10773ndash10779 1993

[42] T A Dar L R Singh A Islam F Anjum A A Moosavi-Movahedi and F Ahmad ldquoGuanidinium chloride and ureadenaturations of 120573-lactoglobulin A at pH 20 and 25 ∘C theequilibrium intermediate contains non-native structures (helixtryptophan and hydrophobic patches)rdquo Biophysical Chemistryvol 127 no 3 pp 140ndash148 2007

[43] M Aatif S Rahman and B Bano ldquoProtein unfolding studiesof thiol-proteinase inhibitor from goat (Capra hircus) musclein the presence of urea and GdnHCl as denaturantsrdquo EuropeanBiophysics Journal vol 40 no 5 pp 611ndash617 2011

[44] A R Singh S Joshi R Arya A M Kayastha and J KSaxena ldquoGuanidine hydrochloride and urea-induced unfoldingof Brugia malayi hexokinaserdquo European Biophysics Journal vol39 no 2 pp 289ndash297 2010

[45] M R Eftink ldquoUse of multiple spectroscopic methods to moni-tor equilibrium unfolding of proteinsrdquoMethods in Enzymologyvol 259 pp 487ndash512 1995

[46] S Sinha and A Surolia ldquoOligomerization endows enormousstability to soybean agglutinin a comparison of the stabilityof monomer and tetramer of soybean agglutininrdquo BiophysicalJournal vol 88 no 6 pp 4243ndash4251 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the same enzyme from E coli Aquifex aeolicus Pyrococcushorikoshii and Archaeoglobus fulgidus was also reportedeither in complex with its ligands or in absence of any [10 13ndash17] Previous reports showed that the N-terminal 461 aminoacid residues are sufficient for the aminoacylation of tRNAAla

[18] Moreover alaRS monomer containing the N-terminal700 amino acids has almost the same 119896cat value for tRNA

Ala

aminoacylation when compared to the full-length dimeric(875 amino acidsmonomer) protein [18] In another study itwas shown that point mutation in the amino acid residue 674(where the glycine has been changed to aspartic acid) resultsin a full-length monomer that has 20-fold reduction in the119896cat value for tRNA aminoacylation [19]

Keeping inmind the unusual features of this enzymewithlarge multidomain dimeric structure in this study we haveinvestigated how the C-terminal domain affects the stabilityof this enzyme In this report we have constructed threemutant proteins The first mutant is a full-length monomerhaving a mutation in the 674 position (G674D previouslydemonstrated to form full-length monomer vide supra)The second mutant contains the N-terminal aminoacylationdomain (1ndash700 N700) The third mutant has N-terminal 461amino acids (1ndash461 N461) aminimum fragment required forthe proper aminoacylation of tRNAAlaThesemutant proteinsand the wild type enzyme were subjected to denaturationin the presence of urea to decipher the unfolding pathwayof these proteins under equilibrium condition This studyreveals that WT-alaRS is more stable than the mutant alaRSsand also have the capacity to refold back from the fullydenatured state unlike the other mutants studied here

2 Materials and Methods

21 Materials The clone of full-length E coli alaRS (WT-alaRS) N700 alaRS and plasmid containing tRNAAla tran-script was a kind gift from Professor Paul Schimmel (SkaggsInstitute for Chemical Biology The Scripps Research Insti-tute California) All the chemicals that are used in thisstudy are of analytical grade and were purchased from Sigma(St Louis MO) SRL (India) E-Merck (Germany) and HI-MEDIA (India)

22 Construction of E coli N461 and G674D alaRS MutantFor construction of N461 truncated enzyme and G674D vari-ant the primer 51015840ATT TAAAGGCTA TGA CTAACTGGAACT GAA 31015840 and 51015840 AGCCGCA CTGATGATAT TGGT 31015840were used respectively Mutagenesis was carried out by usingin vitro site-directed mutagenesis protocol by Stratagene andthe mutation was confirmed by DNA sequencing

23 Purification of E coli Alanyl-tRNA Synthetases Wild typealaRS N461 and G674D alaRS with a N-terminal histidinetag were expressed in BL21 (DE3) after transformation ofplasmid pET28a containing the desired constructs The plas-mid pET20a encoding N-terminal histidine tag N700 alaRSwas also transformed in BL21 (DE3) Single colonies werepicked in all cases inoculated into 10mL of Luria-Bertani(LB) medium containing 50 120583gmL Kanamycin except N700where 100 120583gmL of ampicillin was added and grown

overnight at 37∘C with constant shaking at 250 rpm On thefollowingmorning 1 of overnight culturewas transferred to1 L of LB medium and grown at 37∘C until OD

600= 06 cells

were then induced with 1mM of IPTG and further grownat the same temperature for 4 hrs The cells were pelleted bycentrifugation at 5000 rpm for 15min at 4∘C Except N700alaRS the pelleted cells were lysedwith bufferA (50mMTris-HCl pH 80 containing 500mMNaCl 10mM imidazole 10glycerol and 1mM 120573-mercaptoethanol) For N700 alaRS thebuffer composition was 100mMTris-HCl pH 80 containing10 glycerol and 1mM 120573-mercaptoethanol The suspendedcells were sonicated and centrifuged at 25000 rpm for 1 hThe supernatant was loaded on an Ni-NTA agarose column(preequilibrated with buffer A)The columnwas thenwashedwith 2 column volumes of buffer A finally E coli alaRSand its mutants were eluted with a gradient of imidazoleconcentration ranging from 20 to 200mM in buffer A All theeluted fractions were checked by 12 sodiumdodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis The majoreluted fractions containing desired proteins were pooledand dialyzed against buffer C (100mM Tris-HCl pH 80containing 100mMNaCl and 20 glycerol) at 4∘C After 36ndash40 hrs of dialysis samples were collected from the dialysisbag and protein concentration was determined by measuringabsorbance at 280 nm using the molar extinction coefficientof the protein (71195Mminus1 cmminus1 for WT-alaRS as a monomerand G674D 65320Mminus1 cmminus1 for N700 and 60580Mminus1 cmminus1for N461)

24 Fluorescence Spectroscopy Fluorescence measurementswere performed in a Hitachi F-7000 spectrofluorimeter with1mm quartz cuvette at 25∘C All the experiments wererepeated at least 3 times The fluorescence emission wasrecorded in the wavelength region of 310ndash450 nm after excit-ing the protein solution at 295 nm for selective observationof tryptophan residues The bandpasses were 5 nm for bothexcitation and emission The protein concentrations were4 120583M Proteins were incubated in the presence or absenceof increasing concentration of urea (0ndash8M) for 16ndash18 h at25∘C in 100mM Tris-HCl pH 80 buffer at room tempera-ture before the spectra were recorded Controls containingappropriate concentration of denaturants were prepared andwere subtracted from the corresponding samples spectra Forthe experiments where ANS (30 120583M) had been used theexcitation wavelength was 420 nm to avoid inner filter effectand emission was recorded at 482 nm Appropriate blankswere also subtracted from each experiment

25 Circular Dichroism (CD) Spectroscopy The CD spectrameasurements were carried out in a JASCO J-815 spec-tropolarimeter using 100mM Tris-HCl (pH 80) bufferThe spectra were recorded at 25∘C using 1mm and 10mmpath length cuvettes for far-UV (210ndash260 nm) and near-UV(260ndash320 nm) CD measurement respectively with a scanspeed 50 nmmin The final spectrum was average of fiveindependent measurements For far-UV and near-UV CDexperiments the protein concentrations were kept at 4120583Mand 15 120583M respectively The sample spectra were correctedby subtracting from the buffer spectra containing the same





1 + 119890minus(Δ1198661minus1198981[119863])RT (1)


and 119878119880


=119878119873119878119868119890minus(Δ1198661minus1198981[119863])RT + 119878



(2)


119873 119878119868 and 119878

119880denote the


1= Δ119866

119874+ 119898119897[119863] There are






119868 the value of






3 Result






(Kcalmol)1198981

(KcalmolM)1198982









Abso

rban

ce (O

D)

Radius (cm)

2

18

16

14

12

1

08585 59 595 6 605 61 615

002

001

0

Resid

uals

Radius (cm)585 59 595 6 605 61 615

WT-alaRSG674D


minus002

minus001

06

055

05

045

04

035

03

025

02685 69 695 7 705 71 715

Abso

rban

ce (O

D)

Radius (cm)

685 69 695 7 705 71 715

Radius (cm)

006

004

002

0

Resid

uals

minus002

minus004

minus006

Abso

rban

ce (O

D)

Radius (cm)

12

11

1

09

08

07

06

05635 64 645 65 655 66 665

Radius (cm)635 64 645 65 655 66 665

006

004

002

0Re

sidua

ls

minus002

minus004

minus006

(a)

Figure 1 Continued


0

minus200

minus400

minus600

minus800

minus1000

minus1200

minus1400210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

WT-alaRSG674D


(b)

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

WT-alaRSG674D


10

8

6

4

2

0

260 270 280 290 300 310 320

(c)


12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(a)

12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(b)

120582m

ax(n

m)

352

350

348

346

344

342

340

338

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

(c)

12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(d)


340119865350



WT-alaRSG674D


3

25

2

15

1

05

00 2 4 6 8

F482

Urea (M)



222) was





CD Δ1198661

(Kcalmol)Δ1198662

(Kcalmol)1198981

(KcalmolM)1198982






200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

minus1000210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(a)

200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus1000

minus800

minus1200

minus1000

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(b)200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(c)

1000

500

0

minus500

minus1000

minus1500

0

minus200

minus400

minus600

minus1000

minus1200

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(d)





4 Discussion




50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(a)

12 14 16

50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(b)35

30

25

20

15

10

5

012 14 160 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(c)

30

25

20

15

10

5

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(d)




340119865350





6

4

2

0

minus2

MRE

(deg

middotcm2middotd

molminus1)

260 270 280 290 300 310 320

Wavelength (nm)

(a)

MRE

(deg

middotcm2middotd

molminus1)

7

6

5

4

3

2

1

0260 270 280 290 300 310 320

Wavelength (nm)

(b)

MRE

(deg

middotcm2middotd

molminus1)

6

4

2

0

260 270 280 290 300 310 320

Wavelength (nm)

(c)

MRE

(deg

middotcm2middotd

molminus1)

10

8

6

4

2

0

minus2

minus4

260 270 280 290 300 310 320

Wavelength (nm)

(d)




5 Conclusion


Abbreviations





04

03

02

01

02 4 6 8 10 12


c(s)

0M05M15M2M

25M3M35M8M


Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





1 + 119890minus(Δ1198661minus1198981[119863])RT (1)


and 119878119880


=119878119873119878119868119890minus(Δ1198661minus1198981[119863])RT + 119878



(2)


119873 119878119868 and 119878

119880denote the


1= Δ119866

119874+ 119898119897[119863] There are






119868 the value of






3 Result






(Kcalmol)1198981

(KcalmolM)1198982









Abso

rban

ce (O

D)

Radius (cm)

2

18

16

14

12

1

08585 59 595 6 605 61 615

002

001

0

Resid

uals

Radius (cm)585 59 595 6 605 61 615

WT-alaRSG674D


minus002

minus001

06

055

05

045

04

035

03

025

02685 69 695 7 705 71 715

Abso

rban

ce (O

D)

Radius (cm)

685 69 695 7 705 71 715

Radius (cm)

006

004

002

0

Resid

uals

minus002

minus004

minus006

Abso

rban

ce (O

D)

Radius (cm)

12

11

1

09

08

07

06

05635 64 645 65 655 66 665

Radius (cm)635 64 645 65 655 66 665

006

004

002

0Re

sidua

ls

minus002

minus004

minus006

(a)

Figure 1 Continued


0

minus200

minus400

minus600

minus800

minus1000

minus1200

minus1400210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

WT-alaRSG674D


(b)

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

WT-alaRSG674D


10

8

6

4

2

0

260 270 280 290 300 310 320

(c)


12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(a)

12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(b)

120582m

ax(n

m)

352

350

348

346

344

342

340

338

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

(c)

12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(d)


340119865350



WT-alaRSG674D


3

25

2

15

1

05

00 2 4 6 8

F482

Urea (M)



222) was





CD Δ1198661

(Kcalmol)Δ1198662

(Kcalmol)1198981

(KcalmolM)1198982






200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

minus1000210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(a)

200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus1000

minus800

minus1200

minus1000

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(b)200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(c)

1000

500

0

minus500

minus1000

minus1500

0

minus200

minus400

minus600

minus1000

minus1200

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(d)





4 Discussion




50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(a)

12 14 16

50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(b)35

30

25

20

15

10

5

012 14 160 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(c)

30

25

20

15

10

5

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(d)




340119865350





6

4

2

0

minus2

MRE

(deg

middotcm2middotd

molminus1)

260 270 280 290 300 310 320

Wavelength (nm)

(a)

MRE

(deg

middotcm2middotd

molminus1)

7

6

5

4

3

2

1

0260 270 280 290 300 310 320

Wavelength (nm)

(b)

MRE

(deg

middotcm2middotd

molminus1)

6

4

2

0

260 270 280 290 300 310 320

Wavelength (nm)

(c)

MRE

(deg

middotcm2middotd

molminus1)

10

8

6

4

2

0

minus2

minus4

260 270 280 290 300 310 320

Wavelength (nm)

(d)




5 Conclusion


Abbreviations





04

03

02

01

02 4 6 8 10 12


c(s)

0M05M15M2M

25M3M35M8M


Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


3 Result






(Kcalmol)1198981

(KcalmolM)1198982









Abso

rban

ce (O

D)

Radius (cm)

2

18

16

14

12

1

08585 59 595 6 605 61 615

002

001

0

Resid

uals

Radius (cm)585 59 595 6 605 61 615

WT-alaRSG674D


minus002

minus001

06

055

05

045

04

035

03

025

02685 69 695 7 705 71 715

Abso

rban

ce (O

D)

Radius (cm)

685 69 695 7 705 71 715

Radius (cm)

006

004

002

0

Resid

uals

minus002

minus004

minus006

Abso

rban

ce (O

D)

Radius (cm)

12

11

1

09

08

07

06

05635 64 645 65 655 66 665

Radius (cm)635 64 645 65 655 66 665

006

004

002

0Re

sidua

ls

minus002

minus004

minus006

(a)

Figure 1 Continued


0

minus200

minus400

minus600

minus800

minus1000

minus1200

minus1400210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

WT-alaRSG674D


(b)

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

WT-alaRSG674D


10

8

6

4

2

0

260 270 280 290 300 310 320

(c)


12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(a)

12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(b)

120582m

ax(n

m)

352

350

348

346

344

342

340

338

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

(c)

12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(d)


340119865350



WT-alaRSG674D


3

25

2

15

1

05

00 2 4 6 8

F482

Urea (M)



222) was





CD Δ1198661

(Kcalmol)Δ1198662

(Kcalmol)1198981

(KcalmolM)1198982






200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

minus1000210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(a)

200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus1000

minus800

minus1200

minus1000

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(b)200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(c)

1000

500

0

minus500

minus1000

minus1500

0

minus200

minus400

minus600

minus1000

minus1200

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(d)





4 Discussion




50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(a)

12 14 16

50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(b)35

30

25

20

15

10

5

012 14 160 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(c)

30

25

20

15

10

5

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(d)




340119865350





6

4

2

0

minus2

MRE

(deg

middotcm2middotd

molminus1)

260 270 280 290 300 310 320

Wavelength (nm)

(a)

MRE

(deg

middotcm2middotd

molminus1)

7

6

5

4

3

2

1

0260 270 280 290 300 310 320

Wavelength (nm)

(b)

MRE

(deg

middotcm2middotd

molminus1)

6

4

2

0

260 270 280 290 300 310 320

Wavelength (nm)

(c)

MRE

(deg

middotcm2middotd

molminus1)

10

8

6

4

2

0

minus2

minus4

260 270 280 290 300 310 320

Wavelength (nm)

(d)




5 Conclusion


Abbreviations





04

03

02

01

02 4 6 8 10 12


c(s)

0M05M15M2M

25M3M35M8M


Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Abso

rban

ce (O

D)

Radius (cm)

2

18

16

14

12

1

08585 59 595 6 605 61 615

002

001

0

Resid

uals

Radius (cm)585 59 595 6 605 61 615

WT-alaRSG674D


minus002

minus001

06

055

05

045

04

035

03

025

02685 69 695 7 705 71 715

Abso

rban

ce (O

D)

Radius (cm)

685 69 695 7 705 71 715

Radius (cm)

006

004

002

0

Resid

uals

minus002

minus004

minus006

Abso

rban

ce (O

D)

Radius (cm)

12

11

1

09

08

07

06

05635 64 645 65 655 66 665

Radius (cm)635 64 645 65 655 66 665

006

004

002

0Re

sidua

ls

minus002

minus004

minus006

(a)

Figure 1 Continued


0

minus200

minus400

minus600

minus800

minus1000

minus1200

minus1400210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

WT-alaRSG674D


(b)

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

WT-alaRSG674D


10

8

6

4

2

0

260 270 280 290 300 310 320

(c)


12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(a)

12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(b)

120582m

ax(n

m)

352

350

348

346

344

342

340

338

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

(c)

12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(d)


340119865350



WT-alaRSG674D


3

25

2

15

1

05

00 2 4 6 8

F482

Urea (M)



222) was





CD Δ1198661

(Kcalmol)Δ1198662

(Kcalmol)1198981

(KcalmolM)1198982






200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

minus1000210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(a)

200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus1000

minus800

minus1200

minus1000

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(b)200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(c)

1000

500

0

minus500

minus1000

minus1500

0

minus200

minus400

minus600

minus1000

minus1200

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(d)





4 Discussion




50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(a)

12 14 16

50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(b)35

30

25

20

15

10

5

012 14 160 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(c)

30

25

20

15

10

5

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(d)




340119865350





6

4

2

0

minus2

MRE

(deg

middotcm2middotd

molminus1)

260 270 280 290 300 310 320

Wavelength (nm)

(a)

MRE

(deg

middotcm2middotd

molminus1)

7

6

5

4

3

2

1

0260 270 280 290 300 310 320

Wavelength (nm)

(b)

MRE

(deg

middotcm2middotd

molminus1)

6

4

2

0

260 270 280 290 300 310 320

Wavelength (nm)

(c)

MRE

(deg

middotcm2middotd

molminus1)

10

8

6

4

2

0

minus2

minus4

260 270 280 290 300 310 320

Wavelength (nm)

(d)




5 Conclusion


Abbreviations





04

03

02

01

02 4 6 8 10 12


c(s)

0M05M15M2M

25M3M35M8M


Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

minus200

minus400

minus600

minus800

minus1000

minus1200

minus1400210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

WT-alaRSG674D


(b)

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

WT-alaRSG674D


10

8

6

4

2

0

260 270 280 290 300 310 320

(c)


12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(a)

12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(b)

120582m

ax(n

m)

352

350

348

346

344

342

340

338

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

(c)

12

115

11

105

1

095

09

0850 2 4 6 8

Urea (M)

F340F

350

350

345

340

335

120582m

ax(n

m)

(d)


340119865350



WT-alaRSG674D


3

25

2

15

1

05

00 2 4 6 8

F482

Urea (M)



222) was





CD Δ1198661

(Kcalmol)Δ1198662

(Kcalmol)1198981

(KcalmolM)1198982






200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

minus1000210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(a)

200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus1000

minus800

minus1200

minus1000

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(b)200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(c)

1000

500

0

minus500

minus1000

minus1500

0

minus200

minus400

minus600

minus1000

minus1200

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(d)





4 Discussion




50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(a)

12 14 16

50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(b)35

30

25

20

15

10

5

012 14 160 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(c)

30

25

20

15

10

5

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(d)




340119865350





6

4

2

0

minus2

MRE

(deg

middotcm2middotd

molminus1)

260 270 280 290 300 310 320

Wavelength (nm)

(a)

MRE

(deg

middotcm2middotd

molminus1)

7

6

5

4

3

2

1

0260 270 280 290 300 310 320

Wavelength (nm)

(b)

MRE

(deg

middotcm2middotd

molminus1)

6

4

2

0

260 270 280 290 300 310 320

Wavelength (nm)

(c)

MRE

(deg

middotcm2middotd

molminus1)

10

8

6

4

2

0

minus2

minus4

260 270 280 290 300 310 320

Wavelength (nm)

(d)




5 Conclusion


Abbreviations





04

03

02

01

02 4 6 8 10 12


c(s)

0M05M15M2M

25M3M35M8M


Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


WT-alaRSG674D


3

25

2

15

1

05

00 2 4 6 8

F482

Urea (M)



222) was





CD Δ1198661

(Kcalmol)Δ1198662

(Kcalmol)1198981

(KcalmolM)1198982






200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

minus1000210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(a)

200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus1000

minus800

minus1200

minus1000

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(b)200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(c)

1000

500

0

minus500

minus1000

minus1500

0

minus200

minus400

minus600

minus1000

minus1200

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(d)





4 Discussion




50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(a)

12 14 16

50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(b)35

30

25

20

15

10

5

012 14 160 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(c)

30

25

20

15

10

5

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(d)




340119865350





6

4

2

0

minus2

MRE

(deg

middotcm2middotd

molminus1)

260 270 280 290 300 310 320

Wavelength (nm)

(a)

MRE

(deg

middotcm2middotd

molminus1)

7

6

5

4

3

2

1

0260 270 280 290 300 310 320

Wavelength (nm)

(b)

MRE

(deg

middotcm2middotd

molminus1)

6

4

2

0

260 270 280 290 300 310 320

Wavelength (nm)

(c)

MRE

(deg

middotcm2middotd

molminus1)

10

8

6

4

2

0

minus2

minus4

260 270 280 290 300 310 320

Wavelength (nm)

(d)




5 Conclusion


Abbreviations





04

03

02

01

02 4 6 8 10 12


c(s)

0M05M15M2M

25M3M35M8M


Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

minus1000210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(a)

200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus1000

minus800

minus1200

minus1000

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(b)200

0

minus200

minus400

minus600

minus800

0

minus200

minus400

minus600

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(c)

1000

500

0

minus500

minus1000

minus1500

0

minus200

minus400

minus600

minus1000

minus1200

minus800

210 220 230 240 250 260

MRE

(deg

middotcm2middotd

molminus1)

Wavelength (nm)

0 2 4 6 8

Urea (M)

MRE

222

(deg

middotcm2middotd

molminus1)

(d)





4 Discussion




50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(a)

12 14 16

50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(b)35

30

25

20

15

10

5

012 14 160 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(c)

30

25

20

15

10

5

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(d)




340119865350





6

4

2

0

minus2

MRE

(deg

middotcm2middotd

molminus1)

260 270 280 290 300 310 320

Wavelength (nm)

(a)

MRE

(deg

middotcm2middotd

molminus1)

7

6

5

4

3

2

1

0260 270 280 290 300 310 320

Wavelength (nm)

(b)

MRE

(deg

middotcm2middotd

molminus1)

6

4

2

0

260 270 280 290 300 310 320

Wavelength (nm)

(c)

MRE

(deg

middotcm2middotd

molminus1)

10

8

6

4

2

0

minus2

minus4

260 270 280 290 300 310 320

Wavelength (nm)

(d)




5 Conclusion


Abbreviations





04

03

02

01

02 4 6 8 10 12


c(s)

0M05M15M2M

25M3M35M8M


Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(a)

12 14 16

50

40

30

20

10

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(b)35

30

25

20

15

10

5

012 14 160 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(c)

30

25

20

15

10

5

00 2 4 6 8 10

Time (min)

[3H

]Ala

-tRN

AA

la(p

mol

)

(d)




340119865350





6

4

2

0

minus2

MRE

(deg

middotcm2middotd

molminus1)

260 270 280 290 300 310 320

Wavelength (nm)

(a)

MRE

(deg

middotcm2middotd

molminus1)

7

6

5

4

3

2

1

0260 270 280 290 300 310 320

Wavelength (nm)

(b)

MRE

(deg

middotcm2middotd

molminus1)

6

4

2

0

260 270 280 290 300 310 320

Wavelength (nm)

(c)

MRE

(deg

middotcm2middotd

molminus1)

10

8

6

4

2

0

minus2

minus4

260 270 280 290 300 310 320

Wavelength (nm)

(d)




5 Conclusion


Abbreviations





04

03

02

01

02 4 6 8 10 12


c(s)

0M05M15M2M

25M3M35M8M


Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


6

4

2

0

minus2

MRE

(deg

middotcm2middotd

molminus1)

260 270 280 290 300 310 320

Wavelength (nm)

(a)

MRE

(deg

middotcm2middotd

molminus1)

7

6

5

4

3

2

1

0260 270 280 290 300 310 320

Wavelength (nm)

(b)

MRE

(deg

middotcm2middotd

molminus1)

6

4

2

0

260 270 280 290 300 310 320

Wavelength (nm)

(c)

MRE

(deg

middotcm2middotd

molminus1)

10

8

6

4

2

0

minus2

minus4

260 270 280 290 300 310 320

Wavelength (nm)

(d)




5 Conclusion


Abbreviations





04

03

02

01

02 4 6 8 10 12


c(s)

0M05M15M2M

25M3M35M8M


Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


04

03

02

01

02 4 6 8 10 12


c(s)

0M05M15M2M

25M3M35M8M


Acknowledgments


References
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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