Uversky_2002_Natively Unfolded Proteins a Point Where Biology Waits for Physics

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REVIEW

Natively unfolded proteins: A point

where biology waits for physicsVLADIMIR N. UVERSKYInstitute for Biological Instrumentation, Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia;Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA

Abstract

The experimental material accumulated in the literature on the conformational behavior of intrinsicallyunstructured (natively unfolded) proteins was analyzed. Results of this analysis showed that these proteins

do not possess uniform structural properties, as expected for members of a single thermodynamic entity.Rather, these proteins may be divided into two structurally different groups: intrinsic coils, and premoltenglobules. Proteins from the first group have hydrodynamic dimensions typical of random coils in poorsolvent and do not possess any (or almost any) ordered secondary structure. Proteins from the second groupare essentially more compact, exhibiting some amount of residual secondary structure, although they are stillless dense than native or molten globule proteins. An important feature of the intrinsically unstructuredproteins is that they undergo disorderorder transition during or prior to their biological function. In thisrespect, the Protein Quartet model, with function arising from four specific conformations (ordered forms,molten globules, premolten globules, and random coils) and transitions between any two of the states, isdiscussed.

Keywords: Intrinsically unfolded protein; intrinsically disordered protein; unfolded protein; molten glob-ule; premolten globule; partially folded intermediate; random coil; conformational transition

This review introduces an intriguing protein family of na-tively unfolded proteins, whose existence questions one ofthe cornerstones in protein biology, chemistry and physics,that is, the structurefunction paradigm. This conceptclaims that a specific function of a protein is determined byits unique and rigid three-dimensional (3D) structure. Thisidea, formulated more than 100 years ago as a lock-and-keymodel for explaining the amazing specificity of the enzy-

matic hydrolysis of glucosides (Fischer 1894), proved to beextremely fruitful. Figuratively speaking, the protein struc-turefunction paradigm may be considered as the big bang,creating the universe of modern protein science. Figure 1attempts to illustrate the most obvious scientific conse-quences of this concept.

However, a reappraisal of the protein structurefunctionparadigm is now warranted based on systematic studies ofintrinsically unfolded/disordered proteins (Wright and Dy-son 1999; Dunker et al. 2001; Uversky 2002). There are two

major reasons for such a reappraisal: the results of theamino acid sequence analyses and the accumulation of ex-perimental evidence for the existence of a rather largeamount of protein domains and even entire proteins, lackingordered structure under physiological conditions. Intri-guingly, both of these sets of evidences were gathered usingscientific concepts and approaches originating from the pro-tein structurefunction paradigm, namely, proteomics andprotein self-organization (see Fig. 1).

Reprint requests to: Vladimir N. Uversky, Department of Chemistry andBiochemistry, University of California, Santa Cruz, CA 95064, USA; e-mail: [email protected]; fax: (831) 459-2935.

Abbreviations: NMR, nuclear magnetic resonance; CD, circular dichro-ism; UV, ultraviolet; ORD, optical rotatory dispersion; FTIR, Fourier-transform infrared; SAXS, small-angle X-ray scattering; SANS, small-angle neutron scattering; FRET, fluorescence resonance energy transfer; N,native; MG, molten globule; PMG, premolten globule; U, unfolded; NU,natively unfolded; NUcoil, natively unfolded proteins with coil-like prop-erties; NUPMG, natively unfolded proteins with PMG-like properties.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.4210102.

Protein Science (2002), 11:739756. Published by Cold Spring Harbor Laboratory Press. Copyright 2002 The Protein Society 739


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tertiary structure. It is characterized by a considerable sec-ondary structure, although much less pronounced than thatof the native or the molten globule protein (protein in thepremolten globule state has 50% native secondary struc-ture, whereas in the molten globule state the correspondingvalue is close to 100%). The protein molecule in the pre-molten globule state is considerably less compact than in themolten globule or native states, but it is still more compactthan the random coil (its hydrodynamic volume in the mol-ten globule, the premolten globule, and the unfolded states,in comparison to that of the native state, increases 1.5, 3,and 12 times, respectively). The protein molecule in thepremolten globule state can effectively interact with the hydro-phobic fluorescent probe ANS, although essentially weakerthan in the molten globule state. This means that at least partof the hydrophobic clusters of polypeptide chain accessibleto the solvent is already formed in the premolten globulestate (Uversky and Ptitsyn 1994, 1996a; Ptitsyn 1995; Uver-sky 1997, 1998). It has also been established that in the

premolten globule state the protein molecule has no globu-lar structure (Uversky 1997, 1998; Uversky et al. 1998). Thelast observation indicates that the premolten globule prob-ably represents a squeezed and partially ordered form ofa coil. Finally, it has been shown that the premolten globuleis separated from the molten globule state by an all-or-nonetransition, which represents an intramolecular analog of thefirst-order phase transition (Uversky and Ptitsyn 1994,1996a; Ptitsyn 1995; Uversky 1997, 1998). This means thatthe molten globule and premolten globule represent diversthermodynamic (phase) states.

As native, molten globule, premolten globule, and un-folded conformations possess defined structural differences

along with increasing amounts of disorder, they may beeasily discriminated from one another by several physico-chemical methods (Uversky 1999). These techniques arebriefly considered below.

X-ray crystallography defines missing electron density inmany protein structures, which may correspond to disor-dered region(s). The increased flexibility of atoms in sucha region leads to the noncoherent X-ray scattering, mak-ing them unobserved (Bloomer et al. 1978; Bode et al.1978; Huber 1979, 1987; Schulz 1979; Alber et al. 1982;Spolar and Record 1994; Lewis et al. 1996; Muchmore etal. 1996; Dunker et al. 1997, 2001; Worbs et al. 2000).

Heteronuclear multidimensional NMR is an extremelypowerful technique for protein 3D structure determina-tion in solution and for the characterization of proteindynamics. Recent advances in this technology have al-lowed the complete assignment of resonances for severalunfolded and partially folded proteins, as well as the dis-ordered fragments of folded proteins (Alexandrescu et al.1994; Logan et al. 1994; Zhang et al. 1994; Cho et al.1996; Kriwacki et al. 1996; Lisse et al. 1996; Donne et al.

1997; Gillespie and Shortle 1997a, 1997b; Penkett et al.1997; Daughdrill et al. 1998; Eliezer et al. 1998; Fletcheret al. 1998; Liu et al. 1998; Mogridge et al. 1998; Zhangand Matthews 1998; Bose et al. 1999; Fiebig et al. 1999;Hazzard et al. 1999; Hershey et al. 1999; Love 1999;Bracken 2001; see also Wright and Dyson 1999, andreferences cited therein).

There are two types of optically active chromophores inproteins: side groups of aromatic amino acid residues,and peptide bonds (Adler et al. 1973; Fasman 1996). CDspectra in the near ultraviolet region (250350 nm), alsocalled the aromatic region, reflect the symmetry of theenvironment of aromatic amino acid residues and, con-sequently, are characteristic of protein tertiary structure.Protein denaturation may be easily detected by the sim-plification of near-UV CD spectrum.

Diminishing of ordered secondary structure may be de-tected by several spectroscopic techniques including far-UV CD (Adler et al. 1973; Provencher and Glckner

1981; Johnson 1988; Woody 1995; Fasman 1996; Kellyand Price 1997; Uversky et al. 2000a), ORD, FTIR (seeUversky et al. 2000a, and reference cited therein) andRaman optical activity (Smyth et al. 2001).

Hydrodynamic parameters obtained from techniques suchas gel-filtration, viscometry, SAXS, SANS, sedimenta-tion, and dynamic and static light scattering may help indetermining whether a protein is compact, or it has be-came unfolded. The unfolding of a protein molecule re-sults in an essential increase in its hydrodynamic volume.For instance, there is a well-documented 1520% in-crease in the hydrodynamic radius of globular proteinsupon their transformation into the molten globule state

(Uversky 1993, 1994; Ptitsyn 1995), while the hydrody-namic volume of the premolten globule is even larger(Uversky and Ptitsyn 1994, 1996a; Ptitsyn 1995; Uversky1997, 1998). Moreover, it has been shown that native andunfolded conformations of globular proteins possess verydifferent molecular mass dependencies of their hydrody-namic radii, RS (Tanford 1961, 1968; Uversky 1993). Asa result, intrinsically disordered proteins will have an in-creased hydrodynamic volume relative to native proteins,leading to an increase in their apparent molecular mass(summarized in Uversky et al. 2000a).

Another very important structural parameter is the degreeof globularity, which reflects the presence or absence of

a tightly packed core in a protein molecule. This infor-mation may be extracted from the analysis of SAXS datain the form of a Kratky plot, whose shape is sensitive tothe conformational state of the scattering protein mol-ecules (Glatter and Kratky 1982; Feigin and Svergun1987; Semisotnov et al. 1996; Uversky et al. 1998). It hasbeen shown that a scattering curve in the Kratky coordi-nates has a characteristic maximum for globular proteinsin either their native or molten globule states (i.e., states

Natively unfolded proteins

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with globular structure). However, if a protein is com-pletely unfolded or in a premolten globule conformation(i.e., with no globular structure), such a maximum will beabsent (Glatter and Kratky 1982; Feigin and Svergun1987; Semisotnov et al. 1996; Uversky 1997, 1998; Uver-sky et al. 1998; Tcherkasskaya and Uversky 2001).

Additional knowledge on the intramolecular mobility andcompactness of a protein may be extracted from theanalysis of different fluorescence characteristics. This in-cludes FRET, shape and position of the intrinsic fluores-cence spectrum, fluorescence anisotropy and lifetime, ac-cessibility of the chromophore groups to external quench-ers, and steady-state and time-resolved parameters of thefluorescent dyes. Overall, these techniques add importantinformation to the conformational description of a poly-peptide.

Increased proteolytic degradation in vitro of intrinsicallydisordered proteins indirectly confirmed by their in-creased flexibility (Markus 1965; Mikhalyi 1978; Fon-

tana et al. 1993; Hubbard et al. 1994, 1998; Kriwacki etal. 1996; Lisse et al. 1996; Horiuchi et al. 1997; Hubbard1998; Hershey et al. 1999; Ratnaswamy et al. 1999;Bouivier and Stafford 2000; Iakoucheva et al. 2001; seeDunker et al. 2001, for recent review).

Immunochemical methods may also be applied towardthe elucidation of protein disorder. Important to this dis-cussion, the immunoglobulins obtained against a givenprotein may be specific for different levels of macromol-ecule: the primary structure (Amit et al. 1985; Wilson etal. 1985), the secondary structure (Fujio et al. 1985), orthe tertiary structure (Amit et al. 1985; Fujio et al. 1985;Wilson et al. 1985). In the latter case, the antigenic de-

terminants may reside on either the neighboring residuesin the chain (loops) (Amit et al. 1985; Wilson et al. 1985)or on spatially distant residues (Fujio et al. 1985). Fur-thermore, it has been shown that antibodies in the im-mune serum may possess a high affinity to the internalelements of an antigen (Fujio et al. 1985). Thus, antibod-ies may be successfully used to study the structuralchanges, which a protein-immunogen undergoes uponchanges of the experimental conditions. For example, an-tibodies obtained against the Ca2+-saturated F1-fragmentof prothrombin did not interact with the calcium-free apo-form of this protein (Furie and Furie 1979). An analogouseffect was also observed in the case of osteocalcine (Del-

mas et al. 1984). Finally, intrinsic disorder may be detected by the analysis

of protein conformational stability. For example, the pres-ence or absence of a cooperative transition on the calo-rimetric melting curve for a given protein is a simple andconvenient criterion indicating the presence or absence ofa rigid tertiary structure (Privalov 1979; Ptitsyn 1995;Uversky 1999). Furthermore, it has been shown that thesteepness of urea- or guanidinium chloride-induced un-

folding curves depends strongly on whether a given pro-tein has a rigid tertiary structure (i.e., it is native) or isalready denatured and exists as a molten globule (Ptitsynand Uversky 1994; Uversky and Ptitsyn 1996b). To ex-tend this type of analysis, the values ofeff (which is thedifference in the number of denaturant moleculesbound to one protein molecule in its two states) shouldbe determined. Then this quantity should be compared tothe effNU and

effMGU values corresponding to the

native to coil and molten globule to coil transitions inglobular protein of a given molecular mass, respectively(Uversky and Ptitsyn 1996b).

Application of several techniques mentioned above to agiven protein provides the most unambiguous evidence forthe presence of partially folded intermediates.

It has been shown that a considerable number of proteinspossess some amount of disorder rather than rigid structure.A special term, natively denatured, was introduced in

1994 (Schweers et al. 1994) to emphasize the existence of adrastic structural difference between normal globular pro-tein, with rigid tertiary structure, and an abnormal ex-tremely flexible tau protein. Two years later, a new termnatively unfolded originated as a result of conformationalanalysis of-synuclein, which under physiological condi-tions appeared to lack any secondary structure (Weinreb etal. 1996). Two alternative terms, intrinsically unstruc-tured (Wright and Dyson 1999) and intrinsically disor-dered (Dunker et al. 2001), have also been suggested todescribe these proteins. Because abnormal proteins showan extremely wide diversity in their structural properties, themeaning of the above terms should be clarified. Thus, the

terms denaturedand disorderedmay be considered as syn-onyms, and indicate any set of nonrigid conformations ofpolypeptide chains including different compact partiallyfolded conformations: molten globules and premolten glob-ules, and random coil. The terms unstructuredand unfoldedmay be considered synonymous, and should only be appliedto the subset of disordered proteins characterized by theabsence of any (or almost any) ordered structure. For theremaining of this review, only natively unfolded proteinswill be considered, excluding native molten globules.

Are natively unfolded proteins common?

The number of proteins and protein domains that have beenshown in vitro to have little or no ordered structure underphysiological conditions is rapidly expanding. For example,over the past 10 years there has been a significant increasein publications describing structural properties of intrinsi-cally unstructured (natively unfolded) proteins, startingfrom two papers in 1991 and ending with more than 30 in2000. During the same time other interesting aspects ofnatively unfolded proteins have also been investigated. For

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example, 2382, 1960, and 370 papers were published con-cerning different aspects of amyloid-beta peptide, tau pro-tein, and -synuclein, respectively.

The current list of different natively unfolded proteinsincludes more than 100 entries, with information on 91 ofthem presented in our recent work (Uversky et al. 2000a)and Table 1. Only full-length proteins or domains withchain length greater than 50 amino acid residues have beenconsidered. This list would probably be doubled if shorterpolypeptides 30 to 50 residues long were included. Finally,the set of 100 proteins described in the literature as na-tively unfolded have at least 250 homologs, which are alsoexpected to be natively unfolded. Additionally, a large num-ber of proteins and protein domains have been predicted tobe disordered based on the results of the analysis of aminoacid sequences using the neuronal network predictors(Dunker et al. 1998, 2001; Romero et al. 1998b). All thisshows that polypeptides without ordered structure underphysiological are common, rather than exceptions.

How is unfoldedness encoded in a

protein amino acid sequence?

The existence of at least three different disordered equilib-rium conformations, molten globule (MG), premolten glob-ule (PMG), and unfolded (random coil-like, U), has beenestablished for typical globular proteins (Uversky andPtitsyn 1994, 1996a; Ptitsyn 1995; Uversky 1997, 1998).Apparently, the ability of a protein to adopt different stableconformations is an intrinsic property of a polypeptidechain. Although the correct folding of a protein into its rigidbiologically active conformation is thought to be deter-

mined by its amino acid sequence (Anfinsen et al. 1961), theabsence of rigid structure in natively unfolded proteins maybe reflected in specific features of their amino acid se-quences.

In an attempt to understand the relationship between se-quence and disorder, Dunker and coauthors have developedseveral neuronal network predictors (Romero et al. 1997,1998a, 1998b, 2001a; Dunker et al. 1998, 2001; Garner etal. 1998; Li et al. 1999, 2000). They assumed that if aprotein structure has evolved to have a functional disorderedstate then a propensity for disorder might be predictablefrom its amino acid sequence and composition. The resultsof such analysis were impressive. It was established that

disordered regions shared at least some common sequencefeatures between many proteins, and that more than 15,000proteins in the Swiss Protein database were identified ashaving long regions of sequence that shared these features(Romero et al. 1998b). Interestingly, the Top 20 proteins(i.e., proteins with the highest scores) were shown to havelow sequence complexity, as defined by Wootton (1993,1994; Wootton and Federhen 1996). In other words, se-quences of natively unfolded proteins may be essentially

degenerate. Figure 2A illustrates this idea, comparing thes-antigen from Plasmodium (which was shown to be at thehead of the Top 20) with that of human serum albumin (arigid globular protein of similar molecular mass) in terms oftheir amino acid composition scaled according to McCaldonand Argos (1988). Interestingly, it was later established thatthe distributions of the complexity values for ordered anddisordered sequences overlapped (Romero et al. 2001b),suggesting that low sequence complexity did not representthe only characteristic feature of intrinsically disorderedproteins. However, some general sequence peculiarities of

Fig. 2. How the unfoldedness is encoded in protein amino acid sequence.(A) Comparison of sequences of rigid globular protein, human serum al-

bumin (top) and intrinsically disordered protein s-antigen from Plasmo-dium, which was shown to be at the head of the Top 20 proteins withhighest disorder scores estimated by neuronal network predictors (Romeroet al. 1998b) (bottom) in terms of their composition scaled according toMcCaldon and Argos (1988). (B) The unique property of natively unfoldedprotein sequences is a combination of low overall hydrophobicity and largenet charge. Comparison of the mean hydrophobicity and the mean netcharge for a set of 275 folded (black circles) and 102 natively unfoldedproteins (open circles). The solid line represents the border between in-trinsically unstructured and native proteins calculated using equation 1.Data for this plot are taken from Uversky et al. (2000a).




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Table 1. Major structural characteristics of the natively unfolded proteins

ProteinLength,a. a. r.

[]200,deg cm2 dmol1

[]222,deg cm2 dmol1 Vh,

3 Reference

Thymosin 1 28 15,700 2000 Grottesi et al. 1998c-Jun oncoprotein, basic subdomain 29 19,200 1100 Krebs et al. 1995P19Arf tumor suppressor protein, N-terminal

fragment37 19,000 1500 DiGiammarino et al. 2001

Bacteriophage Pf1 gene 5 protein, C-terminal

domain

38 20,500 800 Fox et al. 2001

WW domain of Yes-associated protein, W17Fmutant

38 16,400 2000 Koepf et al. 1999

50S ribosomal protein L34 46 25,300 2100 Venyaminov et al. 1981-Tubulin, 404451 fragment 48 17,300 1500 Jimenez et al. 1999-Tubulin, 394445 fragment 52 16,600 2000 Jimenez et al. 1999Calpastatin, domain I 53 23,600 2500 Konno et al. 199750S ribosomal protein L33 54 21,300 2000 Venyaminov et al. 1981HIV-1 integrase, N-terminal domain 55 17,500 2000 Zheng et al. 1996RNA polymerase II, C-terminal domain 56 15,000 1500 Bienkiewicz et al. 2000Galline 61 25,000 1000 Nakano et al. 1989B-cell specific transcription coactivator Bob1,

N-terminal domain65 17,500 1700 Chang et al. 1999

Nonhistone chromosomal protein HMG-H6 69 22,000 1000 Cary et al. 198150S ribosomal protein L31 70 20,000 1500 Venyaminov et al. 198130S ribosomal protein S21 70 20,300 3000 Venyaminov et al. 1981Subtilisin BPN pro-domain 75 17,500 2000 Tangrea et al. 2001Phosphodiesterase -subunit 83 14,940 1690 73,622 Uversky et al. 200250S ribosomal protein L27 84 17,700 2000 Venyaminov et al. 1981Vmw65, carboxyl-terminal transactivation domain 87 21,155 1840 91,952 Donaldson and Capone 1992Nonhistone chromosomal protein HMG-17 89 21,500 1300 93,097 Abercrombie et al. 197830S ribosomal protein S19 91 17,000 1200 Venyaminov et al. 1981Em protein 93 18,340 2440 93,937 McCubbin and Kay 1985Nonhistone chromosomal protein HMG-14 100 17,800 1950 Cary et al. 1980Human sperm protamine P2 102 20,000 1200 Gatewood et al. 1990Human sperm protamine P3 103 21,000 1000 Gatewood et al. 1990Apo-cytochrome c 104 103,097 Stellwagen et al. 1972Prothymosin 110 22,000 500 60,105 Gast et al. 1995; Uversky et

al. 199950S ribosomal protein L22 110 22,500 3000 Venyaminov et al. 1981Fibronectin-binding domain B-type, S. dysgalactiae 112 19,200 1500 121,200 House-Pompco et al. 1996

4E-binding protein 1 118 16,500 2250 Fletcher et al. 1998NF-B transcription factor, p65 subunit 120 18,000 800 Schmitz et al. 1994Fibronectin-binding domain A-type, S. dysgalactiae 124 22,000 1200 134,434 House-Pompeo et al. 1996Fibronectin-binding domain, D-type, S. aureus 134 21,000 1200 134,701 House-Pompeo et al. 1996-Synuclein 134 15,495 2124 137,258-Synuclein 134 15,560 2031 117,681-Synuclein 140 15,309 2073 141,155 Weinreb et al. 1996;

Uversky et al. 2001bHelix destabilizing protein (Pf1 gene 5 protein) 144 17,500 700 Bogdarina et al. 1998Stathmin 148 150,533 Belmont and Mitchinson,

1996Calpastatin, domain I 148 16,800 2500 Konno et al. 1997Dessication-related protein 155 19,000 1000 Lisse et al. 1996Thyroid transcription factor-1, N-terminal domain 156 16,700 1350 Tell et al. 1998DFF45 N-terminal domain 116 23,000 500 Zhou et al. 2001

C-terminal domain of bZIP transcription factor Fos 164 16,000 1000 179,594 Campbell et al. 2000Myelin-binding protein 196 15,200 1000 Polverini et al. 1999Nonhistone chromosomal protein HMG-T 204 18,000 2000 Cary et al. 1981Extracellular domain of the low affinity NGF

receptor222 21,500 500 Timm et al. 1992

CREB, truncated form (ACT265) 265 15,300 2000 Richards et al. 1996Salivary proline-rich glycoprotein 293 25,000 3500 Loomis et al. 1985Histidine-rich protein II 327 16,000 2000 Lynn et al. 1999GAGA factor, glutamine-rich domain 383 18,500 2300 Agianian et al. 1999Chromogranin A 439 16,000 2500 838,603 Yoo and Albanesi 1990Caldesmon 771 3,156,551 Lynch et al. 1987Microtubule-associated protein MAP2 1827 19,500 2980 7,606,206 Hernndez et al. 1986

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Table 1. Continued

ProteinLength,a. a. r.

[]200,deg cm2 dmol1

[]222,deg cm2 dmol1 Vh,

3 Reference

Parathyroid hormone-related protein, N-terminalfragment

34 13,500 4000 Willis 1994

Naturally occurring peptide L1-37 37 11,400 3000 Johansson et al. 1998DNA polymerase I, substrate-binding peptide 50 11,000 4000 Mullen et al. 1993Osteocalcin 50 10,000 4500 26,094 Isbell et al. 1993; Dowd et al.

200150S ribosomal protein L32 56 12,400 4050 Venyaminov et al. 1981GCN4, DNA-binding domain 57 9800 4750 Weiss et al. 1990RNase HI, C-terminal domain (88155) 68 12,500 5100 Kanaya and Kanaya 1995Rhodostomin 68 11,629 2350 Guo et al. 2001Lysozyme 1-40/98-127 69 12,500 5500 28,731 Demarest et al. 2001Heat stable protein kinase inhibitor 72 8500 2,662 46,452 Thomas et al. 199130S ribosomal protein S18 74 12,000 4500 Venyaminov et al. 1981SMK killer toxin, -subunit 77 11,000 2500 Suzuki et al. 1997Tropomodulin, N11 fragment 91 8500 3500 Kostyukova et al. 2000Cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1,

N-terminal domain94 10,000 2500 Kriwacki et al. 1996

-Dystroglycan, extracellular region 96 11,500 3,780 Di Stasio et al. 1999HPV16 E7 protein 98 10,800 5200 Pahel et al. 199350S ribosomal protein L23 99 12,700 7100 Venyaminov et al. 1981

50S ribosomal protein L24 103 9900 2700 Venyaminov et al. 1981Reduced RNase T1 104 10,700 2900 Baskakov and Bolen 1998DNA-binding domain of 1,25-Dihydroxyvitamin

D3 receptor110 10,000 5,000 Creig et al. 1997

RNase P 116 11,000 3,000 Henkels et al. 200150S ribosomal protein L14 120 9300 3000 Venyaminov et al. 198130S ribosomal protein S12 123 12,000 3500 Venyaminov et al. 1981Steroidogenic acute regulatory protein, StAR,

N-terminal domain131 11,000 3000 Song et al. 2001

SNase, 131 fragment 131 12,000 3600 65,450 Alexandrescu and Shortle 1994;Shortle and Meeker 1989

Caldesmon, 636771 fragment 136 12,200 3440 89,656 Uversky, unpubl communcGMP-dependent protein kinase inhibitor 155 124,788 Aswad and Greengard 1981MAX-protein 163 10,500 6,100 Horiuchi et al. 1997Cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 164 11,000 3060 Kriwacki et al. 1996

Heat-shock transcription factor, N-terminalactivation domain (S. cerevisiae)167 9500 3500 Cho et al. 1996

Protein phosphatase inhibitor-1 171 141,092 Nimmo and Cohen 1978Glucocorticoid receptor, 77262 fragment 186 8000 3200 Baskakov et al. 1999Heat-shock transcription factor, N-terminal

activation domain (K. lactis)194 10,000 4000 Cho et al. 1996

s-Casein 199 11,000 4500 Bhattacharyya and Das 1999Dopamine- and cAMP-regulated neuronal

phosphoprotein, DARPP-32202 164,636 Nemmings et al. 1984

50S ribosomal protein L3 209 10,100 3300 Venyaminov et al. 1981Histone H1 224 9450 3150 Tarkka et al. 1997Manganese stabilizing protein, L245E mutant 247 10,000 3500 146,464 Lydakis-Simantiris et al. 1999Sec9, SNAP-25-like domain 251 11,000 5500 Rice et al. 199750S ribosomal protein L2 272 10,400 2500 Venyaminov et al. 1981SPARC, BM-40, osteonectin 285 12,000 5150 Engel et al. 1987

GAGA factor, central domain 307 9000 3300 Agianian et al. 1999Calreticulin, human41C fragment 359 413,061 Bouvier and Stafford 2000Calsequestrin 367 8500 4000 381,704 Cozens and Reithmeier 1984;

He et al. 1993Calreticulin, human 400 413,061 Bouvier and Stafford 2000Calreticulin, bovine 407 361,706 Bouvier and Stafford 2000Soluble transducer HtrXI 451 10,300 4500 Larsen et al. 1999Taka-amylase A, reduced 477 335,367 Tanford 1968SdrD protein, B1B5 fragment 562 10,560 4,120 685,568 Josefsson et al. 1998Secretogranin (chromatogranin B) 657 10,500 4250 533,080 Yoo 1995DNA topoisomerase I 765 830,031 Stewart et al. 1996Fibronectin 4772 6,370,626 Pelta et al. 2000




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natively unfolded proteins have been recognized long ago.These include the presence of numerous uncompensatedcharged groups, resulting in a large net charge at neutral pH(Hemmings et al. 1984; Gast et al. 1995; Weinreb et al.1996) and a low content of hydrophobic amino acid residues(Hemmings et al. 1984; Gast et al. 1995).

Recently, we have established that the combination oflow mean hydrophobicity and relatively high net chargerepresents an important prerequisite for the absence of com-pact structure in proteins under physiological conditions,leading to natively unfolded proteins (Uversky et al. 2000a).Figure 2B shows that natively unfolded proteins are spe-cifically localized within a unique region of the charge-hydrophobicity phase space. The solid line in this figurerepresents the border between intrinsically unstructured andnative proteins, satisfying the following relationship:

Hboundary =R + 1.151

2.785(1)

This equation gives the estimation of the boundary meanhydrophobicity value, Hboundary, below which a polypep-tide chain with a given mean net charge R will most prob-ably be unfolded. Thus, sequences of natively unfolded pro-teins may be characterized by a low sequence complexityand/or high net charge coupled with low mean hydropho-bicity.

How unfolded are intrinsically unstructured

proteins? A classification attempt

It is well known that in the presence of large concentrationsof strong denaturants, such as 8 M urea or 6 M GdmCl,normal proteins lose the majority of their specific structure,that is, become essentially unfolded (Anson and Mirsky1932; Mirsky and Pauling 1936; Neurath et al. 1944; Tan-ford 1968). One can expect that under these conditions un-folded proteins will obey the theoretical and empirical rulesthat apply to linear random coils (Tanford 1968). In accor-dance with Tanford, a polymer molecule is randomly coiledwhen internal rotation can take place at about every singlebond of the molecule with the same freedom with which itwould take place in a molecule of low molecular weightcontaining the same kind of bonds (Tanford 1968). The

properties of linear random coils are well understood, assynthetic polymers frequently adopt this conformation(Flory 1953; Tanford 1961). Because the dimensions ofrandom coils depend only on the backbone rotationalangles, the dependence of the hydrodynamic dimensions onmolecular mass (length of polypeptide chain) represents themost effective diagnostic tool for recognition of linear ran-dom coils (Tanford 1961, 1968). Results of early studies onhydrodynamic dimensions of proteins in the presence of 6

M GdmCl were consistent with the conclusion that unfoldedproteins could be described as random coils (Tanford 1961,1968). However, it was later established by heteronuclearNMR that even in high concentrations of strong denatur-ants, when the native state of globular proteins breaks down,the polypeptide chains contained some amount of residualstructure, that is, the polypeptide chain did not reach a ran-dom coil conformation (Dill and Shortle 1991; Logan et al.1994; Zhang et al. 1994; Shortle 1996; Pappu et al. 2000;Baldwin and Zimm 2000). These findings raised severalcompelling biophysical questions related to the structuralcharacteristics of natively unfolded proteins. How unfoldedare these proteins? Are they random coils, or do they pos-sess residual structure? If they have residual structure, howthen should they be classified? Fortunately, the informationaccumulated to date on natively unfolded proteins allows usto make an initial structural classification of these intriguingmembers of the polypeptide kingdom.

As it follows from their definition, intrinsically unstruc-

tured proteins show complete (or almost complete) loss ofany ordered structure under physiological conditions invitro; that is, they should behave as random coils. Structur-ally, this may be manifested by (1) larger hydrodynamicdimensions compared to typical native globular proteinswith corresponding molecular mass, (2) low content of or-dered secondary structure, and (3) high intramolecular flex-ibility. Such anomalous behavior is usually detected by nu-merous hydrodynamic techniques (gel-filtration, viscom-etry, SAXS, SANS, sedimentation, and dynamic and staticlight scattering), far-UV CD, ORD, FTIR, and NMR spec-troscopy (one-dimensional and heteronuclear multidimen-sional). These techniques may also be used for the identi-

fication of residual structure (if any) in an unfolded proteinmolecule. Once again, simultaneous application of severalapproaches should permit one to make more reliable con-clusion.

Flexibility and residual structure

by NMR spectroscopy

NMR spectroscopy of natively unfolded proteins has es-tablished that they contain varied amounts of residual struc-ture. Examples of proteins which are essentially unfoldedunder in vitro physiological conditions include: DFF45 N-terminal domain (Zhou et al. 2001), DNA-binding domainof vitamin D receptor (Craig et al. 1997), C-terminal do-

main of anti-sigma factor FlgM (Daughdrill et al. 1998),p53 regulatory domain of p19Arf tumor suppressor (Di-Giammarino et al. 2001), substrate-binding peptide fromDNA polymerase I (Mullen et al. 1993), poplar apo-plasto-cyanin (Bai et al. 2001), N-terminal domain of StAR (Songet al. 2001), bone sialoprotein and osteopontin (Fisher et al.2001), C-terminal domains of- and -tubulins (Jimenez etal. 1999), N-terminal activation domain of heat-shock tran-scription factors (Cho et al. 1996); 4E-binding proteins I and

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II (Fletcher et al. 1998), cyclin-dependent kinase inhibitorp21Waf1/Cip1/Sdi1 (Kriwacki et al. 1996), SNase, 131 frag-ment (Alexandrescu et al. 1994; Gillespie and Shortle1997a, 1997b), dessication-related protein (Lisse et al.1996), functional domain of eIF4G1 (Hershey et al. 1999),cytoplasmic domain of synaptobrevin (Hazzard et al. 1999),N-terminal domain of prion protein (Donne et al. 1997),C-terminal HMG domain of LEF-1 (Love 1999), N-termi-nal region of TAFII-2301177 (Liu et al. 1998), antitermina-tion protein N (Mogridge et al. 1998), cytoplasmic domainof Snc1 (Fiebig et al. 1999), prothymosin (Uversky et al.1999), nonhistone chromosomal proteins HMG-14 (Cary etal. 1980), HMG-17 (Abercrombie et al. 1978), HMG-T andHMG-H6 (Cary et al. 1981), fibronectin-binding domains(Penkett et al. 1998), DNA-binding domain of GCN4(Weiss et al. 1990), EMB-1 protein (Eom et al. 1996), NEFprotein (Geyer et al. 1999), osteocalcin (Isbell et al. 1993),two-domain fragment of neutral zinc finger factor 1 (Berko-vits and Berg 1999), and several other proteins. On the other

hand, heteronuclear multidimensional NMR analysis pro-vided evidence of some ordered structure in several nativelyunfolded proteins, although the amount and quality of re-sidual structure varied tremendously. In some cases the au-thors were unable to detect any secondary or tertiary con-tacts (e.g., Abercrombie et al. 1978; Cary et al. 1980, 1981;Cho et al. 1996; Eom et al. 1996; Lisse et al. 1996; Penkettet al. 1997; Fletcher et al. 1998; Zhang and Matthews 1998;Bose et al. 1999; Uversky et al. 1999; Campbell et al. 2000;Fisher et al. 2001; DiGiammarino et al. 2001). In other casesit was concluded that the proteins contained mostly dynamicstructure favoring helical or -structural conformation (e.g.,Mullen et al. 1993; Schmitz et al. 1994; Kriwacki et al.

1996; Gillespie and Shortle 1997a, 1997b; Daughdrill et al.1998; Bai et al. 2001, and many others). Thus, NMR analy-sis clearly showed that natively unfolded proteins do notpossess uniform structural properties, as expected for mem-bers of a single thermodynamic entity.

Hydrodynamic dimensions

As previously discussed, the most unambiguous charac-teristic of the conformational state of a globular protein is itshydrodynamic dimension. In fact, it has been shown thatequilibrium conformations of a globular protein (native,molten globule, premolten globule, and unfolded states)

may easily be discriminated by the degree of compactnessof the polypeptide chain (Uversky 1993, 1994, 1997, 1998;Uversky and Ptitsyn 1994, 1996a; Ptitsyn 1995). The equi-librium conformations were characterized by very differentdependencies of their hydrodynamic dimensions on molecu-lar mass (length of polypeptide chain) (Tanford 1961, 1968;Uversky 1993; Tcherkasskaya and Uversky 2001).

To clarify the physical nature of natively unfolded pro-teins, Figure 3 represents the dependencies of their hydro-

dynamic dimensions on the length of their polypeptidechains (see Table 1). The same trends determined for globu-

lar proteins in their native, molten globule, premolten glob-ule, and urea or GdmCl unfolded states are shown for com-parison. The values of the hydrodynamic volumes, Vh, werecalculated from the corresponding Stokes radii, RS, asVh 4/3 RS

3. Data for the different conformations ofglobular proteins were taken from Tcherkasskaya and Uver-sky (2001). For these species, the dependencies ofVh on thelength of the polypeptide chains, N, were described by a setof straight lines (using a logarithmic scale):

logVhN = 2.197 0.037

+ 1.072 0.015logN (2)logVh

MG = 2.46 0.13 + 1.020 0.053 logN

(3)logVh

PMG = 2.41 0.29 + 1.18 0.12 logN

(4)logVh

U(urea) = 1.866 0.026

+ 1.565 0.011 logN (5)

logVhU(GdmCl)

= 1.723 0.041+ 1.655 0.018 logN (6)

Fig. 3. Dependencies of the hydrodynamic volume, VS, on protein poly-peptide length, N, for native (open circles, N), molten globule (gray re-versed triangles, MG), premolten globule (open squares, PMG), 8 M urea-unfolded (open triangles, Uurea) and 6 M GdmCl-unfolded (black triangles,UGdmCl) conformational states of globular proteins and natively unfoldedproteins with coil-like (gray triangles, NUcoil) and PMG-like properties(gray squares, NUPMG). Data used to plot the dependencies for native,molten globule, premolten globule, and GdmCl-unfolded states of globularproteins are taken from Tcherkasskaya and Uversky (2001); data for na-tively unfolded proteins are summarized in Table 1.




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Here, N, MG, PMG, U(urea), and U(GdmCl) correspond tothe native, molten globule, premolten globule, urea, andGdmCl unfolded globular proteins, respectively.

The existence of significant differences in the molecularmass dependencies of the Vh measured for urea and GdmClunfolded proteins should be emphasized. It is well estab-lished that the hydrodynamic dimensions of random coilsessentially depend on the quality of solvent (Flory 1953;Tanford 1961, 1968). A poor solvent induces the attractionof macromolecular segments, resulting in the squeezing of achain. On the other hand, in a good solvent repulsive forcesoccur between segments, leading to the formation of a loosefluctuating coil (Grossberg and Khokhlov 1989). It is as-sumed that solutions of urea and GdmCl are rather goodsolvents for polypeptide chains, with GdmCl being closer tothe ideal one (Tanford 1961, 1968). This difference in sol-vent quality could account for the observed divergence inlog(Vh) versus log(N) dependencies for the globular proteinsunfolded by urea and GdmCl.

Figure 3 shows that natively unfolded proteins are muchless compact compared to native and molten globule pro-teins of similar molecular mass. Surprisingly, under physi-ological conditions in vitro (i.e., in aqueous solution, a poorsolvent for a polypeptide chain), natively unstructured pro-teins are split in two different subclasses (see also Table 1).Proteins from the first subclass, which consists of 17 rep-resentatives, behave as random coils in poor solvent,whereas the 18 proteins of the second subclass are essen-tially more compact, being close to premolten globules as itfollows from their hydrodynamic parameters (cf. equations35):

log(VhNU(coil) = 1.997 0.078+ 1.498 0.035 logN (7)

logVhNUPMG

= 2.33 0.12+ 1.234 0.047 logN (8)

where NU(coil) and NU(PMG) correspond to the nativelyunfolded proteins with coil-like and premolten globule-likehydrodynamic characteristics, respectively. This very in-triguing observation, confirming conclusion on the struc-tural diversity of intrinsically disordered proteins drawnfrom their NMR analysis, may be further verified by theanalysis of far-UV CD spectra.

Residual secondary structure from far-UV CD spectra

Unfolded polypeptide chains are characterized by veryspecific shapes of their far-UV CD spectrum, with an in-tensive minimum in the vicinity of 200 nm and an ellipticityclose to zero in the vicinity of 222 nm (Adler et al. 1973;Provencher and Glckner 1981; Johnson 1988; Woody1995; Fasman 1996; Kelly and Price 1997; Uversky 1999).This is a very useful graphical criterion for the selection of

natively unfolded proteins (see Table 1). To date, a coil-likeshape of far-UV CD spectrum has been reported for 100proteins (see Table 1), which is almost threefold larger thanthe number of proteins shown to be unfolded in accordancewith their hydrodynamic dimensions (35).

Figure 4 represents a double wavelength plot, []222versus []200 that may be used to assort natively unfoldedproteins into two nonoverlapping groups. Fifty-one proteinswere characterized by far-UV CD spectra characteristic ofalmost completely unfolded polypeptide chains: with[]200(18,900 2800) degcm

2dmol1 and []222(1700 700) deg cm2 dmol1. On the other hand, 44other protein spectra were consistent with the existence ofsome residual secondary structure, possessing shape typicalof the premolten globule state of globular proteins (with[]200(10,700 1300) degcm

2dmol1 and []222(39001100) deg cm2 dmol1).

Definitely, the difference in the shape of far-UV CDspectra alone does not allow the unambiguous discrimina-

tion between the two conformations. However, among morethan 100 reported cases, 23 proteins were simultaneouslycharacterized by CD and hydrodynamic methods, makingclassification more certain (see Table 1). Intrinsic premoltenglobules and intrinsic coils studied by both techniques areindicated in Figure 4 as white-dotted and black-dotted sym-bols, respectively. These data are consistent with the im-portant conclusions that more compact polypeptides (withPMG-like hydrodynamic characteristics) possess largeramounts of ordered secondary structure than less compactcoil-like natively unfolded proteins. Thus, the simultaneousapplication of CD and hydrodynamic techniques leaves nodoubts that natively unfolded proteins should be subdivided

Fig. 4. Analysis of far-UV CD spectra in terms of double wavelength plot,[]222 versus []200, allows the natively unfolded proteins division on coil-like (gray circles) and premolten globule-like subclasses (black circles).Intrinsic premolten globules and intrinsic coils for which the hydrodynamicparameters were measured (see Table 1) are marked by white-dotted andblack-dotted symbols, respectively.

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into two structurally distinct groups: intrinsic coils and in-trinsic premolten globules.

Amino acid composition of native coils

and native premolten globules

Figure 5 compares the amino acid compositions of intrin-sic coils and intrinsic premolten globules. Protein sets ana-lyzed in Figure 4 were used to create these graphs. The insetto Figure 5 shows that proteins from both subclasses occupythe same region of the charge-hydrophobicity phase space.Native coils were more dispersed, whereas intrinsic premol-ten globules were localized closer to the border betweenintrinsically unstructured and native proteins (cf. Fig. 2). Toconfirm this idea, the corresponding distances between thegiven sequence and the border between intrinsically un-structured and native proteins were calculated. Resultsof this comparison are shown in Figure 5 asH (Hboundary H) plots. The mean boundary hy-

drophobicity, Hboundary, for a given polypeptide chain witha mean net charge R has been calculated using equation 1.One can see that intrinsic coils are essentially more distantfrom the border than intrinsic premolten globules. Statisticalanalysis shows that the averaged H values are(0.089 0.086) and (0.037 0.033) for the native coilsand native premolten globule, respectively. However, be-cause the sequence characteristics of the two subclassesoverlap, it is difficult to differentiate these proteins by tak-ing into account their mean hydrophobicity and mean netcharge only. Probably, some other sequence features, suchas propensity to form secondary structure should also beconsidered.

Function-related folding of the

intrinsically unstructured proteins

The functional importance of being disordered has beenintensively analyzed (Schulz 1979; Pontius 1993; Dunker et

al. 1997, 2001; Plaxco and Gross 1997; Wright and Dyson1999). It has been established that increased intrinsic plas-ticity represents an important prerequisite for effective mo-lecular recognition (Plaxco and Gross 1997; Wright andDyson 1999; Dunker et al. 2001). The variety diapason ofbiological functions for intrinsically disordered proteins isvery wide, including cell cycle control, transcriptional andtranslational regulation, modulation of activity and/or as-sembly of other proteins, and even regulation of nerve cellfunction (reviewed in Wright and Dyson 1999; Dunker et al.2001). Importantly, it must be emphasized that the majorityof intrinsically disordered proteins undergo a disorder-to-order transition upon functioning (Schulz 1979; Pontius

1993; Spolar and Record 1994; Rosenfeld et al. 1995;Plaxco and Gross 1997; Dunker et al. 1997, 2001; Wrightand Dyson 1999). It has been suggested that the persistenceof natively unfolded proteins throughout evolution may re-side in advantages of flexible structure during disorderorder transitions in comparison with rigid proteins (Dunkeret al. 1997, 1998, 2001; Romero et al. 1998b; Wright andDyson 1999). Among the potential advantages of intrinsiclack of structure and function-related disorderorder transi-tions are (1) the possibility of high specificity coupled withlow affinity (Schulz 1979; Kriwacki et al. 1996; Dunker etal. 1998, 2001); (2) the ability of binding to several differenttargets (Wright and Dyson 1999; Dunker et al. 2001),

known as one to many signaling (Romero et al. 1998b); (3)the capability to overcome steric restrictions, enabling es-sentially larger interaction surfaces in the complex thancould be obtained for the rigid partners (Meador et al. 1992;Choo and Schwabe 1998; Dunker et al. 2001); (4) the pre-cise control and simple regulation of the binding thermo-dynamic (Schulz 1979; Spolar and Record 1994; Rosenfeldet al. 1995; Wright and Dyson 1999; Dunker et al. 2001);(5) the increased rates of specific macromolecular associa-

Fig. 5. Comparison of the amino acid compositions of intrinsic coils (graycircles) and intrinsic premolten globules (black circles) in terms of thedistance of a given sequence from the border between rigid and intrinsi-cally unstructured proteins. The distances were calculated asH (Hboundary H). The mean boundary hydrophobicity,Hboundary, for a given polypeptide chain with a mean net charge R hasbeen calculated using equation 1. Inset shows that proteins from bothsubclasses occupy the same region of the charge-hydrophobicity phasespace, with intrinsic coils being more dispersed.




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tion (Pontius 1993; Dunker et al. 2001); and (6) the reducedlifetime of intrinsically disordered proteins in the cell, pos-sibly representing a mechanism of rapid turnover of theimportant regulatory molecules (Wright and Dyson 1999).There is, however, an alternative explanation for the in-volvement of intrinsic disorder in protein function. By com-puter modeling it has been shown that selective pressure forfunctionality is rather unrelated to that for stability and fold-ability. In this view, a protein that is successfully folded intoone structure would likely be as functional as a protein thatsuccessfully folded into an alternative structure. Includingfunctionality in the model does not greatly alter the distri-bution of the observed structures (Williams et al. 2001).This could mean that metastable proteins are favored duringevolution because there is a tremendously larger amount ofsequences coding for these proteins compared to the veryrigid ones. In other words, the involvement of intrinsic dis-order in protein function may be related to history and evo-lution rather than to functional needs (Dunker et al. 2001).

In their excellent review, Dunker et al. (2001) formulatedthe idea that the protein structurefunction paradigm (whichemphasizes that ordered 3D structures represent the indis-pensable prerequisite to the effective protein functioning)should be altered as The Protein Trinity paradigm (see Fig.6A). According to The Protein Trinity model, native intra-

cellular proteins (or their functional regions) can exist inany of the three thermodynamic states, ordered, moltenglobule, and random coil. Function can arise from any of thethree conformations and transitions between them. In thisview, not just the ordered state, but any of the three statescan be the native state of a protein (Dunker et al. 2001).Experimental results on the conformational behavior of in-trinsically unstructured (natively unfolded) proteins indi-cated, however, that these proteins did not possess uniformstructural properties, as expected for members of one ther-modynamic group, random coils. They were split into twostructurally different subclasses, which, by analogy withconformational states of globular proteins, may be desig-nated as intrinsic coils and intrinsic premolten globules.Moreover, it was already noted that molten globule andpremolten globule might represent different phase states ofthe protein, as they are separated by the first-order phasetransition (Uversky and Ptitsyn 1994, 1996a; Ptitsyn 1995;Uversky 1997, 1998). These observations bring a new

player, the native premolten globule, on the protein func-tioning field. In other words, The Protein Trinity should beextended to The Protein Quartet model, with function aris-ing from four specific conformations (ordered forms, mol-ten globules, premolten globules, and random coils) andtransitions between any two of the states (see Fig. 6B).Experimental evidences for the validity of this extension arepresented below.

Function-related coilpremolten globule transitions

It has been established that the binding of Zn2+ inducespartial folding of intrinsically unfolded proteins, such as

thymosin 1 (Grottesi et al. 1998), prothymosin (Uverskyet al. 2000b), human sperm protamines P2 and P3 (Gate-wood et al. 1996), and phosphodiesterase -subunit (Uver-sky et al. 2002). Human -synuclein was also shown to bepartially folded in the presence of several divalent and tri-valent metal ions (Uversky et al. 2001a). Analysis of struc-tural changes associated with the cation binding showed thatthe transformation of intrinsic coils into premolten globule-like conformations took place in these cases.

Function-related coilmolten globule transitions

The myelin basic protein, MBP, is a major protein of

myelin, the multilamellar membranous sheath surroundingnerve axons. MBP was isolated in water-soluble or deter-gent-soluble form together with endogenous myelin lipids.The water-soluble form is a member of the intrinsic coilfamily. Binding of lipids transformed this protein into themolten globule-like conformation (Polverini et al. 1999).Similar structural rearrangements were induced in the coil-like 77262 fragment of the glucocorticoid receptor (Baska-kov et al. 1999) and in the N-terminal domain of HIV-1

Fig. 6. Extension of The Protein Trinity (A, Dunker et al. 2001) to theProtein Quartet model of protein functioning (B). In accordance with thismodel, function arises from four specific conformations of the polypeptidechain (ordered forms, molten globules, premolten globules, and randomcoils) and transitions between any of the states.

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integrase (Zheng et al. 1996) by TMAO and by Zn2+ bind-ing, respectively. Self-association of an intrinsically un-folded -subunit of phosphodiesterase induced folding ofthis protein into a molten globule-like conformation (Uver-sky et al. 2002).

Function-related coilrigid structure transitions

The N-terminal domain of the caspase-activated DNAfragmentation factor DFF45 was unfolded in solution. Itsfolding into the rigid 3D structure was induced upon inter-action with the N-terminal domain of DFF40 (Zhou et al.2001). Structural analysis revealed that the isolated 50Sribosomal proteins, L22 and L27, and 30S ribosomal proteinS19 were essentially unfolded in solution (Venyaminov etal. 1981). However, they transform into a rigid well-foldedconformation in the functional ribosome (Yusupov et al.2001).

Function-related premolten

globulemolten globule transitions

The human antibacterial peptide LL-37 existed in thepremolten globule like conformation at micromolar concen-trations in aqueous solution. A cooperative transition from adisordered to a helical molten globule-like structure wasobserved in the presence of several anions or with increas-ing protein concentration. The extent of -helicity corre-lated well with the antibacterial activity of LL-37 againstboth Gram-positive and Gram-negative bacteria (Johanssonet al. 1998). Comparably the degree of folding was inducedin osteocalcin as a result of binding of Ca2+, Lu3+ (Isbell et

al. 1993) or Pb3+

(Dowd et al. 2001). Premolten globule tomolten globule transitions accompanied Ca2+ binding toskeletal muscle sarcoplasmic reticulum calsequestrin (Coz-ens and Reithmeier 1984; He et al. 1993) and SPARC, anextracellular glycoprotein expressed in mineralized andnonmineralized tissues (Engel et al. 1987).

The DNA-binding domain of the 1,25-dihydroxyvitaminD3 receptor was shown to undergo a premolten globule-to-molten globule transition as a result of specific Zn2+

binding. This cation-induced folding was an important pre-requisite to the formation of functional complex withosteopontin and several vitamin D response elements (Craiget al. 1997). The first step in steroidogenesis is the move-

ment of cholesterol from the outer to inner mitochondrialmembrane, which is facilitated by the steroidogenic acuteregulatory protein StAR. The interaction of premolten glob-ule-like StAR with dodecylphosphocholine and phospho-lipid liposomes was accompanied by transition of the pro-tein into the molten globule conformation (Song et al.2001). The E7 gene of the human papillomaviruses encodesa 98-amino acid chain of a multifunctional nuclear phos-phoprotein, E7 protein, which cooperates with an activated

ras oncogene to transform primary rodent cells. CD spec-troscopy indicated that Zn2+ and Cd2+ binding by theHPV16 E7 protein induced structural transformations con-sistent with premolten globulemolten globule transition(Pahel et al. 1993).

Transitions of intrinsic premoltenglobules to rigid conformation

It was shown that self-dimerization and DNA bindinginduce rigid 3D structure in the intrinsic premolten globule-like Max protein (Ferre-DAmare et al. 1993; Horiuchi et al.1997). Specific Ca2+ binding initiated folding of the pre-molten globule-like B-repeat segment of SdrD (Josefsson etal. 1998). Similarly, the 50S ribosomal proteins L2, L3,L14, L23, L24, and L32, as well as the 30S ribosomalproteins S12 and S18 were native premolten globules intheir free forms (Venyaminov et al. 1981), but adopted rigidwell-folded conformations during the formation of a func-tional ribosome (Yusupov et al. 2001).

The Escherichia coli RNase HI variant, with the K86Amutation, was purified in two forms: nicked and intact. Thenicked protein, resulting from the cleavage of a Lys87Arg88 peptide bond, was enzymatically active. The N-ter-minal fragment possessed characteristics of molten globule,whereas the C-terminal fragment was essentially disor-dered. The premolten globule-like C-fragment underwent atransition to the rigid 3D structure as a result of RNase HIreconstitution (Kanaya and Kanaya 1995). Comparably, theintrinsically unstructured -subunit of SMK killer toxinfolded into a rigid conformation as a result of interactionwith the -subunit (Suzuki et al. 1997). Finally, the forma-tion of a yeast SNARE complex was accompanied by a

complete folding of the two of its components, Snc1 andSec9 (Rice et al. 1997).

RNase P is the endoribonuclease responsible for the5-maturation of precursor tRNA transcript. Intriguingly,RNase P from Bacillus subtilis, being predominantly un-folded in 10 mM sodium cacodilate at neutral pH, foldedinto a native / structure upon addition of various smallmolecular anions (Henkels et al. 2001). This protein (Hen-kels et al. 2001), as well as the reduced RNase T1 (Baska-kov and Bolen 1998), also underwent a cooperative foldingtransition upon addition of the osmolyte TMAO.

The cyclin-dependent kinase (Cdk) inhibitorp21Waf1/Cip1/Sdi1and its N-terminal fragment lack stable sec-ondary and tertiary structure in the free solution state. Insharp contrast to the disordered free solution state, theseproteins adopted an ordered stable conformation whenbound to Cdk2 (Kriwacki et al. 1996).

Conclusions

Conformational analysis shows that natively unfolded pro-teins do not represent a uniform family, but rather two struc-




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turally different groups. Proteins from the first group havehydrodynamic dimensions typical of random coils in poorsolvent (i.e., they behave as slightly squeezed coils) and donot possess any (or almost any) ordered secondary structure.Proteins from the second group are essentially more com-pact (but still significantly less compact than native or mol-ten globule proteins). They exhibit some amount of orderedsecondary structure being characterized by far-UV CDspectra as typical essentially disordered polypeptide chain,with a pronounced minimum in the vicinity of 200 nm. Byanalogy with the conformational classification of normalglobular proteins, intrinsically unstructured proteins couldbe divided in intrinsic coils and intrinsic premolten glob-ules.

Because the amino acid sequences of native coils aresimilar to native premolten globules (only slightly less hy-drophobic and slightly more charged), some other sequencefeatures (e.g., propensity to form secondary structure) haveto be taken into account for the unambiguous sequence-

based separation of the intrinsic coils from the intrinsicpremolten globules.An intriguing property of intrinsically unstructured pro-

teins is their capability to undergo disorder-to-order transi-tion upon functioning. The degree of these structural rear-rangements varies over a very wide range, from coilpre-molten globule transitions to formation of rigid orderedstructures. Thus, protein functioning may be described bythe Protein Quartet model, with biological activity arisingfrom four unique conformations of the polypeptide chain(ordered forms, molten globules, premolten globules, andrandom coils) and transitions between any of them.

Acknowledgments

I am grateful to Prof. A.K. Dunker for the valuable discussions. Ithank Dr. P. Souillac for the careful reading and editing of themanuscript. I appreciate Prof. J. Goers for his invaluable help withthe manuscript improvement.

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