Sept 14, 2009GMS BI 555/755 Lecture 2.1 Review: Gibbs free energy, enthalpy, entropy Work and energy storage/utilization in biological systems Types of

Sept 14, 2009 GMS BI 555/755 Lecture 2. 1

Review:•Gibbs free energy, enthalpy, entropy•Work and energy storage/utilization in biological systems•Types of chemical bonding (covalent, H-bonding, electrostatic, Van der Waal’s)•The hydrophobic effect•Protein microenvironments•Properties of water, acid-base equilibria


• Reading: Berg 6th ed. Chapter 2 (Supplemental: Creighton: Proteins)• Primary Structure

– Amino acid side chains and classification– The peptide bond

• Secondary structure– Peptide bond angles and rotation– Alpha helix– Beta sheet– Turns

• Tertiary structure– Hydrophobic effect– Effects of solvent– Folding motifs– Protein folding problem– Molecular chaperones

• Quaternary structure

GMS BI 555/755 Lecture 2: Levels of Protein Structure


Protein primary structure

Proteins are polymers of L-amino acids linked by peptide bonds

Amide bond ~60% ~40%• Amide bonds have a substantial degree of

planar character• Chemically unreactive. Hydrolysis at pH

extremes


The peptide bond•Peptide (amide) bond very stable in solution in the absence of a catalyst

Peptide bonds may be trans or cis, trans being favored because there are fewer unfavorable steric interactions


Formation of the peptide bond • Peptide bond is an amide bond• Very stable • Positive ΔH• Peptide bond formation

increases order (negative ΔS)• Not a spontaneous process

(becomes spontaneous when coupled to a process such as ATP hydrolosys)


Glycine and alanine

A, G:• Neutral• Small R group (low accessible surface area)

• Non-polar• FlexibleG: achiral, most flexible AA


Aliphatic amino acidsV,L,I,M:• Neutral• High surface area• Non-polar• Hydrophobic• Van der Waal’s interactions in folded interior

• Structural units with a variety of shapes

• I side chain is chiral

V,L,I: commonM: Rare• Easily oxidized to sulfoxide then sulfone


Methionine oxidation

Methionine residues are susceptible to oxidation in vivo and during protein workup and characterization


Aromatic amino acids

F, Y, W: •Neutral•Very high accessible surface areaF: very non-polar, hydrophobicW: rareW, Y: responsible for 280 nm absorbanceW: Fluorescent properties

A = εBCA = absorbanceε = molar absorbtivityC = concentration


AAs with alphatic hydroxyl group

S, T:•Neutral•Polar, H-bonding donors or acceptors•Hydrophilic or hydrophobic •Sites of post-translational modification•Phosphorylation (S, T, Y)•O-glycosylation ( β-O-GlcNAc, O-glycans)•T side chain chiral


Cysteine • Sulfhydryl (thiol) most reactive group in proteins• Oxidation in presence of oxygen• Very nucleophilic, reactions with electrophiles• Must be alkylated (stabilized) for effective

analysis• Reactions with metal ions• Participates in disulfide bonding with other

cysteine residues. Important secondary structure stabilizing event in proteins.

• Antioxidant, precursor to glutathione

Cystine: disulfide bonded Cys residues

NH2

CH

C

H2C

OH

O

SH I

O

OH

NH2

CH

C

H2C

OH

O

S

O

OH

pH 8.3

Cys is nucleophilic and must be alkylated for analysis (reaction with iodoacetic acid)


Cysteine alkylation

Fluoresceine-5-maleimide

I

O

OHI

O

NH2

N

O

NH2

iodoacetic acid iodoacetamide

4-vinylpyridine acrylamide

Derivatizing groups for cys stabilization Fluorescent alkyl groups


Homocysteine: analog of Cys and Met, metabolic intermediate

• Elevations of homocysteine occur in the rare hereditary disease homocystinuria and in the methylene-tetrahydrofolate-reductase polymorphism genetic traits. The latter is quite common (about 10% of the world population) and it is linked to an increased incidence of thrombosis and cardiovascular disease and that occurs more often in people with above minimal levels of homocysteine (about 6 μmol/L)

• Risk factor for vascular disease, Alzheimer’s Disease

Darvesh, S., Walsh, R., and Martin, E. (2007) Homocysteine Thiolactone and Human Cholinesterases. Cell Mol Neurobiol 27, 33-48.


The basic amino acids

Histidine Ionization. Histidine can bind or release protons near physiological pH.

pKa ~ 6pKa ~ 12

R, K:•Positively charged at pH 7•Most basic protein groups (also N-term)H:•Can participate in acid/base reactions at pH 7

nucleophilic


Hydroxylysine

•Biosynthesized from lysine oxidation by lysyl oxidase•Found only in animal proteins, mostly in collagen as a PTM•6-67 of 1000 AA residues of collagen are hydroxylysine•17-90% of collagen lysyl residues are hydroxylated•Hydroxylysine is typically found in triple-helical regions almost exclusively in the Y positions of the repeating -X-Y-Gly- sequences in various collagens.

•Embryonic tissues contain much more hydroxylysine than adult tissues.•Hydroxylation of lysyl residues in collagens prevents deposit of minerals between fibrils

•Lysine hydroxylation seems to be increased as well in some diseases, for example, lipodermatosclerosis, osteoporosis, and osteogenesis imperfecta

•Precursor to collagen crosslinking •Hydroxylated lys residues may be glycosylated

http://herkules.oulu.fi/isbn9514267990/html/x319.html


Ornithine lactamization: ornithine is unstable in peptide chains due to its propensity to form 6-membered cyclic lactams

Ornithine is one of the products of the action of the enzyme arginase on L-arginine, creating urea. Therefore, ornithine is a central part of the urea cycle, which allows for the disposal of excess nitrogen.

Ornithine is not an amino acid coded for by DNA, and, in that sense, is not involved in protein synthesis. However, in mammalian non-hepatic tissues, the main use of the urea cycle is in arginine biosynthesis, so as an intermediate in metabolic processes, ornithine is quite important (wikipedia)

Ornithine (analog of Lys, product of arginase)


AAs with side chain carbonyls

D, E are acidic, hydrophilic Neutral, hydrophilic

D,E:• pKa~5• Very polar• Usually charged in

proteins• Esterification

reactions possible

N,Q:•Neutral•Polar, H-bonding•Deamidation reactions (protein ageing)


Protein deamidation • Deamidation is a common post-translational modification

• Conversion of Asn to a mixture of Asp and isoaspartate (aka beta-aspartate).

• Occurs to a lesser extent with Gln• Deamidation may cause loss of

protein activity• An important consideration for

recombinant protein-based drugs and therapeutics

• Occurs in vivo, especially among proteins with long life times.

• Highest frequency for Asn-Gly sequences

• Intermediate frequency for Asn-X where X = polar (Ser, Thr, Asp)

• Low frequency for Asn-X where X = hydrophobic residue

• Asn must be on flexible portion of protein

• Alkaline pH accelerates deamidation

• Change in protein acidity


Proline• Cyclic imino acid• No rotaton about N-Cα bond• No backbone N-H H-bonding. • No resonance stabilization of amide bond

• Peptide bond more likely to be in cis-conformation

Trans and Cis X-Pro Bonds. The energies of these forms are relatively balanced because steric clashes occur in both forms.


• Hydroxyproline is produced by hydroxylation of the amino acid proline by the enzyme prolyl hydroxylase following protein synthesis (as a post-translational modification). The enzyme catalysed reaction takes place in the lumen of the endoplasmic reticulum.

• Hydroxyproline is a major component of the protein collagen. • Hydroxyproline and proline play key roles for collagen stability. They

permit the sharp twisting of the collagen helix. In the canonical collagen Xaa-Yaa-Gly triad (where Xaa and Yaa are any amino acid), a proline occupying the Yaa position is hydroxylated to give a Xaa-Hyp-Gly sequence. This modification of the proline residue increases the stability of the collagen triple helix.

• It was initially proposed that the stabilization was due to water molecules forming a hydrogen bonding network linking the prolyl hydroxyl groups and the main-chain carbonyl groups. It was subsequently shown that the increase in stability is primarily through stereoelectronic effects and that hydration of the hydroxyproline residues provides little or no additional stability.

• Hydroxyproline is found in few (animal) proteins other than collagen. The only other mammalian protein which includes hydroxyproline is elastin. For this reason, hydroxyproline content has been used as an indicator to determine collagen and/or gelatin amount. (wikipedia)

There are 28 types of collagen, over 90% of the collagen in the body are of type I, II, III, and IV.

Collagen One - bone (main component of bone)

Collagen Two - cartilage (main component of cartilage)

Collagen Three - reticulate (main component of reticular fibers)

Collagen Four - floor – key component of basement membranes

Twisted, left handed helix due to high Pro, Gly content.

Hydroxyproline


Lesk: Introduction to Protein Science, chap 3, Fig. 1

Space filling amino acid side chain structures



Codon usage and protein structure evolution


Table of the frequency with which one amino acid is replaced by others in the amino acid sequence of the same protein in different organisms


Rotation of peptide bonds in a polypeptide

Dihedral angles in a polypeptide Rotation About Bonds in a Polypeptide. The structure of each amino acid in a polypeptide can be adjusted by rotation about two single bonds. (A) Phi (φ) is the angle of rotation about the bond between the nitrogen and the α-carbon atoms, whereas psi (y) is the angle of rotation about the bond between the α-carbon and the carbonyl carbon atoms. (B) A view down the bond between the nitrogen and the α-carbon atoms, showing how φ is measured. (C) A view down the bond between the α-carbon and the carbonyl carbon atoms, showing how y is measured.

The dihedral angles of a sequence of amino acid residues defines the three dimensional structure of the protein backbone

A Ramachandran Diagram Showing the Values of φ and Ψ. Not all φ and Ψ values are possible without collisions between atoms. The most favorable regions are shown in dark green; borderline regions are shown in light green. The structure on the right is disfavored because of steric clashes.


Protein secondary structure: alpha helix

•Structure of the α Helix. (A) A ribbon depiction with the α-carbon atoms and side chains (green) shown. (B) A side view of a ball-and-stick version depicts the hydrogen bonds (dashed lines) between NH and CO groups. (C) An end view shows the coiled backbone as the inside of the helix and the side chains (green) projecting outward. (D) A space-filling view of part C shows the tightly packed interior core of the helix.•3.6 res/turn•H-bonding to i+4



Proteins with high α-helical content

A Largely α Helical Protein. Ferritin, an iron-storage protein, is built from a bundle of α helices.

Myoglobin: first protein structure reconstructed by X-ray crystallography (Kendrew and Perutz), proved prediction of α-helix structure by Corey and Pauling


Protein secondary structure: β-sheets

An Antiparallel β Sheet. Adjacent β strands run in opposite directions. Hydrogen bonds between NH and CO groups connect each amino acid to a single amino acid on an adjacent strand, stabilizing the structure.

A Parallel β Sheet. Adjacent β strands run in the same direction. Hydrogen bonds connect each amino acid on one strand with two different amino acids on the adjacent strand.



A mixed β-sheet

Ribbon diagrams of twisted β-sheets



Loops on a Protein Surface. A part of an antibody molecule has surface loops (shown in red) that mediate interactions with other molecules

Structure of a Reverse Turn. The CO group of residue i of the polypeptide chain is hydrogen bonded to the NH group of residue i + 3 to stabilize the turn


What determines whether a particular protein sequence (sub-sequence) forms an α-helix, β-sheet, or a turn?

Amino acid residues have varying propensities to be present in secondary structures.

• α-helix (default), branched R-groups disfavored (V,T,I); H-bond donating R-groups disfavored (S, D, N)

• β-strands more tolerant of bulky R groups

• Proline disrupts α-helices and β-sheets, found in turns.Values diff’t in 5th ed


Hydropathicity/hydrophobicity index

AA residue K&D Hydrophobicity

Arginine -4.5Lysine -3.9Asparagine -3.5Aspartic acid -3.5Glutamine -3.5Glutamic acid -3.5Histidine -3.2Proline -1.6Tyrosine -1.3Tryptophan -0.9Serine -0.8Threonine -0.7Glycine -0.4Alanine 1.8Methionine 1.9Cysteine 2.5Phenylalanine 2.8Leucine 3.8Valine 4.2Isoleucine 4.5

Kyte, J., and Doolittle, R. F. (1982) J Mol Biol 157, 105-132.

Kyte and Doolittle hydrophobicity K&D Hydrophobicity plot for human rhodopsin

Expasy (http://ca.expasy.org)


Prediction of protein secondary structure from AA sequence

K&D

Chou and FasmanComputed scale of alpha helix forming properties for the 20 AA based on known protein structures Chou P.Y., Fasman G.D. Adv. Enzym. 47:45-148(1978).


Tertiary structure: the overall three dimensional fold of a polypeptide chain

Diagram depicting the amino acid backbone of myoglobin as a ribbon (8 helices) but no side chains

Ball and stick model showing all myoglobin atoms but not showing the amount of space each occupies


Tertiary structure of proteins: driven by the hydrophobic effect

Distribution of Amino Acids in Myoglobin. (A) A space-filling model of myoglobin with hydrophobic amino acids shown in yellow, charged amino acids shown in blue, and others shown in white. The surface of the molecule has many charged amino acids, as well as some hydrophobic amino acids. (B) A cross-sectional view shows that mostly hydrophobic amino acids are found on the inside of the structure, whereas the charged amino acids are found on the protein surface.

Figure 3.46. “Inside Out” Amino Acid Distribution in Porin. The outside of porin (which contacts hydrophobic groups in membranes) is covered largely with hydrophobic residues, whereas the center includes a water-filled channel lined with charged and polar amino acids.


Protein sequence motifs: structural elements (folds) found in different proteins

Greek key motif of beta strands

• Protein motifs are three dimensional structures (folds) found in a diversity of proteins and protein families. Their presence may imply a certain class of function (structural, enzymatic, or adhesive)

• Algorithms exist for predicting the presence of motifs from the primary sequence.


Protein folding motifs

Richardson, J. S. (1994) Introduction: protein motifs. Faseb J 8, 1237-9.


The role of solvent in secondary/tertiary structure formation

Reduction and denaturation of ribonuclease

Anfinson, 1950s: Reduced, denatured ribonuclease regains enzymatic activity when urea and β-mecaptoethanol are removed by dialysis

• Chaotropic (denaturing) agents form H-bonds with water, disrupt the normal structure of water, change the energetic balance that favors sequestering hydrophobic sequences in interior domains. In the absence of the entropic driving force behind protein folding, unfolding occurs.

SDS


Protein folding

• Proteins have the capacity to fold and become active based on the information contained in their amino acid sequence.

• Thermodynamically spontaneous• Proteins fold in buffered water; • Chaotropic agents disrupt the structure of water by participating in hydrogen

bonding. As a result, the hydrophobic driving force that makes a folded structure energetically favorable is disrupted– Guanidine salts, urea, detergents

• Proteins also denature at pH values deviating significantly from neutral. • Water miscible organic solvents are able to participate in hydrogen bonding.

Their presence also alters the thermodynamic driving force behind protein folding. As the percent of organic solvent in a solution increases, the tendency of proteins to unfold increases.

• Heat increases molecular motion. As proteins heat they fold and unfold rapidly. Intermolecular interactions of hydrophobic domains may cause proteins to precipitate (cooking an egg).


Proteins fold cooperatively, not randomly

Lodish

A current view of protein folding. Each domain of a newly synthesized protein rapidly attains a “molten globule” state. Subsequent folding occurs more slowly and by multiple pathways, often involving the help of a molecular chaperone. Some molecules may still fail to fold correctly. These are recognized and degraded by specific proteases.

Components of a Partially Denatured Protein Solution. In a half-unfolded protein solution, half the molecules are fully folded and half are fully unfolded. (Berg). There are too many possible structures for a random process (Levinthal’s paradox, 5 x 1047 structures for 100 aa protein). Progressive stabilization of intermediates results in correctly folded proteins.



Molecular chaperones stabilize hydrophobic sequences of newly synthesized polypeptides to enable orderly folding

•Improperly folded proteins do not exit the ER•HSPs: heat shock proteins, so named because their expression increases in response to heat and other cellular stresses that result in buildup of mis-folded proteins•HSPs require energy. •Polypeptides carry the information to fold in their sequences with assistance from chaperones


The GroEL/GroES (Hsp60/Hsp10) chaperone machine

Richardson, A., Landry, S. J., and Georgopoulos, C. (1998) Trends Biochem Sci 23, 138-43.


Molecular chaperones and protein folding quality control example: calnexin and protein N-glycosylation

Lodish

The role of N-linked glycosylation in ER protein folding. The ER-membrane-bound chaperone protein calnexin binds to incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides, trapping the protein in the ER. Removal of the terminal glucose by a glucosidase releases the protein from calnexin. A glucosyl transferase is the crucial enzyme that determines whether the protein is folded properly or not: if the protein is still incompletely folded, the enzyme transfers a new glucose from UDP-glucose to the N-linked oligosaccharide, renewing the protein's affinity for calnexin and retaining it in the ER. The cycle repeats until the protein has folded completely. Calreticulin functions similarly, except that it is a soluble ER resident protein. Another ER chaperone, ERp57 (not shown), collaborates with calnexin and calreticulin in retaining an incompletely folded protein in the ER.


The protein folding problem: can we predict the three dimensional structure of a protein from its amino acid sequence

• The sequence contains the information necessary for folding

• Useful predictions of secondary structure can be made (numerous tools on web, Expasy)

• A given peptide sequence may produce more than one fold in different proteins

• Conformational preferences of AAs not absolute

• Tertiary interactions among residues far apart in sequence influence the formation of secondary structure.

• The integration of secondary structures is a very computationally intensive problem.

• There is steady progress in understanding polypeptide properties but no clear solution to the protein folding problem (Nobel Prize!)


Free energy change of protein folding:• Unfolded proteins are random, folding entails considerable increase in order (so, why does it occur spontaneously?)

• Water molecules must form highly ordered cages around hydrophobic aa residues. Folding shields these residues from water, balancing the apparent increase in order.

• H-bonding, electrostatic and Van der Waal’s interactions results in a release in heat (negative enthalpy, ΔH)


Lesk, chap 5 Fig. 8


Quaternary structure: spatial arrangement of multi subunit proteins (made of more than one polypeptide chain)

A simple dimer: Quaternary Structure. The Cro protein of bacteriophage λ is a dimer of identical subunits.

A hetero-tetramer: hemoglobin is composed of 2 α and 2 β chains, each with a heme group.

Quaternary structure results from numerous interactions between the surfaces of the protein molecules. Structural plasticity allows cooperative oxygen binding in hemoglobin. Molecular machines are multiprotein complexes that execute many of the important functions in the cell (ribosome, nuclear pore complex, etc)


Protein quaternary structure


Atomic structure of the 50S Ribosome Subunit. Proteins are shown in blue and the two RNA strands in orange and yellow. The small patch of green in the center of the subunit is the active site. (Wikipedia)

Micro-environments and macromolecular complexes

The eukaryotic membrane, showing lipid bilayer, integral membrane proteins, protein channel, glycolipids


X-Ray CrystallographySteps:1. Recombinant expression of protein2. Formation of large, pure crystals,

regular in structure, no imperfections3. X-ray exposure, measurement of

diffraction pattern, as crystal is rotated

4. Computation on raw data, refinement, model building

5. Repeat

• ~36,000 protein structures solved to date using X-ray crystallography

• Crystal formation difficult for membrane proteins

• Very bright X-ray source needed (synchrotron) to produce the highest resolution (national labs)

Wikipedia


NMR Spectroscopy

6000 protein structures solvedWuthrich, K. (1990) J Biol Chem 265, 22059-22062.

•Nuclear magnetic resonance measures the environment of protons and other nuclei (13C, 15N, 31P)•CH, NH, OH, COOH, etc•NMR experiments determine distance constraints between NMR active nuclei in biomolecules•High concentration, high purity protein needed •Able to measure protein dynamics•Limited ability to solve structures of very large proteins•Expression of isotope enriched proteins to maximize NMR sensitivity•Requires a very large magnet (900 MHz NMR spectrometer, above)•Resource and computationally intensive

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Sept 14, 2009GMS BI 555/755 Lecture 2.1 Review: Gibbs free energy, enthalpy, entropy Work and energy storage/utilization in biological systems Types of