Flexibility of Protein Structure

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1 Flexibility of Protein Structure Proteins show varying degree of conformational flexibility Due to movements of atoms in molecules vibration in bond length and angles Reflects the existence of populations of alternative conformations Severe constraints on the extent of conformational variations Integral proteins are less flexible than watersoluble proteins

2 Hydrogen Exchange The best evidence for extensive mobility of protein structure Hydrogen atoms bonded covalently to various atoms exchange with solvent at different intrinsic rates, depending on the tendency of that atom to ionize H atoms on oxygen, nitrogen, or sulfur exchange rapidly H atoms on carbon change at slow rate Intrinsic rate of exchange is temperature dependent 3 fold increase with 10 C increase Also, influenced by the environment and by inductive and charge effects on the amide

3 Hydrogen Exchange Local unfolding by hydrogen exchange in interior group atoms By diffusion of solvent molecules to various sites Rates are correlated with the solvent accessibility of the residue and of its neighbors, not distance Rate is decreased by increased pressure Wide variations in processes depending on the proteins and conditions

4 Stability of the Folded Conformation The folded conformations of proteins are only marginally stable even under the best conditions Can be easily denatured by changes in environmental conditions Temperature increase ph changes Pressure increase Addition of denaturants

5 Reversible folding & unfolding Anfinsen (1950 s) showed reversibility of denaturation with urea for RNAse A amino acid sequence encodes structure: thermodynamic hypothesis Folding is cooperative S-S bonds in brown Denaturation-studied by various methods is reversible

6 Stability of Proteins Stability of the folded structure of a globular protein depends on the interplay of three factors. 1. The unfavorable conformational entropy change, which favors random chains instead. 2. The favorable enthalpy contribution arising from intramolecular side group interactions. 3. The favorable entropy change arising from the burying of hydrophobic groups within the molecule. Factor 1 works against folding, whereas 2 and 3 help stabilize folding.

7 Denaturation Do need not changes in covalent structure Usually reversible if it is due to unfolding Proteins can be unfolded by Breaking any disulfide bonds present Removing any essential cofactors Mutation in certain crucial residues Deleting residues from the primary structure

8 Reversible Unfolding Transition No decrease in biological activity Unfolding is a two-state phenomenon, with only fully folded (F) and fully folded (U) states Partially folded structures are unstable relative to U or F Under conditions that favor unfolding, The folded conformation changes very little if at all Increase in flexibility Localized conformational alterations Average structure not changed Then protein unfolds completely within a limited range of conditions

9 Nature of the Unfolded State Unfolded states produced under various unfolding conditions have different physical properties Unfolded proteins do not contain cooperative folded structure But, unfolded and folded proteins are indistinguishable thermodynamically

10 Molten Globule State Conformation that are neither fully folded nor fully unfolded The overall dimensions of the polypeptide chain are much less than those of a random coil Average content of secondary structure is similar to folded The side chains are in homogeneous surroundings Many amide groups exchange hydrogen atoms with the solvent much rapidly than the folded The enthalpy is nearly the sale as the folded state Interconversions of the molten globule state with the fully folded state are rapid

11 Extreme ph Physical Basis for Protein Denaturation Non-ionized His and Tyr buried in folded structure can be ionized Disruption of salt bridges Denaturants: Urea and Guanidinium salts Break H bonds Have hydrogen bonding capabilities comparable to that of water, but different geometries Increase the solubilities of both polar and nonpolar molecules

12 Thermodynamics of Unfolding Elevated temperature increase unfolding Proteins from thermophiles are more heat stable Unfolded state has a greater heat capacity than the folded state Large temperature dependence of the changes in enthalpy and entrophy The more hydrophobic the interior of a protein, the lower its net stability

13 Forces that hold protein structure together Dispersion (van der Waals) forces Electrostatic interactions Hydrogen bonds Hydrophobic interactions What pulls it apart? Conformational entrophy

14 Stabilizing Forces of Folded Hydrogen bonds Conformations The major stabilizing force for folded conformation H-bonds in the folded conformation is stronger than those in unfolded ones H-bonds with water and a protein are present a fraction of a time while that within protein present are present all of the time (more negative enthalpy) Electrostatic interactions More important van der Waals interactions Van der Waals interactions Hydrophobic interactions Unique in folded structure Driving force for folding

15 Contributions to protein stability type of interaction total contribution* hydrophobic group burial ~200 kcal/mol hydrogen bonding small?? ion pairs/salt bridges <15 kcal/mol disulfide bonds 4 kcal/mol per link *For globular protein of 150 residues hydrophobic burial is the chief interaction favoring protein stability, but this is balanced by a huge loss of conformational entropy that opposes folding. Consequently typical net protein stabilities are 5-20 kcal/mol--> so even minor interactions can make a difference!

16 Hydrophobicity and the Stability of Folded Conformation Hydrophobicity is not the Primary Force to the Stability of Folded Conformation. Why? Although the interior of proteins are densely packed, they are not closely packed with all adjacent atoms in Van der Waals contact simultaneously Imperfect Van der Waals interactions Van der Waals interactions in proteins are in less than optimal conditions and weaker than those occur in liquid

17 Stability of the Folded conformation Folded state has only marginal net stability because of the large conformational entrophy of the unfolded state Depends on its primary structure Single amino acid replacement can alter the stability of a folded protein quite drastically

18 Alanine scanning A way of assessing the importance of amino-acid side chains for structure/stability etc. Remove each residue one by one by replacement with Ala Many Ala mutations have no effect on stability--> about half! A large group also cause significant effects-->several kcal/mol Occasional a mutant will stabilize the protein--> natural proteins not maximally stable!

19 The interior is more important for stability than the exterior side chains with stability-neutral Ala mutations side chains with destabilizing Ala mutations

20 Side-chain packing in the hydrophobic core Protein interiors have a jigsaw puzzle -like aspect. Their packing densities are similar to those of crystals of organic molecules. This dense packing are importance for stability and a precise threedimensional structure Over or under filling can be detrimental to stability However, not all cores with equal volume are equally stable.

21 Effect of nondisruptive hydrophobic core mutations nondisruptive means not causing any steric clashes or uncompensated buried charges, H-bonds etc leucine in core mutation to alanine difference in water-octanol transfer free energies of leucine and alanine is ~2 kcal/mol. Effects of Leu-->Ala mutations are typically larger than this. Why? Not all buried Leu-->Ala mutations give the same destabilization. Why?

22 The cost of cavity formation in protein cores A Leu-->Ala core mutation leaves a cavity in the hydrophobic core. In addition to the ~2 kcal/mol transfer free energy difference between Leu and Ala, there is a penalty of 20 cal-mol/å 2 for forming this cavity. This is due to loss of van der Waals interactions with the mutated side chain. This increases the cost of a Leu-->Ala mutation from 2 to about 5 kcal/mol! Since proteins are only stable toward unfolding to the extent of 5 to 15 kcal/mol, such mutations are potentially devastating This suggests that having good packing in terms of not having cavities is important to stability.

23 Disruptive mutations in hydrophobic cores Three kinds: steric mutants extreme volume mutants polar mutants change in shape, not volume increase core volume put polar/charged residue in core Polar/charged core mutants are almost invariably very destabilizing, for obvious reasons. Charged groups or groups that can form hydrogen bonds that are isolated within a protein interior are bad for stability Energetic effects of increased volume are often hard to predict-- subtle backbone shifts often occur to accommodate the extra volume

24 Mutations of surface (solvent-exposed) residues Although the surface of proteins are very polar overall, individual surface positions can usually be replaced by many other residues including hydrophobics without much effect on stability. average effect on stability of surface mutations is small little stability penalty for change of individual surface polars to hydrophobics However: too many hydrophobics on a protein s surface will reduce solubility and promote aggregation surface polar-to-hydrophobic mutations can reduce structural specificity by favoring alternative conformations in which the introduced hydrophobic side chain becomes buried.

25 Hydrogen bond inventory Definition: Although hydrogen bonds probably do not stabilize proteins per se, it is nonetheless important that all potential hydrogen bond donors and acceptors be hydrogen bonded to something - solvent, protein backbone, or protein side chains. Removal of one partner of a hydrogen bonded pair by mutation can be quite destabilizing if the remaining partner is not able to satisfy its hydrogen bond potential by interacting with solvent. Also applicable to ion pairing/salt bridge interactions. Even though ion pairs don t contribute much to stability, charged groups which are neither paired with oppositely charged groups nor solvated by water can be very destabilizing!

26 Role of solvent-exposed salt bridges Typical mutations of surface salt bridges are destabilizing by less than 1 kcal/mol, but there are cases where larger effects are observed. Surface salt bridges are thus not large contributors to protein stability. However, some salt bridges may be important at the level of specifying a particular precise structure, much in the way that hydrophobic packing interactions are. Buried salt bridges (and buried polar interactions in general) not important for stability per se, but removal of individual partners can be hugely destabilizing. Buried polar interactions serve more to impart specificity to the structure rather than stability, due to the strict requirement for satisfaction of H-bond potential (H-bond inventory) and compensation of charge.

27 Stability-activity trade-offs? Proteins have not evolved to maximize stability. They generally evolve to preserve adequate stability. However, sometimes stability and activity are selected at the expense of the other. Some mutations in the active sites are more stable but less active. For instance, the active site of barnase is highly positively charged because it has to bind a negatively charged pentacoordinate phosphate at the transition state. When substrate is not bound, the positively charged side chains repel each other, reducing stability.

28 Mutation of catalytic residues in T4 lysozyme Asp 20 active site cleft protein ΔT m ΔΔG f, activity, C kcal mol -1 (relative) wild-type E11F < E11M < E11A < E11H < E11N < D20N < D20T < D20S < D20A Glu 11 Replacement with some amino acids increases stability but strongly diminishes activity. This same phenomenon was found to occur for residues involved in substrate binding. Glu11 and Asp20 are examples of what has been referred to as electrostatic strain in enzyme active sites. However, not all mutations which remove the charge stabilize the protein, emphasizing that the situation is complex. Shoichet et al. PNAS 92, 452 (1995)

29 Factors Determining Secondary and Tertiary Structure 29

30 The Information for Protein Folding The information for determining the 3-dimensional structure of a protein is carried entirely in the amino acid sequence of that protein. Native structure is the natural 3-dimensional structure of a protein. Denaturation is a process for the lost of natural structure of a protein, along with many of its specific properties. In a cell, a newly synthesized polypeptide chain will spontaneously fold into the proper conformation. 30

31 Protein Folding Proteins normally want to fold into their native, or lowest-energy, conformation the most stable Christian B. Anfinsen won the Nobel Prize in 1973 for finding that this folding is based on directions from the amino acid sequence and from its hydrophobic/phillic nature He started by unfolding proteins into random coils, and found the proteins folded back into their native shape spontaneously, without any other catalysts

32 Thermodynamics of Folding The overall free energy change on folding must be negative. Conformational Entropy ΔG = ΔH TΔS Charge-Charge Interactions Salt bridges Internal Hydrogen Bonds van der Waals Interactions The Hydrophobic Effect 32

33 Gibbs Free Energy (ΔG) A thermodynamic potential that measures the "useful" or process-initiating work obtainable from an isothermal, isobaric thermodynamic system The maximum amount of non-expansion work that can be extracted from a closed system Gibbs free energy (ΔG) or available energy is the chemical potential that is minimized when a system reaches equilibrium at constant pressure and temperature.

34 Contributions to ΔG of folding What features of protein folding produce large, negative ΔH or large positive ΔS changes, to compensate for the conformational entropy loss?

35 The Role of Disulfide Bonds The formation of disulfide bonds between cysteine residues further stabilize the 3-D structure of proteins. Formation of the disulfide bonds is depend on the protein primary structure. Disulfide bonds are not essential for correct protein folding, but for contribution to the stability of the structure once it is folded. Site-directed mutagenesis, a powerful method for the test of the effect of changing one or more amino acid residues or adding or removing disulfide bonds. 35

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37 Kinetics of Disulfide Bond Formation The process of refolding of a protein from a state in which disulfide bonds have been cleaved is complicated and slower. Some disulfide bonds that are not in the native structure are formed in the intermediate stages of the folding. The protein can utilize a number of alternative pathways to fold but ultimately finds both its proper tertiary structure and the correct set of disulfide bonds. It is aided in vivo by enzymatic catalysis of -s-sbond rearrangement. 37

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39 When Folding Goes Wrong Protein Misfolding: Proteins fold into conformations which are local energy minima, but not the global minimum States reaching local, but not global, minima are sometimes called intermediate states Misfolding is responsible for many diseases due to the accumulation of plaque of misfolded proteins Alzheimers, Parkinsons, Mad Cow Disease, etc. chaperone proteins are present to help the target protein reach the desired functional (native) conformation

40 Prions - Protein Folding and Mad Cow Disease A class of diseases that is transmitted by a protein. Bovine spongiform encephalopathy or mad cow disease is the best known of these diseases. The infectious agent is called prion, and the protein is called prion-related protein, or PrP. PrP protein is normally present in many animals including human in a nonpathological form of PrP c (prion-related protein cellular). Under certain circumstances (not yet known), PrP c, can change the conformation to a different structure called PrP sc (or prion-related protein scrapie). The N-terminal portion of PrP sc is partially folded into a β- sheet which wreaks havoc with the nervous system. It can induce conversion of PrP c in the recipient to PrP sc.

41 The Protein Folding Problem: Levinthal s paradox How many conformations could a protein chain adopt? there are about 3-5 combinations of φ/ψ for each amino acid that would be acceptable to its neighbour. so its 3-5 n conformations that a protein can adopt (n = number of amino acids) 100 amino acids = i.e; about 1 x conformations

42 Levinthal paradox If a protein can sample conformations per second (average frequency of bond rotation is about s, 0.01 ps) Then it would take our 100 residue protein 1 x seconds, or 5 x years to sample all its options. Folding cannot simply occur by the protein trying every conformation until it finds the most stable one. There isn't enough time. HOW DO PROTEINS EVER FOLD????

43 Time-scales for folding cytochrome b562: lambda repressor: rat IFABP: CRABP 1: tryptophan synthase β2-subunit: 5 μs 0.67 ms 33 ms 24.5 sec 992 sec (396 aa)

44 Thermodynamic vs. Kinetic control? Folding is driven by thermodynamic considerations. The native state must be thermodynamically stable. But the kinetics of folding is also important. Folding must occur rapidly. Do folded structures represent true global energy minimum, or just kinetically accessible local minima? What causes slow folding: a high transition-state barrier, or just a large space to search?

45 Kinetics of Protein Folding Folding takes place through a series of intermediate states, unfolded protein U, nucleation protein, partially folded I I, nearly folded I N and the final rearrangement to become native protein F. The funneling model for nucleation of proteins proposes that there is not just one but many possible paths from the denatured state to the folded state, and each path leads downhill in energy. The molten globule, a compact structure in which much of the secondary and tertiary folding has occurred, but the internalized hydrophobic residues have not yet formed. There is also off-path state in which some key element is incorrectly folded, and the final folding time will be delayed.

46 Lowest energy state? The forces of macromolecular interaction: Covalent bonds Electrostatics Hydrogen bonds van der Waals Hydrophobic effect If not optimal then each of these forces contributes an energy cost to the system. If optimal then the forces above are satisfied and the energy is minimal

47 Balls roll downhill, and Levinthal is satisfied Levinthal s problem is removed if the folding pathway is biased towards the correct structure. i.e. once you start moving in the right direction, you ll get there correctly folded state is the lowest energy state

48 Folding theory: Energy landscapes formation of local structure stabilizes An intermediate called the transition state ensemble is thought to exist Metastable Key contacts Same topology as fully folded structure energy enables collapse to native fold

49 Energy Landscapes in 2-D E Local minima Transition states Global minimum In the process of folding from a random coil to the native state (at the global minimum), proteins pass through intermediate states (at local minima) Sometimes the proteins can get stuck in these intermediate states and cannot get over the energy hump needed (at transition states) to get to the final native conformation The study of protein folding from the denatured to the native state of vice versa, over time, is kinetics

50 How does force modify the unfolding landscape? TS When you add FORCE (F): Relative to the native state, N, G TS G TS-N the barrier to unfolding ( G TS-N ) is lowered by: Fx u -N x f D and the free energy of unfolding ( G D-N ) is lowered by: F(x u + x f ) N x u G D-N The protein is less stable and unfolds more rapidly - the unfolding rate (k u ) is a measure of the height of the barrier between N and TS

51 The Funnel (3-D) Landscape Model Protein Folding actually proceeds (as down a funnel) through multiple kinetic pathways Unstable (High Energy) unfolded conformations at rim can follow many energy gradients to get to the single global minimum (Lowest Energy) The steepest path is the fastest folding trajectory, but other slower trajectories to the minimum exist Note the diverse multitude of unfolded conformations, but only one conformation for the native state

52 Energy Landscapes How does an energy landscape solve Levinthal s paradox? a) A random walk to a level energy surface has little chance of finding N b) The barriers surrounding the minimum energy paths that connect the different local minima are very steep c) A funnel lead to N via many paths: some straight and others with intermediates d) Not a single conformation of the chain which optimizes all the interactions at the same time a) Golf course, b) Ant-trail, c) energy funnel, d) realistic rugged funnel

53 Molten Globule An intermediate state in the folding of protein pathway of a protein that has some secondary and tertiary structure, but lacks the well packed amino acid side chains that characterize the native state of a protein. Observed for many protein under both equilibrium and non-equilibrium conditions. By contrast, for fast folding proteins without intermediates, the search for a core or nucleus is likely to be the rate-determine step; once the core is formed, folding to the native state is fast

54 Chaperonins A class of proteins which "chaperone" a protein to help keep it properly folded and non-aggregated. Aggregation is a problem for unfolded proteins The hydrophobic residues, which normally are deep inside of a protein, may be exposed when the protein is released from the ribosome. If they are exposed to hydrophobic residues in other strands, the two strands may associate with each other hydrophobically (to aggregate) instead of folding properly. It provides a central cavity (shelter) in which new protein chains can be "incubated" until they have folded properl ATP is required to drive the process in one direction. 24

55 Chaperonins

56 Enzymes that Speed Folding Protein disulphide isomerase Facilitates formation of correct disulphide bridges Peptidyl proline isomerase Catalyses cistrans isomerisation of peptide bonds involving proline Molecular chaperones Help folding, especially of large proteins, by preventing interaction with other proteins

57 Prediction of Protein Structure Can we predict the protein structure? Yes, if we could obtain all the necessary information, including the amino acid and understanding the rules of folding. Secondary structure can be predicted fairly well to-date, but not for the tertiary structure.

58 Problem of protein structure prediction For over 30 years, there has been an ardent search for methods to the predict threedimensional (3D) structure from the sequence Many methods were found which looked initially very promising - but always the hope has been dashed

59 Techniques of Structure Prediction Computer simulation based on energy calculation Based on physio-chemical principles Thermodynamic equilibrium with a minimum free energy Global minimum free energy of protein surface Knowledge Based approaches Homology Based Approach Threading Protein Sequence Hierarchical Methods