* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download ppt file
Gene expression wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Genetic code wikipedia , lookup
Expression vector wikipedia , lookup
Magnesium transporter wikipedia , lookup
Ancestral sequence reconstruction wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Biochemistry wikipedia , lookup
Homology modeling wikipedia , lookup
Bimolecular fluorescence complementation wikipedia , lookup
Protein purification wikipedia , lookup
Western blot wikipedia , lookup
Metalloprotein wikipedia , lookup
Interactome wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Proteolysis wikipedia , lookup
Basic protein structure and stability VII: Determinants of protein stability and structure Biochem 565, Fall 2008 09/12/08 Cordes Obvious interactions in native protein structures hydrophobic interactions R R1 2 NH disulfide crosslinks S R3 O S NH3 CO2 polar interactions (hydrogen bond/salt bridge) Contributions to protein stability type of interaction hydrophobic group burial hydrogen bonding ion pairs/salt bridges disulfide bonds total contribution* ~200 kcal/mol small?? <15 kcal/mol 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! 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! The interior is more important for stability than the exterior side chains with stabilityneutral Ala mutations side chains with destabilizing Ala mutations 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 can have importance both in maintaining stability and in maintaining a precise three-dimensional structure which is optimized for activity. One issue with the cores of proteins is simply volume. Given a particular backbone configuration, there is a certain amount of space that has to be filled, and over or underfilling it can be detrimental to stability Another constraint is sterics--not all cores with equal volume are equally stable. For instance, a simple switch of two residues in Gene V protein, Leu35/Val47-->Val35/Leu47, results in a 4 kcal/mol reduction in stability (Sandberg & Terwilliger, 1991) For a good review of packing, see Richards & Lim, Q Rev Biophys 26, 423 (1993). 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, however. Why? Not all buried Leu-->Ala mutations give the same destabilization. Why? 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/Å2 for forming this cavity. This is due to loss of van der Waals interactions with the mutated side chain. This increases the “vertical” (i.e. assuming the structure of the mutant is the same) 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, and this suggests that having good packing in terms of not having cavities is important to stability. The plasticity of protein cores Matthews and coworkers solved the crystal structures of a number of T4 lysozyme core mutants and found that the protein structure often adjusts to reduce the cavity size, and that this reduces the energetic penalty, restoring some stability...note that this doesn’t happen in all cases slope is penalty for cavity formation y-intercept is just the Leu/Ala transfer free energy difference! Eriksson et al. Science 255, 178 (1992) 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 The V111I mutant of lysozyme at right illustrates a typical backbone shift to accommodate increased volume. Lambda repressor V36L/M40L/ V47I is more stable than wild-type despite a 50 Å3 increase in core volume. A crystal structure shows that the backbone adjusts to accommodate the mutations. However, the mutant does not bind target DNA as well as wild type. Thus, despite increased stability and despite none of the residues being directly involved in function, the mutation is not tolerated. Thus, side chain packing is not only a determinant of stability, It can also be a key determinant of the precise structure of the protein [Lim et al. PNAS 91, 423 (1994)] 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 (though there are definitely exceptions) 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 • at least two studies have shown that surface polar-to-hydrophobic mutations can reduce structural specificity by favoring alternative conformations in which the introduced hydrophobic side chain becomes buried. This is another type of effect which may impact function. Cordes et al., Protein Sci 8, 318 (1999); Schwehm et al. Biochem 37, 6939 (1998); Cordes et al. Nat Struct Biol 7, 1129 (2000). Hill & DeGrado Struct Fold Des. (2000); Pakula & Sauer Nature 344, 363 (1990). Sickle-cell hemoglobin: a surface polar-to-hydrophobic mutation that lowers solubility Glu b6-->Val mutation causes self-association and polymerization mutation leads to hydrophobic interaction between hemoglobin tetramers Phe 85 Val 6 Leu 88 fibril formation at high concentration source: Biochemistry by Voet & Voet. picture of sickle-cell hemoglobin fibrils spilling out of a distorted, ruptured erythrocyte Energetics of hydrogen bonding in proteins The relevant situation for protein folding is arrow 3 or 5, depending upon how solvent-exposed the hydrogen bond is in the native state. Buried hydrogen bonds (5) can actually destabilize proteins, while solventexposed ones (3) may be slightly stabilizing. The same is true of ion pairs/salt bridges. unfolded protein exposed H-bond in folded protein buried H-bond in folded protein “Hydrogen bond inventory” 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, be it solvent, protein backbone, or protein side chains. Alan Fersht has called this concept “hydrogen bond inventory”. This is important when trying to understand the effect of mutations that impact hydrogen bonding, because removal of one partner of a hydrogen bonded pair can be quite destabilizing if the remaining partner is not able to satisfy its hydrogen bond potential by interacting with solvent. Essentially this same logic is 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! In fact, one observes very few “uncompensated” buried polar or charged groups in proteins, and mutation of one partner of a salt bridge or hydrogen bond is usually very destabilizing. 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. (His 31-Asp 70 in lysozyme is an example). 0.0 kcal/mol 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. Strop & Mayo, Biochemistry 39, 1251 (2001) P. furiosis rubredoxin 1.5 kcal/mol Structural role of buried salt bridges wild-type Arc R31-E36-R40 “Arc-MYL” M31-Y36-L40 Substitution of of Arg31, Glu36 or Arg40 by Ala destabilizes Arc repressor by 3 to 6 kcal/mol. Mutation of all three by the “MYL” triad, however, stabilizes the protein by 4 kcal/mol!! [Waldburger et al. Nature Struct Biol 2, 122 (1995)] Buried salt bridges (and buried polar interactions in general) not important for stability per se, but removal of individual partners can be hugely destabilizing. It has been hypothesized that 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. This has been directly shown to be true for some proteins [e.g. Lumb & Kim, Biochemistry, 34, 8642 (1995)] 2TRX.pdb Buried polar residues/interactions in thioredoxin D26 water-mediated H-bond to C32 carbonyl + T66-G74 schmch hydrogen bond + = water C32-C35 disulfide T77-D9 sch-sch hydrogen bond Effects of mutating buried polar residues in thioredoxin IAALV means D26I/C32A/C35A/T66L/T77V AALV means C32A/C35A/T66L/T77V Bolon D & Mayo SC Biochemistry 40, 10047 (2001). IAALV found to have “less specific” native state-can’t remove all buried polar residues H-bonding motifs: N-termini of alpha helices Many helices have side-chain to main-chain hydrogen bonds at their N-termini. Mutations to alanine of side chains involved in such interactions have effects ranging from +0.5 to +2.0 kcal/mol These residues usually occupy the position immediately before the helix starts. “N-cap” side-chain to main-chain hydrogen bond Ser, Thr, Asp and Asn are most stabilizing here. (small side chains that can act as acceptors) Asp better than Asn, possibly because solvent-exposed of “helix-dipole” effects. amide hydrogens Gly is also OK at N-cap. Why? serine side chain Relative stability of helix “N-cap” variants of barnase amino acid Asp Thr Ser Asn Gly Gln Glu His Ala Val Pro DDGu (kcal/mol) 2.02 2.05 1.64 0.86 0.69 0.42 0.25 0.16 0.00 -0.15 -0.87 from Fersht AR, “Structure and Mechanism...” Chapter 17, p. 527. The numbers represent the average of two positions in the protein. The N-cap is defined as the first residue the carbonyl of which makes an i,i+4 hydrogen bond to an amide. These are relative free energies of unfolding, so a higher number means greater stability. Residues with unusual backbone conformation preferences: glycines at alpha-helix C-termini left-handed (aL) conformation here leads to capping of carbonyls here while terminating the helix and causing a change in chain direction these carbonyls hydrogen bond to solvent Many helices terminate this way, and glycine is favored at the left-handed position because of its backbone flexibility and because large side chains here would point upward and interfere with solvation of carbonyls. About one-third of all helices terminate in glycine! “Schellman motif” Relative stability of mutants at C-terminal ends of helices in barnase amino acid Gly His Asn Arg Lys Ala Ser Asp from Fersht “Structure and Mechanism.. DDGu (kcal/mol) in Protein Science”, Ch. 17, 2.23 p. 526 0.67 Gly--> Ala mutations 0.47 have ranged from +1 to +3 kcal/mol in a number 0.47 of proteins. The 2.2 kcal/mol 0.01 number observed here is 0.00 typical -0.16 -0.27 The residue being mutated is the left-handed (aL) residue at the Cterminal end of the a-helix. Since these are relative free energies of unfolding, a higher number means higher stability. Glycines can contribute to stability (at certain positions, relative to other residues) because of their unique backbone conformation characteristics. Would the average glycine be stabilizing, though? Intrinsic secondary structure propensities and stability All effects listed relative to Ala amino acid Ala Arg Leu Met Lys Trp Gln Ser Ile Phe Cys Glu Tyr Asn Thr Val His Asp Gly Pro DDGu (kcal/mol), alpha-helix 0.00 -0.17 based on effects of -0.17 surface mutations -0.19 in helices in a -0.31 variety of proteins. -0.31 Some residues like Ala consistently -0.33 stabilize proteins -0.44 relative to other -0.43 residues, when -0.47 they occur in -0.54 helices. -0.56 -0.56 -0.61 -0.61 -0.63 -0.65-0.88 (0 or + charge) -0.68 -0.90 -3.47 in helices, effect of average substitution is very small. In beta-sheets can be larger but depends upon the sequence/structure context. DDGu (kcal/mol), beta-sheet 0.00 0.40 based on effects of 0.45 surface mutations 0.90 at Thr 53 in beta0.35 sheet of B1 domain 1.04 of protein G. These effects depend very 0.38 strongly upon 0.87 context, e.g. what 1.25 side chains interact 1.08 with the mutated 0.78 position on the same face of the 0.28 beta-sheet. 1.63 It could be argued, 0.52 therefore, that there 1.36 is no such thing as 0.94 “intrinsic” betasheet propensity. 0.37 -0.85 -1.21 > -5 from Fersht book Chapter 17, p. 528 Stability-activity trade-offs? It has been shown for many proteins that it is possible to engineer higher stability by introducing mutations. In many cases, this does not appear to impair activity in in vivo and/or in vitro assays. Moreover, comparable proteins from thermophilic organisms have higher stability than those from mesophilic counterparts. This shows that proteins have not evolved to maximize stability. Rather, it is likely that they generally evolve to preserve adequate stability. However, sometimes stability and activity are directly at odds with one another, and one is selected at the expense of the other. Many thermophilic proteins have low activities at lower temperatures. Some mutations in the active sites of enzymes (barnase, T4 lysozyme) have been shown to give more stable but less active proteins. 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. [source: Chapter 17 of Fersht. “Structure and Mechanism in Protein Science”] Mutation of catalytic residues in T4 lysozyme Asp 20 protein wild-type E11F E11M E11A E11H E11N D20N D20T D20S D20A DTm , °C 0 4.3 4.1 2.6 0.1 -0.6 3.1 2.2 1.6 -0.8 DDGf, kcal mol-1 0 1.7 1.6 1.1 0.1 -0.1 1.3 0.9 0.7 -0.3 active site cleft activity (relative) 1 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0005 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) “Intrinsically disordered”/”Natively denatured” proteins protein sequences with high net charge, low hydrophobicity tend not to be stable Not all natural proteins have stable folded structures! In your average organism 10-20% do not, by various estimates! Folding sometimes depends upon binding activity. Oldfield CJ et al Biochemistry 44, 1989 (2005). review: Wright PE, Dyson JH J Mol Biol 293, 321 (1999)