Basic protein structure and stability VII: Determinants of protein stability and structure Biochem 565, Fall /12/08 Cordes

Obvious interactions in native protein structures disulfide crosslinks polar interactions (hydrogen bond/salt bridge) hydrophobic interactions

Contributions to protein stability type of interactiontotal contribution* hydrophobic group burial~200 kcal/mol hydrogen bondingsmall?? ion pairs/salt bridges<15 kcal/mol disulfide bonds4 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 stability- neutral 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 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? nondisruptive means not causing any steric clashes or uncompensated buried charges, H-bonds etc

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 “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 y-intercept is just the Leu/Ala transfer free energy difference! slope is penalty for cavity formation Eriksson et al. Science 255, 178 (1992)

Disruptive mutations in hydrophobic cores Three kinds: steric mutantschange in shape, not volume extreme volume mutantsincrease core volume polar mutantsput 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 Å 3 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 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). 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.

Sickle-cell hemoglobin: a surface polar-to-hydrophobic mutation that lowers solubility Glu  6-->Val mutation causes self-association and polymerization source: Biochemistry by Voet & Voet. fibril formation at high concentration mutation leads to hydrophobic interaction between hemoglobin tetramers Val 6 Leu 88 Phe 85 picture of sickle-cell hemoglobin fibrils spilling out of a distorted, ruptured erythrocyte

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 solvent- exposed ones (3) may be slightly stabilizing. The same is true of ion pairs/salt bridges. Energetics of hydrogen bonding in proteins unfolded protein buried H-bond in folded protein exposed 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.

0.0 kcal/mol 1.5 kcal/mol Role of solvent-exposed salt bridges P. furiosis rubredoxin 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). 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)

wild-type Arc R31-E36-R40 “Arc-MYL” M31-Y36-L40 Structural role of buried salt bridges 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)]

Buried polar residues/interactions in thioredoxin 2TRX.pdb C32-C35 disulfide T77-D9 sch-sch hydrogen bond T66-G74 sch- mch hydrogen bond D26 water-mediated H-bond to C32 carbonyl + + = water

Effects of mutating buried polar residues in thioredoxin Bolon D & Mayo SC Biochemistry 40, (2001). IAALV means D26I/C32A/C35A/T66L/T77V AALV means C32A/C35A/T66L/T77V IAALV found to have “less specific” native state-- can’t remove all buried polar residues

“N-cap” side-chain to main-chain hydrogen bond 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. Ser, Thr, Asp and Asn are most stabilizing here. (small side chains that can act as acceptors) Asp better than Asn, possibly because of “helix-dipole” effects. Gly is also OK at N-cap. Why? solvent-exposed amide hydrogens serine side chain

amino acid  G u (kcal/mol) Asp2.02 Thr2.05 Ser1.64 Asn0.86 Gly0.69 Gln0.42 Glu0.25 His0.16 Ala0.00 Val-0.15 Pro-0.87 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. Relative stability of helix “N-cap” variants of barnase from Fersht AR, “Structure and Mechanism...” Chapter 17, p. 527.

left-handed (  L ) conformation here leads to capping of carbonyls here while terminating the helix and causing a change in chain direction Residues with unusual backbone conformation preferences: glycines at alpha-helix C-termini About one-third of all helices terminate in glycine! these carbonyls hydrogen bond to solvent “Schellman motif” 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.

The residue being mutated is the left-handed (  L ) residue at the C- terminal end of the  -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? amino acid  G u (kcal/mol) Gly2.23 His0.67 Asn0.47 Arg0.47 Lys0.01 Ala0.00 Ser-0.16 Asp-0.27 Relative stability of mutants at C-terminal ends of helices in barnase Gly--> Ala mutations have ranged from +1 to +3 kcal/mol in a number of proteins. The 2.2 kcal/mol number observed here is typical from Fersht “Structure and Mechanism.. in Protein Science”, Ch. 17, p. 526

Intrinsic secondary structure propensities and stability amino acid  G u (kcal/mol), alpha-helix  G u (kcal/mol), beta-sheet Ala Arg Leu Met Lys Trp Gln Ser Ile Phe Cys Glu Tyr Asn Thr Val His (0 or + charge) 0.37 Asp Gly Pro-3.47 > -5 based on effects of surface mutations in helices in a variety of proteins. Some residues like Ala consistently stabilize proteins relative to other residues, when they occur in helices. based on effects of surface mutations at Thr 53 in beta- sheet of B1 domain of protein G. These effects depend very strongly upon context, e.g. what side chains interact with the mutated position on the same face of the beta-sheet. It could be argued, therefore, that there is no such thing as “intrinsic” beta- sheet propensity. All effects listed relative to Ala in helices, effect of average substitution is very small. In beta-sheets can be larger but depends upon the sequence/structure context. 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”]

protein  T m  G f, activity, °C kcal mol -1 (relative) wild-type001 E11F4.31.7< E11M4.11.6< E11A2.61.1< E11H0.10.1< E11N < D20N3.11.3< D20T2.20.9< D20S1.60.7< D20A Mutation of catalytic residues in T4 lysozyme Glu 11 Asp 20 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. active site cleft Shoichet et al. PNAS 92, 452 (1995)

“Intrinsically disordered”/”Natively denatured” proteins 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) protein sequences with high net charge, low hydrophobicity tend not to be stable