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

S

S

R3

R1R2

CO2

NH3

ONH

disulfide crosslinks polar interactions (hydrogen bond/salt bridge)

hydrophobic interactions

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!

Alanine scanningA way of assessing the importance of amino-acid side chains for structure/stability etc.“Remove” each residue one by one by replacement with Alamany Ala mutations have no effect on stability--> about half!A large group also cause significant effects-->several kcal/molOccasional 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”-likeaspect. Their packing densities are similar to thoseof crystals of organic molecules. This densepacking can have importance both in maintainingstability and in maintaining a precise three-dimensional structure which is optimized for activity.

One issue with the cores of proteins is simplyvolume. 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 equalvolume are equally stable. For instance, asimple switch of two residues in Gene V protein,Leu35/Val47-->Val35/Leu47, results in a 4 kcal/molreduction 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

leucinein core

mutation to alanine

difference in water-octanol transferfree energies of leucine and alanineis ~2 kcal/mol. Effects of Leu-->Alamutations are typically larger thanthis, however. Why?

Not all buried Leu-->Ala mutationsgive the same destabilization. Why?

nondisruptivemeans not causingany steric clashesor uncompensatedburied charges, H-bondsetc

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 betweenLeu 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 mutatedside chain. This increases the “vertical” (i.e. assuming the structure ofthe mutant is the same) cost of a Leu-->Ala mutation from 2 to about5 kcal/mol! Since proteins are only stable toward unfolding to the extentof 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 coworkerssolved the crystal structures of a number of T4 lysozyme coremutants and found that the protein structure often adjusts to reduce thecavity size, and that this reduces the energetic penalty, restoring some stability...note that this doesn’t happen in all cases

y-intercept is justthe Leu/Ala transfer freeenergy difference!

slope is penaltyfor cavity formation

Eriksson et al. Science 255, 178 (1992)

Disruptive mutations in hydrophobic cores

Three kinds:steric mutants change in shape, not volumeextreme volume mutants increase core volumepolar mutants 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 rightillustrates a typical backboneshift to accommodate increasedvolume.

Lambda repressor V36L/M40L/V47I is more stable than wild-typedespite a 50 Å3 increase in core volume. A crystal structure showsthat the backbone adjusts to accommodate the mutations. However, the mutant doesnot bind target DNA as well as wild type. Thus, despite increased stability and despitenone of the residues being directly involvedin 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 andpromote 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-hydrophobicmutation that lowers solubility

Glu 6-->Val mutation causesself-association and polymerization

source: Biochemistry by Voet & Voet.

fibrilformationat highconcentration

mutation leads tohydrophobicinteractionbetweenhemoglobintetramers

Val 6

Leu 88

Phe 85

picture ofsickle-cell hemoglobinfibrils spilling out ofa distorted, rupturederythrocyte

The relevant situationfor 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 ArcR31-E36-R40

“Arc-MYL”M31-Y36-L40

Structural role of buried salt bridges

Substitution of of Arg31, Glu36 orArg40 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 polarinteractions in general) not importantfor 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-bondto C32 carbonyl

+ + = water

Effects of mutating buried polar residues in thioredoxin

Bolon D & Mayo SC Biochemistry 40, 10047 (2001).

IAALV means D26I/C32A/C35A/T66L/T77VAALV means C32A/C35A/T66L/T77V IAALV found to have

“less specific” native state--can’t remove all buried polarresidues

“N-cap” side-chain to main-chainhydrogen bond

H-bonding motifs:N-termini of alpha helices

Many helices have side-chain to main-chainhydrogen bonds at their N-termini. Mutationsto alanine of side chains involved in such interactions have effects ranging from +0.5 to +2.0 kcal/mol These residues usuallyoccupy the position immediately before thehelix starts.

Ser, Thr, Asp and Asn are most stabilizing here. (small side chainsthat can act as acceptors)Asp better than Asn, possibly becauseof “helix-dipole” effects.Gly is also OK at N-cap. Why?

solvent-exposedamide hydrogens

serineside chain

amino acid Gu (kcal/mol)

Asp 2.02

Thr 2.05

Ser 1.64

Asn 0.86

Gly 0.69

Gln 0.42

Glu 0.25

His 0.16

Ala 0.00

Val -0.15

Pro -0.87

The numbers represent the average of two positions in the protein. TheN-cap is defined as the first residue the carbonyl of which makesan i,i+4 hydrogen bond to an amide. These are relative free energies ofunfolding, 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 conformationpreferences: glycines at alpha-helix C-termini

About one-third of all helicesterminate in glycine!

these carbonylshydrogen bondto solvent

“Schellmanmotif”

Many helices terminate this way,and glycine is favored at the left-handedposition because of its backbone flexibility and because large side chainshere 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 Gu (kcal/mol)

Gly 2.23

His 0.67

Asn 0.47

Arg 0.47

Lys 0.01

Ala 0.00

Ser -0.16

Asp -0.27

Relative stability of mutants at C-terminal endsof helices in barnase

Gly--> Ala mutationshave ranged from +1 to +3kcal/mol in a numberof proteins. The 2.2 kcal/molnumber observed here istypical

from Fersht“Structure and Mechanism..in Protein Science”, Ch. 17,p. 526

Intrinsic secondary structure propensities and stabilityamino acid Gu (kcal/mol), alpha-helix Gu (kcal/mol), beta-sheet

Ala 0.00 0.00

Arg -0.17 0.40

Leu -0.17 0.45

Met -0.19 0.90

Lys -0.31 0.35

Trp -0.31 1.04

Gln -0.33 0.38

Ser -0.44 0.87

Ile -0.43 1.25

Phe -0.47 1.08

Cys -0.54 0.78

Glu -0.56 0.28

Tyr -0.56 1.63

Asn -0.61 0.52

Thr -0.61 1.36

Val -0.63 0.94

His -0.65-0.88 (0 or + charge) 0.37

Asp -0.68 -0.85

Gly -0.90 -1.21

Pro -3.47 > -5

based on effects of surface mutations in helices in avariety 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 bookChapter 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 Tm Gf, activity , °C kcal mol-1 (relative)

wild-type 0 0 1E11F 4.3 1.7 <0.0001E11M 4.1 1.6 <0.0001E11A 2.6 1.1 <0.0001E11H 0.1 0.1 <0.0001E11N -0.6 -0.1 <0.0001D20N 3.1 1.3 <0.0001D20T 2.2 0.9 <0.0001D20S 1.6 0.7 <0.0001D20A -0.8 -0.3 0.0005

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 hydrophobicitytend not to be stable