Transcript Document

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)