IBS 601 - Section on Protein Folding

Warning: This is a informal summary of some of the material covered in class. These notes
are NOT a substitute for attending class or the reading of the review paper listed at the
bottom.

Protein Stability

Proteins are usually only marginally stable

• Protein stability is intimately connected with protein folding - proteins have to be folded
into their final active state (and maintain it) to be stable.
• D -> F or U -> F (Denatured or Unfolded state to folded state; D is not the same as U,
and there can be a variety of different D or U states). D can often be different states
depending on the denaturing condition; Dphys is often a denatured physiological state of a
protein (usually a molten globule state, with extensive residual secondary structure). U is
sometimes defined as the protein at a very high chemical denaturant concentration.
• Delta(G) = Delta(H) - T delta(S)
• Free energy of folding is the sum of enthalpic and entropic forces. These are large forces,
and the resulting delta(G) is often a small number (-5 to -15 kcal/mol is typical for many
proteins).
• This delta(G) is comparable to the energy of a few hydrogen bonds, even though a
protein may have hundreds of H bonds. Proteins are thus only marginally stable.
• The favorable enthalpic gains (H bonds, salt bridges, electrostatics, etc.) gains during the
folding of a protein are offset by the substantial loss of entropy as the protein goes from
an extended chain into a compact structure.
• This loss of entropy, called Delta (S conf) or conformational entropy is quite large (of the
order of RlnN, where N is the number of states; approx 1.67 kcal/mol/residue at 25
degrees C)
• There is also gain of entropy on folding from the hydrophobic effect (from the burial of
hydrophobic groups)
• For a typical 100 aa protein being folded:
○ Loss of conformational entropy: +167 kcal/mol
○ Hydrophobic effect: -95 kcal/mol
○ H-bonding and other enthalpic effects: -83 kcal/mol
○ Total delta G of folding: -11 kcal/mol
Forces driving protein folding (and hence stability)
• Hydrophobic effect
○ Major effect in protein stability - "driving force"
 ○ Non-polar side chains (leucine, isoleucine, phenylalanine, tryptophan (max
hydorphobic effect) ....)
○ Water cannot form H bonds with these side chains; thus water molecules form
ice-like "cages" around these side chains, resulting in a loss of entropy if these
side chains are solvent exposed.
○ Clustering of these side chains reduces this entropic loss (fewer water molecules
in a ice-like state). These side chains thus prefer to cluster or get buried into the
interior of the protein in "hydrophobic cores"
○ This can be energetically quite favorable; the burial of a single -CH2 group has a
delta G of about -1 kcal/mol (close to energy of a H bond).

• H-bonds
○ Within the protein backbone, these are usually between N-H and O=C (amide
hydrogen and the carbonyl oxygen; ~70% of H-bonds in a globular
protein)
○ Delta G of -1 to -5 kcal/mol
Conditions that can denature proteins (optional reading)
• Thermal denaturation
○ As temperature increases, entropy of unfolding becomes higher and overcomes
enthalphic forces holding the protein together. There is also breakdown of the "ice
cages" around non-polar side chains, weakening the hydrophobic effect and
favoring the unfolded state.
○ Since proteins are only marginally stable, even a little temperature increase can
destabilize proteins. This is the reason that point mutations can often yield
"temperature sensitive" proteins that denature with just a few degree increase in
temperature.
○ Proteins in thermophilic organisms are often seen to include more H-bonds and
salt bridges (more delta H), as well as greater hydrophobic interactions.

• Cold Denaturation
○ Some proteins denature in cold temperatures as well
○ At lower temperature, the hydrophobic effect weakens - bulk water becomes more
ordered and the hydration of non-polar groups (ice cage formation) is not as
unfavorable as under more reasonable temperatures.
○ Delta H also decreases at lower temperatures.
○ These changes favor the unfolded state for some proteins.
○ Cold adapted proteins often show fewer hydrophobic forces (reduced aromatic
interactions; fewer salt-briges; lower number of prolines & arginines for more
flexibility; ....).
 ○ There is a trade-off between stability and flexibility in most proteins. Most
enzymes require some flexibility (for activity), while also needing structural
rigidity (for stability).
○ Proteins are stable and active in only a narrow range of temperatures; thus, even
though life can exist between -50 and 110 degrees C, most species are active only
in a much narrower band of temperatures (usually in tens of degrees C).

• Chemical denaturants
○ e.g. Urea, guanidinium chloride.
○ chemical denaturants often act by increasing solubility of hydrophobic side chains
in water, thus decreasing the hydrophobic effect.
○ Urea can also form H-bonds with the protein backbone, and aromatic sidechains.
○ effect is usually linearly propotional to the concentration of the denaturant.

• High or low pH
○ Proteins have buried groups with highly perturbed pKa's.
○ High or low pH affects protonation levels, and electrostatic charges between
groups.
○ Often destabilization of protein is because of increased electrostatic repulsion.

• High pressure

Protein Folding
Why do proteins fold
• The folded state is more stable (lower delta G) than the unfolded state.
• While delta G of folding is negative, most proteins are only marginally stable - (delta G
of only a few kcal/mol).
○ One reason for the marginal stability is that both the folded and unfolded states
form a large number of H-bonds & other non-ionic interactions; gain of H-bonds
as protein folds is often offset by the loss of H-bonds between the protein side-
chains and the solvent.
○ Another reason is that delta G = delta H - T delta S (enthalpy - entropy terms). So
gain in enthalpic contributions is offset by loss of entropy as the protein folds.
How do proteins fold (in-vitro)
• Protein folding is spontaneous. [Anfinsen's principle (1973) - all information necessary to
specify the native 3D fold of a protein is contained in its amino acid sequence].
• Reversible for small proteins, under ideal conditions.
 • Protein folding is non-random & highly cooperative [Levinthal's paradox (1968) -
proteins fold in time scale of milliseconds-seconds, even though a systematic search of all
possible conformations in even a small protein (10^100 conformations for a 100 aa
protein) will take a very-very long time].
• There is no single model or specific pathway for folding - protein folding is better
described in terms of multi-dimensional energy landscapes or folding funnels, with
different pathways possible depending on the sequence, conditions, etc.
• Folding can generally be described by a two state transition model for small (<100 aa)
proteins: U -> F.
• For small proteins, a general pattern of rapid nucleation and hydrophobic collapse is seen
(formation of a molten globule state), followed by a slower compaction into the native
state [referred to also as a "nucleation-condensation" reaction].
○ The unfolded state (U) is generally "expanded & loose" with most of the local
secondary structural features (helices, beta sheets, loops, etc.) in place, but with
only a few long-range interactions.
○ The folded state (F) is more "compact and ordered", and has a large number of
long range interactions between different parts of the protein chain, apart from the
local secondary structure.

• For larger proteins, there are often multiple structural domains which each fold by
mechanisms similar to that for smaller proteins. Once these fold, the different domains
reshuffle slightly to form the final native structure.
How do proteins fold in-vivo ?
• While most of the principles described above also apply in-vivo, protein folding within
cells in complicated by two major factors:
○ Proteins are synthesized in a sequential manner at a rate much slower (4-20
aa/second) than typical folding rates (< 1 second). so the n terminal of the protein
may start to fold before the c terminal end has even been synthesized by the
ribosome.
○ Cells are very crowded with all kinds of macromolecules (typical densities in E.
coli are about 340 mg/ml of protein), leading to increased possibilities for the
nascent polypeptide chain to interact with other molecules and hydrophobic
surfaces. This can lead to aggregation or misfolding.

• Folding of over half of the proteins in cells is assisted by molecular chaperones.

○ "Molecular chaperones are proteins whose role is to mediate the folding of
certain other polypeptides and, in some instances, their assembly into oligomeric
structures, but which are not components of these final structures" (definition
from RJ Ellis, 2000).
○ Chaperone assist or mediate folding and assembly (non-covalent) of proteins, and
also inhibit "off-pathway" folds. They increase the efficiency of protein folding
within the cell.
 ○ Chaperone assisted protein folding appears to be a universal mechanism in all
cells that "enables the crowded state of the cellular interior to be compatible with
life" (RJ Ellis, 2000).
○ Over 20 families of chaperone molecules have been identified.
○ While most chaperones are proteins, there are hints that ribosomal RNA and some
phospholipids may also play a chaperone type function.
○ Chaperones are often stress or heat shock factors
○ Another class of chaperones in the "steric chaperones" - where the chaperones
provide essential steric information during folding. An example of this is proteins
with a "prosequences" - an N-terminal part that is required for correct folding, but
is subsequently cleaved off to get the functional protein (e.g. prosubtilisin).
○ The best understood families of chaperones are the HSP70 and HSP60 family of
chaperones [HSP stands for Heat Shock Protein]. The HSP70 family proteins bind
short exposed hydrophobic stretches on unfolded proteins, and assist in folding by
preventing aggregation. The HSP60 family (also called the chaperonins), form a
"folding cage" with a large central cavity which provides a protective
environment for other proteins to fold.
○ See Figure 5 & description in the Radford review paper for more details.

• Apart from chaperones, other enzymes are also involved in proper folding of some
proteins within the cell. For example, protein disulfide isomerases (PDI) are involved in
the proper formation of some disulfide bonds. Another set of such enzymes are the
peptidyl prolyl cis-trans isomerases (PPI).
○ PDI's - Protein disulfide isomerases catalyse formation of disulfide bonds (-S-S-)
 disulfide bonds act like "staples" in a protein structure
 These can often form in a complex pathway - e.g. BPTI (Bovine
pancreatic trypsin inhibitor)
 kinetics of disulfide bond formation are largely independent and much
slower than conformational folding kinetics
 disulfide bond formation usually requires an oxidative environment
(periplasm in bacteria, ER in eukaryotes), while the cytoplasm is usually a
reducing environment

○ PPI's - Peptidyl prolyl cis-trans isomerases

 These are ubiquitious enzymes - isomerases or rotamases that catalyse the
cis-trans isomerization without breaking bonds
 In proteins most peptidyl bonds are trans (omega = torsion along the C-N
bond = 180) and this conformation is heavily favored in both denatured or
folded forms. However, in extended chains, the peptide bond preceding a
proline can be either in trans or cis forms, with the trans form only slightly
 more favored than the cis form. In folded proteins, on the other hand, only
about 7&percnt; of all prolyl-peptide bonds are cis
 Isomerization about the prolyl peptide bond from the cis to trans
conformation is very slow - t1/2 of 10-100 seconds with activation energy
of almost 20 kcal/mol. PPIs catalyse this to a timescale of a second or less.
This isomerization is often the rate limiting step in protein folding.
 Three unrelated families of PPI's - cyclophilins, FK506 binding proteins
(FKBP), and Parvulins. The Trigger Factor in bacteria is a 48 kDa
ribosome associated PPIase with a FKBP type catalytic domain

• The endoplasmic reticulum (ER) is an important site for protein folding in eukaryotes.
About 1/3 of all proteins in eukaryotes fold within the ER, especially all secretory and
membrane proteins. ER is especially rich in chaperones, such as BIP which belongs to the
Hsp70 family.
• Protein folding quality control - unfolded proteins cause a response called UPR (unfolded
protein response). This uses three mechanisms to enhance overall level of protein folding:
○ Attenuation of translation levels in the cell
○ Induction or transcriptional upregulation of chaperones
○ degradation of malfolded proteins - degradasome

• Suggested reading:
○ Mori, K. (2000) Tripartite management of unfolded proteins in the endoplasmic
reticulum. Cell, 101, 451-454.
○ Ellis, R. J. (2000) Introduction. Seminars in Cell & Developmental Biology, 11, 1-
5.
Amyloid Formation
• Many proteins do not have a single low energy folded state, and can adopt very different
conformations. For example, serpins (a family of protein protease inhibitors) undergo a
large structural transformation on binding their target protease.
• Amyloid formation is a consequence of this "structural plasticity" seen in many proteins.
• Certain protein folds are "metastable" (these often include beta sheets) and can associate
with each other in a "catalytic" manner to form amyloid fibrils (the prion hypothesis).
• There is usually a dynamic equilibrium between the "native" and the "metastable" states
of amyloidogenic proteins. This equilibrium can shift towards the metastable amyloid
state due to mutations, protein damage or unfolding, as well as changes in the cellular
environment or protein levels.
• Suggested reading:
○ Dobson, C. M. (2003) Protein folding and misfolding. Nature, 426, 884-890.
Abstract
How can we measure or observe proteins folding
 • While the underlying molecular events in protein folding occur in the nanosecond or
faster time scale, global folding of typical proteins occurs in the millisecond - second
time scale.
• Protein folding/unfolding is usually initiated in experiments by a temperature jump
(thermal denaturation) or changes in the chemical environment [pH jump, chemical
denaturation or unfolding by addition of urea or guanidinium chloride (GdmCl)].
• Spectroscopic techniques are often used to follow protein folding. Measurement of
changes in intrinsic fluorescence (typically of tryptophan residues in the protein) or
circular dichroism (CD) are commonly used. These techniques can measure changes in
the millisecond time scale using stopped-flow type instruments.
○ Fluorescence measurements monitor the state of aromatic sidechains within the
protein (often tryptophans, which when excited at 280 nm, emit energy at around
320 nm). As a protein unfolds and the aromatic sidechains are exposed, the
fluorescence signal goes up.
○ Circular Dichroism (CD) measures the unequal absorption of left and right
handed circularly polarized light by optically active protein molecules. Folded
proteins have a characteristic CD spectra which changes as the protein unfolds.

• NMR can be used follow changes in the environment of individual side-chains in the
millisecond to second time scale.
• See Table I in the Radford review paper for a list of many other techniques.
Protein Dynamics and Simulation (Optional Reading)
• Apart from proper folding and rigidity for stability, proteins need flexibility for their
function
• Protein dynamics is important to understand motions in proteins, and the pathway of
protein folding
• X-ray crystallographic structures include a column with the B or thermal factor, apart
from the X,Y,Z coordinates of each atom. While these are often just measures of disorder
in the crystal, they can provide hints about regions of a protein that may be flexible
• NMR based relaxation methods are an excellent approach to probe dynamics in proteins
• Simulations of molecular dynamics (MD) are often necessary to understand the fast steps
in protein folding (especially for following transitions faster than the resolution of
experimental techniques)
Coupling between protein folding and binding (Optional Reading)
Based on Dyson and Wright (2002) Curr. opinions in Structural Biology, 12, 54-60
• A significant fraction of protein in cells may be unstructured, especially in eukaryotes
○ Drosophilla: about 17&percnt; of proteins predicted to be wholly denatured
○ Four eukaryotic genomes: about 30&percnt; of proteins expected to have
disordered segments of 50 aa or more
 • These "unstructured" proteins may fold only on finding their binding targets. Some
example may include:
○ ribosomal proteins
○ transcriptional factors
○ proteins involved in complexes
○ inducible "snap-lock" folding activated by DNA binding

• "folding process for any protein can be thought of as a 'binding reaction' that involves
binding of distant parts of the polypeptide chain, as tertiary structure is formed. However
in some cases, there is a requirement for external factors as well ...."
Why is any of this important?
• Correct folding is required for proteins to function. Aberrant folding of proteins is
involved in many diseases. For example:
1. Cystic fibrosis - A deletion mutation (Phe 508) in a chloride channel protein
(CFTR) leads to improper folding & reduced Cl- conductance in diseased cells
2. Amyloid diseases (such as Alzheimer's, prion diseases, mad-cow, etc.) involve
protein misfolding or defective processing, leading to aggregation & formation of
insoluble plaques.

• Understanding the mechanism of protein folding will allow better ab-initio prediction of
protein 3D structures from their amino acid sequences.
Required Reading
Radford, S. E. (2000) Protein folding: progress made and promises ahead. Trends in Biochemical
Sciences (TIBS), 25, 611-618. Abstract
Optional Reading
Baker, D. (2000) A surprising simplicity to protein folding. Nature, 405, 39-42. Abstract
Dobson, C. M. (2003) Protein folding and misfolding. Nature, 426, 884-890. Abstract
Fersht, A. R. & Daggett, V. (2002) Protein folding and unfolding at atomic resolution. Cell, 108,
573-582. Abstract
Daggett, V. & Fersht, A. R. (2003) Is there a unifying mechanism for protein folding? Trends in
Biochemical Sciences (TIBS), 28, 18-25. Abstract
Mayor U., Guydosh N. R., Johnson C. M., Grossmann J. G., Sato S., Jas G. S., Freund S. M.,
Alonso D. O., Daggett V. & Fersht A. R. (2003) The compete folding pathway of a protein from
nanoseconds to microseconds. Nature, 421, 863-867. Abstract

Co-chaperones are nonclient-binding partners of Hsp90 or Hsp70 and may be

loosely defined as proteins that participate in the function of other chaperones.
Co-chaperones are proteins that assist chaperones in protein folding and
other functions.

[edit]List of Co-chaperones

 GroES
 Aha1  Hch1
 auxilin  Hip (Hsc70-interacting
 BAG1 protein)/ST13

 CAIR-1/Bag-3  Hop (Hsp70/Hsp90 organizing

protein)/STIP1
 CDC37/p50
 Mrj
 Chp1
 PP5
 Cysteine string
protein (CSP)  SGT

 Cyp40  Snl1

 Djp1  SODD/Bag-4

 DnaJ  Swa2/Aux1

 E3/E4-ubiquitin ligase  Tom34

 FKBP52  Tom70

 GAK  UNC-45

 WISp39
[edit]See also
 Chaperone (protein)

 Heat shock protein

[edit]Source

 Young JC, Barral JM, Hartl FU (October 2003). "More than folding: localized
functions of cytosolic chaperones". Trends Biochem. Sci. 28 (10): 541–
7. doi:10.1016/j.tibs.2003.08.009. PMID 14559183.
Pharmacological chaperone
From Wikipedia, the free encyclopedia

A pharmacological chaperone is a relatively new concept in the treatment of

certain genetic disease. Small molecules which stabilize the correct folding of a
protein are administered to the patient which results in a recovery of function
lost due to mutation. Pharmacological chaperones present an attractive
alternative to transplantation due to its low risk and an alternative to enzyme
replacement therapy due to lowered expense and oral availability. The
development of these drugs has become possible as a result of our improved
understanding of cell biology.

Targets for the pharmacological chaperoning must have a mutation which

renders the protein unstable but not inactive. Pharmacological chaperones are
particularly effective if a small amount of the protein can make a big difference
in terms of palliating the negative effects of the mutation.

All pharmacological chaperones so far have targeted proteins along the

secretory pathway; this is because the function of the mutated enzymes is
sequestered from the location of its synthesis. The following sequence describes
the general technique for this set of proteins:

The mutated protein is unstable (in the thermodynamic sense) and

in retrotranslocation to the cytoplasmis degraded by the proteasome. The
pharmacological chaperone intercepts the mutated protein and stabilizes its
fold, evading the retrotranslocation machinery. The protein can then be shuttled
to its target destination (usually beyond the ER), where it performs its
appropriate function.

Ironically, most pharmacological chaperones are inhibitors of the enzyme

targeted; due to differing conditions in the target's destination (pH, metal ions,
etc) the inhibitor is ejected, and the enzyme functions; or, the relatively high
concentration of substrate outcompetes the inhibitor; or a combination of both
effects enables the enzyme to function. Even if the chaperone continued to
inhibit, for most of these diseases an inhibited amount of enzyme would be
much better than quantitative degradation.
Hemchandra Jha
 who folds the molecular chaperons????

who folds the molecular chaperons????

ok....lemme keep it as simple as i can.........
if molecular chaperons or chaperonins help in folding other proteins.....who folds the
chaperons themselves???

plz plz tell me the ans any1!!

Ans 1
that is found that chaperons contains some motif...i.e. required for prtein folding...
and there are autofolding occurs when chaperons are translated...
in some disease the target site is in folding of chaperons......
in these cases chaperons are misfolded......

Ans 2:

chaperon families are also a kind of protein so obviously it is possible to regulate the
proper folding of one chaperon sp by other chaperon sps.again some proteins do not
need the help of chaperons,may chaperon be itself of that kind.folding of protein in
vivo is a dynamic process which can be effected by both thermodynamically and
chaperons's assistance,thus without chaperons protein folding is possible.

thanks.

Apr 5

Santosh Kumar

Hi Hem, nice to see ur mail.

As you might also appreciate, many proteins in the cell fold to correct conformation, in
a mechanism independent of chaperones. So chaperones also fall into this category. So
chaperone requirement is not universal. But since some essential porteins require
chaperones to fold to correct confirmation, some of the chaperones, like GroEL, are
essential.

But in some organism, like urecoplasma urecotellum, complete set of chaperone

proteins are absent. And it is predicted that all the proteins are translated a bit slowly
and are having auto-folding propensities to overcome this deficiency.

So chaperones, like many different chaperone-independent proteins, fold by

themselves.

Regards,
Santosh.