288 Chapter 8. Chemical forces and self-assembly[[Student version, January 17, 2003]]
contributions to the force driving protein folding using ideas from this chapter and Chapter 7.
Relatively small disturbances in the protein’s environment, for example change of temperature,
solvent, or pH, can disrupt the native conformation,denaturingthe protein. H. Wu proposed
in 1929 that denaturation was in fact precisely the unfolding of the protein from “the regular
arrangement of a rigid structure to the irregular, diffuse arrangement of the flexible open chain.” In
this view unfolding changes the protein’s structure dramatically, and destroys its function, without
necesarily breaking any chemical bonds. Indeed, restoring physiological conditions returns the
balance of driving forces to one favoring folding; for example, M. Anson and A. Mirsky showed that
denatured hemoglobin returns to a state physically and functionally identical to its original form
when refolded in this way. That is, the folding of a (simple) protein is aspontaneousprocess, driven
bythe resulting decrease in the free energy of the protein and the surrounding water. Experiments
of this sort culminated in the work of C. Anfinsen and coauthors, who showed around 1960 that,
for many proteins,
- The sequence of a protein completely determines its folded structure, and
- The native conformation is the minimum of the free energy.
The thermodynamic stability of folded proteins under physiological conditions stands in sharp
contrast to the random-walk behavior studied in Chapter 4. The discussion there pointed out
the immense number of conformations a random chain can assume; protein folding thus carries
acorrespondingly large entropic penalty. Besides freezing the protein’s backbone into a specific
conformation, folding also tends to immobilize each amino acid’s side chain, with a further cost in
entropy. Apparently some even larger free energy gain overcomes these entropic penalties, driving
protein folding. At body temperature, the balance between forces driving folding and unfolding
rarly exceeds 20kBTr,the free energy of just a few H-bonds.
What forces drive folding? Section 7.5.1 on page 240 already mentioned the role of hydrogen
bonds in stabilizing macromolecules. W. Kauzmann argued in the 1950s that hydrophobic inter-
actions also give a major part of the force driving protein folding. Each of the twenty different
amino acids can be assigned a characteristic value of hydrophobicity, for example by studying how
it partitions into water. Kauzmann argued that a polypeptide chain would spontaneously fold to
bury its most hydrophobic residues in its interior, away from the surrounding water, similarly to the
formation of a micelle. Indeed, structural data, not available at the time, has borne out this view:
The more hydrophobic residues of proteins tend to be located in the interior of the native confor-
mation. (The exceptions to this rule turn out to be important for helping proteins stick to each
other; see below.) In addition, the study of analogous proteins from different animal species shows
that, while they may differ widely in their precise amino-acid sequences, still the hydrophobicities
of the core residues hardly differ at all—they are “conserved” under molecular evolution. Similarly,
one can create artificial proteins by substituting specific residues in the sequence of some natural
protein. Such “site-directed mutagenesis” experiments show that the resulting protein structure
changes most when the substituted residue has very different hydrophobicity from the original.
Kauzmann also noted a remarkable thermal feature of protein denaturation. Not only can high
temperature (typicallyT> 55 ◦C)unfold a protein, but in many caseslowtemperature does too
(typicallyT< 20 ◦C). Denaturation by heat fits with an intuitive analogy to melting a solid, but
cold denaturation was initially a surprise. Kauzmann pointed out that hydrophobic interactions
weaken at lower temperatures (see Section 7.5.3 on page 246), so that the phenomenon of cold
denaturation points to the role of such interactions in stabilizing protein structure. Kauzmann