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Encyclopedia of Physical Science and Technology EN013H-614 July 27, 2001 10:29

182 Protein Folding

the aqueous solvent. Solvation of nonpolar side chains
by aqueous solutions causes a decrease in the entropy
of solution. To avoid this entropic penalty, proteins typ-
ically bury their nonpolar residues in the interior of a
protein. 4

III. FOLDING PATHWAYS

The intramolecular interactions discussed above stabilize
the final folded structure of a protein. However, knowl-
edge of the end states, N and U, tells us nothing of the
path taken between them. Proteins fold on the time scale
FIGURE 2 Illustration of cooperative vs. noncooperative unfold-
of microseconds to hundreds of seconds. It is impossible
ing transitions. If the native state of a protein (N) is denatured into
the unfolded state (U) in a single transition (pathway 1), then it is a to sample all possible conformations during this time and
two-state or cooperative unfolding transition. Alternatively, the na- it is clear that there is a preferred order of events leading
tive state may be converted into one or more intermediate states to the final tertiary fold. Determining this order of events
(pathway 2). For example, if a protein is comprised of multiple do- is an area of active inquiry. The questions that experimen-
mains, one of the domains may be unfolded first. It is also possible
to form a completely different intermediate before unfolding com- talists are attempting to answer are “Do autonomously
pletely. The presence of intermediate species may be observed folding substructures nucleate the folding of other regions
using kinetic or equilibrium techniques. However, intermediates of the protein?” or “Do neighboring substructures fold and
detectable by kinetic methods may or may not be observable by then collide to make the tertiary structure?” There is ex-
equilibrium methods.
perimental evidence that hydrophobic amino acid residues
collapse into a “hydrophobic core” and then the secondary
structural units form around the core. It is likely that a
G o un = G H-bond + G ionic + G vdW combination of these scenarios leads to a correctly folded

+ G S −S + G H phob (4) protein.
It is clear that the kinetics of protein folding is protein
Each term in Eq. (4) will be discussed separately. As dependent. Some fold in a distinctly cooperative fashion,
mentioned earlier, an important stabilizing factor for the such that one can detect only the unfolded and native end
tertiary fold of a protein is its intramolecular hydrogen states (U ↔ N), being two-state in a kinetic as well as
bonds ( G H-bond ). Secondary structures are stabilized by equilibrium sense. This is equivalent to saying that there
hydrogen bonds between backbone amide atoms (Fig. 1). is a single rate-limiting step, and intermediate species are
The side chains of neighboring secondary structural units not populated. Alternatively, some proteins fold by pop-
can interact through hydrogen bonding. Ionic interactions ulating one or more distinct intermediate species (e.g.,
( G ionic ) between acidic and basic side chains may stabi- U ↔ I ↔ N; see Fig. 2). Thus, formation of the interme-
lize the tertiary structure of proteins and are pH dependent. diate species is fast, often formed in the dead-time of the
The actual pKa of an ionizable side chain is influenced by instrument, and formation of the native species from the
the microenvironment in which it resides. Nonpolar and intermediate is relatively slow and easily monitored ex-
polar, but uncharged, amino acids interact through van der perimentally. It has been shown that this slow phase in
Waals interactions ( G vdW ). In some proteins, cysteine some cases may be due to proline isomerization. 5
residues (side chain is a sulfhydryl) form disulfide link-
ages that can increase the overall stability of the protein
( G S–S ). Other possible factors not considered explic- IV. EMPIRICAL APPROACHES
itly here are the effects of metals, nucleotides, prosthetic
groups, and cofactors on protein structure and stability.
A. General Experimental Strategies
By far the most important noncovalent factor that
determines protein stability is hydrophobic interactions As discussed above, experimental studies of protein fold-
( G H phob ). In globular proteins, hydrophobic amino ing reactions fall into the category of either equilibrium or

acids are buried in the interior where they create a “hy- kinetics studies, with the former yielding thermodynamic
drophobic core.” Although these nonpolar residues par- information about the energy differences between the na-
ticipate in van der Waals interactions, the primary driving tive and denatured structural states and the latter stud-
force for the formation of the hydrophobic core is to avoid ies providing information about the folding pathway and