Desolvation is a Likely Origin of Robust Enthalpic
Barriers to Protein Folding
Zhirong Liu and Hue Sun Chan
*
Protein Engineering Network of
Centres of Excellence
Department of Biochemistry
and Department of Medical
Genetics and Microbiology
Faculty of Medicine, University
of Toronto, Toronto, Ont.
Canada M5S 1A8
Experimental data from global analyses of temperature (T) and denaturant
dependence of the folding rates of small proteins led to an intrinsic
enthalpic folding barrier hypothesis: to a good approximation, the
T-dependence of folding rate under constant native stability conditions is
Arrhenius. Furthermore, for a given protein, the slope of isostability
folding rate versus 1/T is essentially independent of native stability. This
hypothesis implies a simple relationship between chevron and Eyring plots
of folding that is easily discernible when both sets of rates are expressed as
functions of native stability. Using experimental data in the literature, we
verify the predicted chevron–Eyring relationship for 14 proteins and
determine their intrinsic enthalpic folding barriers, which vary approxi-
mately from 15 kcal/mol to 40 kcal/mol for different proteins. These
enthalpic barriers do not appear to correlate with folding rates, but they
exhibit correlation with equilibrium unfolding enthalpy at room tempera-
ture. Intrinsic enthalpic barriers with similarly high magnitudes apply as
well to at least two cases of peptide–peptide and peptide–protein
association, suggesting that these barriers are a hallmark of certain general
and fundamental kinetic processes during folding and binding. Using a
class of explicit-chain C
a
protein models with constant elementary
enthalpic desolvation barriers between C
a
positions, we show that small
microscopic pairwise desolvation barriers, which are a direct consequence
of the particulate nature of water, can act cooperatively to give rise to a
significant overall enthalpic barrier to folding. This theoretical finding
provides a physical rationalization for the high intrinsic enthalpic barriers
in protein folding energetics. Ramifications of entropy–enthalpy compen-
sation in hydrophobic association for the height of enthalpic desolvation
barrier are discussed.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: activation enthalpy; activation entropy; heat capacity; chevron
plot; Arrhenius
*Corresponding author
Introduction
Deciphering the physico-chemical forces that
drive proteins to fold is one of the central
unresolved questions in molecular biology. As a
challenge to the human intellect, this problem is
difficult because many degrees of freedom, of the
protein as well as the surrounding solvent mol-
ecules, need to be taken into account, but a precise
description of the interactions involved is currently
lacking. Mathematically, it is natural to formulate
the folding process in terms of a high-dimensional
energy landscape that provides the free energy for
every protein conformation (with the energetics
associated with the solvent degrees of freedom pre-
averaged). It is clear from general polymer physics
principles that the conformational search for the
native structure, while stochastic to a certain extent,
has to be partially directed by incremental energetic
favorabilities
1–8
so as to allow the protein chain to
circumvent the Levinthal paradox.
9–11
The energy
landscape of protein folding is necessarily “funnel”-
like in this particular respect.
In contrast to this funnel perspective, “barriers”
or empirically defined transition states along free
energy profiles (often functions of a single reaction
0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
Abbreviations used: FPT, first passage time; PDB,
Protein Data Bank.
E-mail address of the corresponding author:
chan@arrhenius.med.toronto.edu
doi:10.1016/j.jmb.2005.03.084 J. Mol. Biol. (2005) 349, 872–889