Energy landscape in protein folding and unfolding

Francesco Mallamacea,b,c,1, Carmelo Corsaroa,d, Domenico Mallamacee, Sebastiano Vasid, Cirino Vasia, Piero Baglionif,
Sergey V. Buldyrevg, Sow-Hsin Chenb, and H. Eugene Stanleyc,1
a
CNR-Istituto per i Processi Chimico Fisici Messina, I-98166 Messina, Italy; bDepartment of Nuclear Science and Engineering, Massachusetts Institute of
Technology, Cambridge, MA 02139; cCenter for Polymer Studies and Department of Physics, Boston University, Boston, MA 02215; dDipartimento di
Fisica e di Scienze della Terra, Università di Messina, I-98166 Messina, Italy; eConsorzio per lo Sviluppo dei Sistemi a Grande Interfase, Unità di Catania,
I-95125 Catania, Italy; fDipartimento di Chimica, Università di Firenze and Consorzio per lo Sviluppo dei Sistemi a Grande Interfase, I-50019 Florence,
Italy; and gDepartment of Physics, Yeshiva University, New York, NY 10033

Contributed by H. Eugene Stanley, December 22, 2015 (sent for review March 17, 2015; reviewed by Anders Nilsson and Michele Parrinello)

We use 1H NMR to probe the energy landscape in the protein fold- simulation (9, 10) and different experimental techniques (11–16).
ing and unfolding process. Using the scheme ⇄ reversible unfolded Whereas MD simulations directly model peptide conformational
(intermediate) → irreversible unfolded (denatured) state, we study transitions in terms of the energy landscape, experiments supply
the thermal denaturation of hydrated lysozyme that occurs when useful but limited information, revealing some details in the
the temperature is increased. Using thermal cycles in the range structure and the collective dynamics of protein, both dry and in
295 < T < 365 K and following different trajectories along the protein solution. Examples of this include the spectroscopic techniques
energy surface, we observe that the hydrophilic (the amide NH) and (e.g., NMR, neutron, X-ray, Raman, and FTIR) that supply data
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hydrophobic (methyl CH3 and methine CH) peptide groups evolve on structure and dynamical modes of the protein, and the
and exhibit different behaviors. We also discuss the role of water calorimetric measurements that follow the reversible folding–
and hydrogen bonding in the protein configurational stability. unfolding as far as irreversible denaturation (9, 10). The protein
dynamics have been studied from the glass state in the deep

APPLIED PHYSICAL
protein folding | proton NMR | energy landscape | hydration water supercooled regime (T < 200 K) to the completely denatured

SCIENCES
state (T ’ 350 K) by proving the essential role of the hydration

A n intriguing problem of statistical physics concerns the

evolutionary pathways that molecular systems follow as they
form mesoscale structures and exhibit new functional behaviors
water. As for bulk water, hydrogen bond (HB) interactions
strongly determine the properties of these systems, for which
water is not simply a solvent but is also an integral and active
(1). An example of this problem is the self-organization of bio- component, i.e., it is itself an important “biomolecule” that plays
systems that evolve from basic molecules. This challenging sub- both a dynamic and structural role (17). Hence, HB interactions

COMPUTATIONAL BIOLOGY
ject is studied by using a variety of theoretical methods (2–4). are the key to understanding water’s properties and how water

BIOPHYSICS AND
The free-energy landscape model is nowadays the most used to functions in biological environments (18).
describe such phenomena and especially the aging of the protein Among the different experimental methods, calorimetry, by
folding mechanism (1, 5, 6), i.e., the way in which proteins fold to monitoring the process reaction rates, focuses directly the energetic
their native state and then unfold (protein denaturation) (6, 7). properties of the hydrated proteins folding–unfolding mechanism
The model is based on the idea that in complex materials and and measures the enthalpy and entropy behaviors. Unlike common
systems there are many thermodynamical configurations in which chemical reaction rates, which increase as the temperature is in-
the free-energy surface exhibits a number of local minima sepa- creased, the rate constant of the protein folding reaction initially
rated by barriers, i.e., as the system explores its phase space the increases on increasing T, by following an Arrhenius law goes to a
trajectory of its evolution is an alternating sequence of local maximum, and then decreases as the temperature continues to
energy minima and saddle points (transition states), which are increase (19). This latter situation is also well-described in MD
associated with the positions of all of the system particles. A simulations (20). The activation enthalpy is thus T-dependent and
trajectory thus specifies the path of the system as it evolves by the corresponding protein specific heat, CP ðTÞ, exhibits large
moving across its energy landscape.
A peptide is a linear chain of amino acids, and globular pro- Significance
teins are polypeptide chains that fold into their native confor-
mation. During the folding process a polypeptide undergoes
many conformational changes and there is a significant decrease Protein folding represents an open question in science, and the
free-energy landscape framework is one way to describe it. In
in the system configurational entropy as the native state is
particular, the role played by water in the processes is of spe-
approached. To understand folding we focus on how proteins
cial interest. To clarify these issues we study, during folding–
search conformational space. The process is accompanied by
unfolding, the temperature evolution of the magnetization for
many microscopic reactions, the nature of which is determined
hydrophilic and hydrophobic groups of hydrated lysozyme
by the specifics of the energy surface. Thus, the characteristics of
using NMR spectroscopy. Our findings confirm the validity of
the energy surface of a polypeptide chain are the key to a quan-
the theoretical scenario of a process dominated by different
titative understanding of folding. Although the degrees of freedom
energetic routes, also explaining the water role in the protein
of a polypeptide chain allow an enormously large number of
configuration stability. We also highlight that the protein native
possible configurations, “constraints” on the energy decrease these
state limit is represented by the water singular temperature that
configurations visited in the folding reaction to a limited number
characterizes its compressibility and expansivity and is the origin
(8). Understanding the free-energy surface (“landscape”) enables
of the thermodynamical anomalies of its liquid state.
us to understand the folding process. A balance between the
potential energy and the configurational entropy leads to a free- Author contributions: F.M., C.C., P.B., S.V.B., and H.E.S. designed research; F.M., C.C., D.M.,
energy barrier that generates the two-state folding behavior usu- S.V., and C.V. performed research; F.M., D.M., S.V., P.B., S.V.B., and S.-H.C. analyzed data;
ally observed in small proteins. The potential energy decreases as and F.M., C.C., and H.E.S. wrote the paper.
the native state is approached and favors folding, but decreasing Reviewers: A.N., SLAC National Accelerator Laboratory; and M.P., ETH Zurich.
the entropy of the configuration is unfavorable to folding. The authors declare no conflict of interest.
The thermodynamics and kinetics of folding and unfolding 1
To whom correspondence may be addressed. Email: [email protected] or
have been intensively studied by molecular dynamics (MD) [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1524864113 PNAS | March 22, 2016 | vol. 113 | no. 12 | 3159–3163

 changes: first it increases with T, reaches a maximum, and then precisely, to quantify the behavior of the protein hydrophilic (the
decreases. Hence a CP ðTÞ plot against T exhibits an endothermic amide NH) and hydrophobic (methyl and methine) groups, during
peak whose area is related to the enthalpy of transformation of the folding–unfolding process, we use as control variable the
the protein. Such a behavior is also strongly influenced by the temperature, which slows the process kinetics to many hours.
protein hydrophobic side chains and hydration water (19, 21, 22). The goal is to determine the topography of the protein energy
NMR spectroscopy instead can be used to follow folding (or landscape by following different trajectories along the energy
unfolding) processes by probing interactions as they form at surface. This allows us also to consider explicitly the role of water
the level of individual residues, and to compare the findings and hydrogen bonding in protein configurational stability.
obtained with simulations (13, 23–25). NMR studies involve in-
vestigation of the equilibrium conversion between native and Results
denatured states resulting from a protein being subjected to heat, Thermal denaturation of lysozyme occurs according to the
extremes of pH, or chemical denaturants. The kinetics of the scheme, native state (N) ⇄ reversible unfolded (intermediate)
process may limit the observations. If the folding occurs on a state (RU) → irreversible unfolded (denatured) state (IU); N ⇄
time scale for which spectra can be measured and recorded se- RU → IU (21, 22). This is consistent with the general view that
quentially, the spectral changes accompanying the reaction can the first step in the denaturation of small one-domain globular
be monitored at the individual residue level and thus be accu- proteins, e.g., lysozyme, is a reversible conformational (unfolding)
rately studied. Recently, new methods have been developed to transition, and the second step is an irreversible denaturation.
probe very fast processes or by slowing down the protein folding Studies performed in the 290 < T < 370 K range have revealed that
kinetics by means of proper reactants. the observed CP ðTÞ peak is caused by heat absorption when the
One example of 1H NMR spectroscopy in which the protein
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equilibrium constant between the native lysozyme state and a

folding can be properly detailed is represented by a real-time study conformational different intermediate state increases with T (26,
of bovine α-lactalbumin (BLA) refolding, at 293 K, where spectra P ðTÞ peak temperature is TD = 347 K and seems to
27). The Cmax
show significant resonance changes from methyl and methine represent the reversibility limit. Lysozyme at a hydration level of
groups of aromatic residues (25). In this case the experimental h = 0.3 has a water monolayer covering its surface (27). As de-
strategy was in the control of the Ca2+ concentration for which the scribed in Methods, we have conducted the experiments using dif-
folding kinetics can vary over several orders of magnitude. The ferent heating/cooling cycles exploring completely or partially the
BLA refolding was initiated by a pH jump (in the absence of Ca2+) folding process N ⇄ RU → IU. Briefly, cycles A (295 K →
by injecting a solution at pH 8.8 containing a proper buffer into a 365 K → 297 K) and B (296 K → 366 K → 298 K) follow the complete
protein solution at pH 2.0. In such a way the 1H NMR spectra denaturation starting from the native state with steps of ΔT = 2 K.
were recorded at incremented time points (between 1.2 s and Cycle C (295 K → 320 K → 298 K) operates only inside the native
10.3 min, with steps of 10 ms) after initiation of refolding. Such an N state with ΔT = 1 K. Cycles D (310 K → 349 K → 310 K) and
approach, in enlarging the folding kinetics, represents the poten- E (310 K → 343 K → 310 K) work inside the N ⇄ RU → IU and
tiality of the NMR methodology in its capability of monitoring N ⇄ RU regions, respectively, with ΔT = 1 K.
specific aspects of the folding, like the protein structural changes, Fig. 1 shows the NMR spectra of the hydrated protein for two
the energetic configurations, and the role of the hydration water. thermal cycles after the water contribution has been extracted. In
Here, to study the folding–unfolding of hydrated lysozyme we case A the thermal evolution moves from the native state (N) to
use a different approach based on its thermal denaturation. More the fully denatured state (295–365 K), and vice versa. In case E

Fig. 1. Protein 1H NMR spectra (magnetization versus the chemical shift) of cycles A (Left) and E (Right). (Top) Spectra in the heating phase; (Bottom) spectra
in the cooling phase. Cycle A regards the complete thermal denaturation of lysozyme, whereas cycle E deals with the reversible evolution from the native to
the unfolded (intermediate) state. In both cycles the spectral evolution from the native to the denatured state is reported in different colors just to clarify the
protein thermal behavior. The spectra of the native state are reported in green, those of the RU region before the onset of the CP ðTÞ peak (320 − 336 K) in
dark yellow, the spectra above this region up to the CPmax ðTÞ (337 − 347 K) are in red, and finally spectra in the irreversible denatured region (IU) are in blue.

3160 | www.pnas.org/cgi/doi/10.1073/pnas.1524864113 Mallamace et al.

 the protein is in the N ⇄ RU phase (310–343 K). From the
spectra in cycle A in the heating phase (shown in red), a marked
change in the T region around Cmax P ðTÞ (337–347 K) occurs.
Thus, Fig. 1 shows that the folding–unfolding reaction at the
individual residue level can be quantitatively monitored, and it
supplies the details of the system energy configurations. Among
the several residues observable in the 1H NMR spectra in Fig. 1,
we considered the hydrophilic (the amide NH) and hydrophobic
(CH3 and CH) side-chain groups centered at chemical shifts
δ ’ 6.7 ppm, δ ’ 0.8 ppm, and δ ’ 0.94 ppm, respectively (28).

Data Analysis and Discussion. The hydrogens attached to the amide

nitrogen atoms of peptide via HB (29–31) rapidly exchange with
solvent hydrogen atoms in unfolded states, but are often protected
from exchange when the protein folding is the result of the in-
volvement of amides in the HBs and burial in the protein interior.
It is well known that the HBs of water molecules—with the car-
bonyl oxygen (C = O) and an amide N–H molecular group—
trigger the biomolecular activity of the protein peptides. The most
stable water–protein configuration has two HBs: (i) a water
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proton donor bond to the carbonyl oxygen and (ii) an amide N–H
proton donor bond to the water oxygen (29–31). In protein
folding, the water HBs play a role in protein–protein binding and

APPLIED PHYSICAL
in molecular recognition. In short, water acts as an HB “glue”
between the carbonylic and amidic groups in a protein (31), and

SCIENCES
during the folding phase the formation of hydrophobic clusters
compensates for the loss of system configurational entropy.
Hence, the protein stability is strongly dependent on the HB
strength (or lifetime) that decreases by increasing temperature.
It is just this change in the HB strength that determines the

COMPUTATIONAL BIOLOGY
thermodynamic properties of the hydrated protein and the cor-
responding heat capacity effects (30). All of this is reflected in

BIOPHYSICS AND
Fig. 1. Figs. 2–4 show an Arrhenius plot [the log of the measured Fig. 2. AR representation of the measured magnetization values of the
hydrophilic amide groups (NH). Data for all five different studied thermal
magnetization MI ðTÞ vs. 1=T] that provides a detailed analysis of
cycles (A, B, C, D, and E) are illustrated. The characteristic temperatures T *
the different energetic behaviors of the protein groups, namely and TD are also reported. Lines represent AR behaviors; the corresponding
hydrophilic (the amide NH) and hydrophobic (methyl and methine) activation energies EA are indicated in kcal/mol. Cycles A, B, and D deal with
groups, evaluated using the procedure described in Methods. a complete denaturation; C operates in the native protein state (N), whereas
Fig. 2 deals with the Arrhenius representation of the measured E refers to the native and intermediate states (N⇄ RU).
magnetization values [MI ðTÞ] of the hydrophilic amide groups.
All five different thermal cycles (A, B, C, D, and E) studied are
illustrated. There is a large T interval in A and B for which the behavior shown in cycle E reverses at T = 343 K, i.e., 4 K below
complete protein denaturation can be studied, and a smaller T TD, but MI ðTÞ in the heating phase exhibits the same behavior as
interval in C and D. All figures show the TD and T * tempera- the corresponding behavior in cycles A, B, and D and, in the
tures. T * is the compressibility minimum temperature of bulk cooling phase, differs completely because it recovers its native
water. It is invariant with increasing pressure, and coincides with behavior as it nears T *. All of this demonstrates the energetic
the cross-over point at which thermal expansion is found to be behavior of the hydrophilic protein group NH in a cycle that
constant with pressure (32). T * also signals the breakdown of the operates reversibly between the N native state and the RU state
tetrahedral structure of water (33) and the limit of the protein (as in cycle E). We evaluate the energy (EA) and enthalpy dif-
native state (34). Above T * water becomes a “simple” liquid and ference between the native and unfolded state (in cycles A, B, and
thus a bad solvent (32). Fig. 2 shows the T region of the lyso- D) and the energy of the reversible unfolding (in cycle E). We
zyme-water configurational CP ðTÞ peak (dotted line) (26, 27). find ’50 kcal/mol for the first case and ’12 kcal/mol for the
The overall behavior of cycles A, B, and D is essentially the second, values that agree with those calculated for lysozyme
same. By increasing T, MI ðTÞ exhibits a pure Arrhenius (AR) (58 kcal/mol and 14 kcal/mol) and for globular proteins (35).
behavior (with an activation energy EA ’ 4.58 kcal=mol) up to Fig. 3 shows the thermal behavior of the methyl (CH3) lysozyme
the onset of the CP ðTÞ peak. Above that it presents a marked groups and reports all of the studied cycles. Note that there are
increase [super-Arrhenius (SA) in character] that stops at ap- qualitative similarities with the amide groups but that the differing
proximately TD, after which it evolves again according to the AR energies indicate a different pathway in both the irreversible de-
law (EA ’ 7.38 kcal=mol) up to 365 K, where the thermal cycle naturation and the reversible unfolding. In the first case the cooling
is inverted. During the cooling phase the energetic behavior of phase appears to be fully AR across the wide T range with EA ’ 8.69
this amide group is about the same as the heating phase up to TD. kcal/mol. In the cooling side of the RU region EA ’ 12 kcal=mol. In
At the lowest temperatures, MI ðTÞ shows two other AR behav- the heating phase the N region has an EA ’ 8.3 kcal=mol, the AR
iors: (i) one that stops near T * with EA ’ 9.91 kcal=mol and (ii) behavior stops at T *, and in the RU region a weak maximum appears
one at T < T * that differs somewhat from that of the heating at ’330 K, after which MI ðTÞ assumes the values of the irreversible
phase in the same T range. Note that cycle D inverts (in T after denatured phase and rapidly increases to TD.
the heating phase) above TD and denatures in the same way as A Fig. 4 shows MI ðTÞ for methine (CH) that in the heating phase is
and B. Cycle C operating in the native state shows complete larger than that of the cooling phase. Unlike the amide and methyl
reversibility and thus has the same activation energy as the other polypeptide groups, which are more mobile in the fully denatured
cycles during the early heating phase. Note that the energy phase (IU), i.e., the protein is an open polyelectrolyte and the

Mallamace et al. PNAS | March 22, 2016 | vol. 113 | no. 12 | 3161
 there is a violation of the Stokes–Einstein relationship for T > T * in
the first NMR measurement of the proton diffusion in water as a
function of T (33). This physical effect of T * in bulk water is also
found in confined water. In protein hydration and internal water
it is the internal water that “drives” the protein structure from a
globular configuration to an open, unfolded configuration. The
mechanism for this is the HB structure shared by water, carbonyl
oxygen (C = O), and amidic proton (N–H). Figs. 2–4 show that our
findings confirm this picture. They also show that above T * as TD is
approached the role of hydration water becomes increasingly im-
portant and causes irreversible unfolding in the biopolymer. In the
reversibility interval between T * and TD that defines the interval of
the reversibility, the HB structure of the protein side chains enables
the internal water to impose a persistent folded structure. All of the
measured activation energies quantitatively confirm this picture.
In summary, this study reports the energetic evolution that
occurs during the folding and unfolding of separate peptide hy-
drophilic and hydrophobic groups in single-layer hydrated lyso-
zyme. After studying different thermal cycles, including those
that are reversible and those involving complete denaturation,
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we find that protein properties during this process are strongly

affected by different energetic routes.
A comparison between the behavior of these three peptide groups
confirms that HBs play a role in protein folding. Evidence for this
rests not only in the activation energy values that are of the same
order of magnitude as in the HBs, but also in the thermal behavior
of the methine groups, specifically in their magnetization MI ðTÞ
behavior in the N and the RU phases. The higher molecular mobility
is caused by the burial effect of the HBs on the hydrophobic groups,
which is particularly strong in the case of the methyl groups.
The two forms of protein water, hydration water and internal
Fig. 3. Thermal evolution of the magnetization, MI ðT Þ, of the protein
water, are essential in protein folding. Because all water in the
methyl (CH3) groups for all of the studied thermal cycles. Lines and symbols
are the same as used in Fig. 2.

water a bad solvent, the methine groups are more mobile during
the heating phase of the RU region than in the IU phase. This
confirms that the hydrophobic groups are buried during protein
folding—more so in CH3 than in CH—and that they are affected
by the presence of solvent hydrogen atoms. However, in cycles A,
B, and C the methine groups exhibit AR behavior in the cooling
phase (EA ’ 6.41 kcal/mol), and their thermal evolution during the
heating phase is more complex. Within the native region the
heating phase has an AR evolution (EA ’ 9.09 kcal/mol) that stops
near T *, MI ðTÞ has a maximum at T ’ 330 K and then decreases
to a minimum at TD and further evolves, exhibiting AR behavior
that is nearly identical to that of the cooling phase. In cycle D the
methine MI ðTÞ recovers (from the RU phase) the thermal trend of
the native state, which is an AR behavior with EA ’ 5 kcal/mol.
T * strongly affects protein folding; it marks the cross-over in
bulk water from “normal” liquid behavior when T > T * to be-
havior characterized by thermodynamic anomalies when T < T *.
In particular, T * indicates a “singular and universal expansivity
point” related to the balance between the entropy and volume
cross-correlations hδSδV i. In normal liquids δS and δV fluctua-
tions become smaller as T decreases and they are positively
correlated, but in water they become more pronounced at T * and
are anticorrelated at the density maximum (at ambient pressure).
Structurally T * is identified as the onset temperature of HB
clustering (32). Such transport properties as self-diffusion data in
the high-temperature regime of bulk water Ds ðTÞ indicate that T *
signals a new dynamic cross-over. Specifically, when T deceases
the dynamics change and there is a shift from AR to SA be-
havior. We used the Adam–Gibbs approach, connected Ds to the
configurational entropy Sc, and found that at this temperature Fig. 4. Protein methine (CH) magnetization, MI ðTÞ, for all of the different
the local order of water becomes more structured (36). Note that thermal cycles. Lines and symbols are the same as previously used.

3162 | www.pnas.org/cgi/doi/10.1073/pnas.1524864113 Mallamace et al.

 unfolded state belongs to the solvent, hydrogens in the amide groups To avoid unwanted abrupt T changes, heating and cooling steps were
with peptide bonds interact rapidly via HBs with water. When the executed slowly and were ∼20 min in duration. Fig. 1 shows all of the spectra
protein folds these interactions involve only internal water, which is (magnetization intensity MI versus chemical shift δ in the −1.2–10-ppm
range) of cycle A (Left) and cycle E (Right) after subtracting from the spectra
linked with amides and buries the protein interiors. Thus, clusters the central contribution due to water. Both the spectra in the heating phase
of hydrophobic residues are formed in the folded protein and the (Top) and the cooling phase (Bottom) are shown. Fig. 1 also shows the many
methine groups are more mobile than the methyl groups. different contributions made by different protein chemical groups when MI
is increased by increasing T. During the cooling phase the reverse is true.
Methods Note that in cycle A the starting spectra and the final spectra (at the same
We used hen egg white lysozyme at the hydration level h = 0.3 prepared temperature, 297 K) differ to the extent that the spectral evolutions during
according to a precise procedure (27). The enzymatic activity in lysozyme is the heating and cooling phases differ. To clarify the temperature spectral
very low up to h ∼ 0.2, but when h is increased from 0.2 to 0.5 the activity evolution, the different phases are color-coded: the native state spectra in
increases sharply. The hydration level was determined by thermogravimetric green, the RU region before the CP ðT Þ peak at 320–336 K in dark yellow,
analysis and also confirmed by directly measuring the weight of the absor- the spectra above this region up to the CPmax ðT Þ at 337–347 K in red, and
bed water. This hydration level corresponds to a monolayer coverage on the the irreversible denatured (IU) region in blue. Note that in the heating
protein surface. For each experimental run we used different samples. phase of the cycle A spectra the corresponding MI rapidly increases in the
Hydrated lysozyme has been studied at ambient pressure and different interval 339 < T < 351 K immediately inside the temperature range of the
temperatures (essentially in the 290 < T < 370 K range) by using a Bruker CP ðTÞ peak, and in the cooling phase MI seems to continuously evolve with
AVANCE 700-MHz NMR spectrometer. We focused on the 1H NMR spectra T. In contrast, cycle E exhibits a different thermal evolution in which the
obtained from free-induction decay, and explored the hydrated protein as a initial and final spectra are essentially the same, indicating that we were
function of temperature in heating–cooling cycles with an accuracy of ±0.1 K in the N ⇄ RU region, that TD represents the limit of the reversibility, and
by using the T dependence of the chemical shift of ethylene glycol as a that the thermal evolution appears to be continuous in both the heating
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T standard. The spectroscopic experimental technique was “magic angle and cooling phases. Note that in the cooling phase we collected spectra
spinning” (37). The NMR signal intensity MI is directly related to the system in steps of ΔT = 1 K.
equilibrium magnetization M0, which is related to the susceptibility χ 0, To obtain the thermal behavior of hydrophilic (the amide NH) and hy-
drophobic (methyl CH3 and methine CH) groups, we performed a spectral

APPLIED PHYSICAL
which depends linearly on the total number of mobile spins per unit volume,
on the mean-square value of the nuclear magnetic moment, and on 1=T (the deconvolution in terms of their Lorentzian contributions by examining all of

SCIENCES
Curie law); hence, the spectra were corrected for the Curie effect. the spectra contributions shown in Fig. 1. For all of the measured spectra we
used an AR plot to analyze the MI of the three groups centered at chemical
We used different heating and cooling cycles to explore, completely or
shifts NH ’ 6.7 ppm, CH3 ’ 0.8 ppm, and CH ’ 0.94 ppm. The results in Figs.
partially, the folding process N ⇄ RU → IU. (i) In cycles A and B we began in
2–4 are shown for the same MI -reduced temperature (1,000=T) intervals, and
the native state and studied the entire process up to complete denaturation.
are explicitly indicated for the N and IU regions.
In cycle A the hydrated lysozyme was heated from 295 to 365 K and then
cooled down to 297 K, and in cycle B cycle from 296 to 366 K and down to
ACKNOWLEDGMENTS. The authors acknowledge the Consiglio Nazionale

COMPUTATIONAL BIOLOGY
298 K. In both the warming and the cooling cycles the spectra were mea-
delle Ricerche for its support. The Boston University research is supported by
sured using steps of ΔT = 2 K. (ii) Cycle C operates inside the native N state.

BIOPHYSICS AND
the National Science Foundation, Grants CHE 1213217, CMMI 1125290, and
(iii) Cycle D operates inside the N ⇄ RU → IU region and cycle E inside the PHY 1505000. The research at Massachusetts Institute of Technology is
N ⇄ RU region. We started both cycles at the sample heating temperature funded by US Department of Energy Grant DE-FG02-90ER45429. S.V.B.
(310 K), but cycle D was inverted immediately above TD at T = 349 K and the acknowledges the support of this research through the Dr. Bernard
cooling in cycle E was initiated at 343 K (4 K below TD). W. Gamson Computational Science Center at Yeshiva College.

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