Temperature dependence of amino
acid hydrophobicities
Richard Wolfenden1, Charles A. Lewis Jr., Yang Yuan, and Charles W. Carter Jr.
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599
Contributed by Richard Wolfenden, April 20, 2015 (sent for review February 16, 2015; reviewed by Robert L. Baldwin, George D. Rose, and D. Peter Tieleman)
The hydrophobicities of the 20 common amino acids are reflected
in their tendencies to appear in interior positions in globular
proteins and in deeply buried positions of membrane proteins. To
determine whether these relationships might also have been valid
in the warm surroundings where life may have originated, we
examined the effect of temperature on the hydrophobicities of the
amino acids as measured by the equilibrium constants for transfer
of their side-chains from neutral solution to cyclohexane (Kw>c).
The hydrophobicities of most amino acids were found to increase
with increasing temperature. Because that effect is more pro-
nounced for the more polar amino acids, the numerical range of
Kw>c values decreases with increasing temperature. There are also
modest changes in the ordering of the more polar amino acids.
However, those changes are such that they would have tended to
minimize the otherwise disruptive effects of a changing thermal
environment on the evolution of protein structure. Earlier, the
genetic code was found to be organized in such a way that—with
a single exception (threonine)—the side-chain dichotomy polar/
nonpolar matches the nucleic acid base dichotomy purine/pyrimi-
dine at the second position of each coding triplet at 25 °C. That
dichotomy is preserved at 100 °C. The accessible surface areas of
amino acid side-chains in folded proteins are moderately corre-
lated with hydrophobicity, but when free energies of vapor-to-
cyclohexane transfer (corresponding to size) are taken into consid-
eration, a closer relationship becomes apparent.
hydrophophobicity | protein folding | anticodon | temperature |
genetic code
T he equilibrium conformations of proteins in neutral solution
are strongly influenced by interactions between their con-
stituent amino acids and solvent water. Early work on the crystal
structure of hemoglobin and related proteins showed that the
side-chains of the more polar amino acid residues tend to be
exposed to solvent, whereas less polar side-chains tend to be
buried within the interior of globular proteins (1). Later, those
tendencies were put to a quantitative test by measuring equilibria
of transfer of amino acid side-chains from neutral aqueous so-
lution into less polar environments, such as the vapor phase (2,
3) or a nonpolar solvent such as cyclohexane (4), which dissolves
only ∼2 × 10−3 M water at saturation (5) and appears to be
devoid of specific interactions with solutes. The water-to-cyclo-
hexane distribution coefficients (Kw>c) of the 20 common side-
chains [here termed “hydrophobicities” (6, 7) and expressed in
concentration units of mol/L in each phase; SI Appendix] were
found to span a range of 15 orders of magnitude at pH 7 and
25 °C. Values of Kw>c have been shown to be related to their
outside-to-inside distributions in globular proteins (4, 8) and to
their tendencies to appear within the buried sequences of trans-
membrane proteins (9–11).
Those solvent distribution experiments were conducted at what
we would consider ordinary temperatures. However, there is
widespread (12, 13)—if not universal (14)—agreement that life
originated when the earth was warmer—perhaps much warmer—
than it is today, and some modern organisms thrive at tempera-
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tures approaching the boiling point of water. The literature dis-
closes little concrete information about how the hydrophobicities
7484–7488 | PNAS | June 16, 2015 | vol. 112 | no. 24
of the individual amino acids respond to changing temperature.
Would the rules of protein folding be expected to differ signifi-
cantly for an organism such as Pyrococcus horikoshii, which grows
optimally at 98 °C (15), and might such differences have affected
the early stages of evolution of life on Earth?
Earlier work also uncovered the existence of a pronounced
bias in the relationship between amino acid hydrophobicity and
the genetic code. Thus, a pyrimidine at the second codon position
signals amino acids whose average hydrophobicity is much greater
than those coded by a purine at the same position (2, 3). The
values reported here allow a more detailed analysis, described in
a companion paper (16), which reveals that two separate codes
for amino acid size and hydrophobicity appear to be embedded in
different parts of their tRNA sequences, with size (represented by
vapor-to-cyclohexane equilibria) encoded in the acceptor stem,
and hydrophobicity (represented by water-to-cyclohexane equi-
libria) embedded in the anticodon. Would one expect these re-
lationships to be maintained at elevated temperatures?
In the present work, we sought to address these questions by
determining the effect of temperature on equilibria of transfer of
molecules representing the side-chains of each amino acid from
neutral aqueous solution to cyclohexane (Kw>c).
Materials and Methods
For molecules representing the side-chains of Gly, Ala, Val, Leu, and Ile
(hydrogen, methane, propane, butane, and isobutane), values of Kw>c were
obtained by combining the best values for the molar solubilities of the gases
in water and in cyclohexane, compiled from the literature in the Solubility
Data Series (17). These values are considered accurate to within ±0.2 kcal/mol
in ΔG at 25 °C, ±0.4 kcal/mol in ΔH, and ±0.6 kcal/mol in TΔS at 25 °C.
For molecules representing most of the other amino acid side-chains, we
used 1H-NMR as described earlier (4) to determine values of Kw>c for the
uncharged forms. In most cases, the solute was dissolved at a known con-
centration, established gravimetrically (0.01–0.1 M), in the more-favored
solvent (water or cyclohexane). An aliquot (1.5 mL) was introduced into a
scintillation vial and mixed vigorously with the less-favored solvent (15 mL)
for 30 min using a magnetic stirring bar, immersed in a Haake K20 water
Significance
Systematic relationships have long been recognized between
the hydrophobicities of amino acids and (i) their tendencies to
be located at the exposed surfaces of globular and membrane
proteins and (ii) the composition of their triplets in the genetic
code. Here, we show that the same coding relationships are
compatible with the high temperatures at which life is widely
believed to have originated. An accompanying paper reports
that these two properties appear to be encoded separately by
bases in the acceptor stem and the anticodon of tRNA.
Author contributions: R.W. and C.W.C. designed research; R.W., C.A.L., Y.Y., and C.W.C.
performed research; R.W. analyzed data; and R.W., C.A.L., and C.W.C. wrote the paper.
Reviewers: R.L.B., Stanford University; G.D.R., Johns Hopkins University; and D.P.T., Uni-
versity of Calgary.
The authors declare no conflict of interest.
1
To whom correspondence should be addressed. Email: water@med.unc.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1507565112/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1507565112
 bath in which the temperature was maintained within 0.1 °C as measured
with an ASTM International thermometer. The mixture was allowed to settle
for 15 min, and a sample (0.1 mL) of the aqueous layer was removed using
a pipette with a gel-loading tip and mixed with 99% D2O (0.8 mL) to which a
known concentration of pyrazine (0.01 M, δ 8.60 ppm) had been added as
a proton integration standard for determining the concentration of solute.
1
H-NMR spectra were acquired using a Bruker 500-MHz spectrometer
equipped with a cryoprobe, using a water suppression pulse sequence with
two transients. When the favored solvent was cyclohexane, Kw>c was cal-
culated from the concentrations of solute in the aqueous phase before and
after extraction. When the favored solvent was water, the concentration of
solute in cyclohexane was determined by back-extraction from the cy-
clohexane phase (10 mL) with D2O (1 mL).
Values of Kw>c for the uncharged side-chains of Asp and Glu (acetic and
propionic acids) were determined in the presence of 0.03 M HCl to suppress
ionization. Values for the uncharged side-chain of His (1-methylimidazole)
and Lys (1-butylamine) were determined in the presence of 0.03 M KOH to
suppress ionization. Values for the uncharged side-chain of Arg (N-pro-
pylguanidine) were obtained by conducting measurements in the presence
of 0.3, 1, and 3 M KOH to suppress ionization, and no significant variation
was observed. To circumvent problems associated with the volatility of
methanethiol (the side-chain of Cys) at elevated temperatures, distribution
measurements were conducted in the presence of potassium phosphate
buffer (0.1 M, pH 12.35), to maintain this solute in a constant state of ion-
ization (99% thiolate) (pKa 10.35, assumed to be equivalent to that of eth-
anethiol at 25 °C) (18). Because the heat of ionization of HPO4-2 (4 kcal/mol)
is similar to that of thiols (6 kcal/mol) (19), minimizing variations in the
relative abundance of the charged and uncharged species of methanethiol
within the 40 °C range of temperatures over which distribution experiments
were conducted (Table 1), potassium phosphate buffers were chosen for
these experiments. To obtain the distribution coefficient of uncharged
methanethiol, these results were corrected to reflect the fraction of un-
charged thiol that was present in the aqueous phase at various tempera-
tures. In the case of methanol, distribution measurements were conducted
using 1-14C-methanol by determining the concentrations of radioactivity in
both the aqueous and the cyclohexane phase.
In each case, experiments were conducted over at least a fivefold range of
solute concentrations. The results showed no significant variation in Kw>c value,
such as would have been expected if self-association of the solute had been
present in either phase. Table 1 shows the temperature range over which the
positions of transfer equilibria were measured. In all cases, van’t Hoff plots
were linear within experimental error over the ∼50 °C temperature range ex-
amined, yielding values with an estimated error of ±0.3 kcal/mol in ΔG at 25 °C,
±0.5 kcal/mol in ΔH, and ±0.8 kcal/mol in TΔS at 25 °C. Differences in heat
capacity appeared not to exert an important influence on the response of these
transfer equilibria to temperature, in view of the absence of significant cur-
vature in the van’t Hoff plots over the 40–50 °C temperature range examined.
The side-chains of Asp, Glu, His, Lys, and Arg enter cyclohexane in de-
tectable amounts only in their uncharged forms (4), as expected from the
extremely large heats of solution of ions by water (20). Thus, the overall
equilibria of transfer [Kw>c(total)] of those ionizable solutes (involving both
the charged and uncharged forms of the side-chains of Asp, Glu, His, Lys, and
Arg that are present at pH 7) include a term for the water-to-cyclohexane
transfer of the uncharged species, divided by the fraction (α) of the solute
molecules that are present in the uncharged form in aqueous solution at pH 7.
For an amine side-chain (A), that fraction was calculated from the pKa value of
the amine’s conjugate acid (AH+) using Eq. 1. For a carboxylic acid side-chain
(CH), α was calculated using Eq. 2
 
fraction uncharged = α = ðAÞuncharged ðAÞuncharged + ðAH+ Þ = 1=½1 + ðH+ Þ=Ka ,
[1]
 
fraction uncharged = α = ðCHÞuncharged ðCHÞuncharged + ðC- Þ = 1=½1 + Ka =ðH+ Þ.
[2]
To estimate values of α at elevated temperatures, enthalpies of ionization
of molecules representing each side-chain (19) (e.g., acetic acid representing
Asp and n-butylamine representing Lys) were used to estimate pKa values of
the side-chain models at elevated temperatures from the corresponding
values (21) at 25 °C (Table 2). The resulting values of the equilibrium con-
stant Kw>c(total), which describes the distribution of the solute in both its
charged and uncharged forms from neutral aqueous solution to cyclohex-
ane, are shown in Table 3.
Values of Kv>c (SI Appendix, Table S1) needed to complete the vapor
phase thermodynamic cycle (SI Appendix, Fig. S1) were calculated by mul-
tiplying the values of Kv>w (3) by the present values of Kw>c(total).
Results
Table 1 summarizes the present values of log10 Kw>c(uncharged) for
water-to-cyclohexane transfer of uncharged molecules representing
side-chains of the 20 common amino acids at 25 °C and 100 °C

COMPUTATIONAL BIOLOGY
BIOPHYSICS AND
Table 1. Thermodynamics of transfer (kcal/mol at 25 °C) of molecules representing uncharged amino acid side-chains from water to
cyclohexane
Amino acid Solute Log Kw>c 25 °C ΔG 25 °C ΔH TΔS 25 °C Log Kw>c 100 °C Range, °C Footnote

Ile Butane 4.24 −5.77 1.03 6.80 4.39 10–50 *

Leu Isobutane 4.24 −5.77 0.60 6.37 4.33 10–50 *
Val Propane 4.09 −5.56 1.48 7.04 4.31 10–50 *
†
Pro Triangulation 3.75 −5.10 0.54 5.64 3.83 8–37
‡
Phe Toluene 2.64 −3.59 −0.25 3.34 2.60 14–54

CHEMISTRY
Ala Methane 2.11 −2.87 2.55 5.42 2.49 10–50 *
‡
Met Ethylmethylsulfide 1.91 −2.60 0.80 3.40 2.03 10–50
‡
Trp 3-Methylindole 1.83 −2.49 −0.13 2.36 1.81 14–54
‡
Cys Methanethiol 1.53 −2.08 3.12 5.20 1.99 10–49
Gly Hydrogen 0.20 −0.27 2.24 2.52 0.53 10–50 *
‡
Lys n-Butylamine -0.27 0.37 5.31 4.94 0.51 15–45
‡
Tyr p-Cresol -0.31 0.42 4.35 3.93 0.33 14–64
‡
Thr Ethanol -1.83 2.49 6.72 4.23 −0.84 10–52
‡
Glu Propionic acid -2.26 3.07 10.35 7.28 −0.74 10–50
‡
Ser Methanol -2.82 3.84 6.21 2.37 −1.88 15–50
‡
Asp Acetic acid -3.29 4.47 8.34 3.86 −2.06 10–40
‡
His 4-Methylimidazole -3.49 4.75 11.59 6.84 −1.78 15–45
‡
Gln Propionamide -4.07 5.54 8.79 3.25 −2.78 10–45
‡
Arg n-Propylguanidine -4.32 5.88 13.74 7.86 −2.30 10–50
‡
Asn Acetamide -4.88 6.64 7.14 0.51 −3.83 10–55

*Data from Goldberg et al. (19).

†
Values for proline, an amino acid that does not have a side-chain in the ordinary sense, were estimated indirectly by first determining equilibrium constants
(Kv>w and Kw>c) for transfer of N-acetylpyrrolidine and N-butylacetamide. To obtain Kw>c, the ratio between those equilibrium constants was multiplied by
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the values observed for propane, the side-chain for norvaline (38).
‡
This work.

Wolfenden et al. PNAS | June 16, 2015 | vol. 112 | no. 24 | 7485
 Table 2. Fractions (α) of molecules representing ionizable amino acid side-chains that remain uncharged in neutral
solution at 25 °C and 100 °C
Amino acid Solute pKa (25 °C) log10 α (25 °C) ΔH ionization (kcal/mol) pKa (100 °C) log10 α (100 °C)

Asp Acetic acid (39) 4.76 (39) −2.24 −0.33 (19) 4.78 −1.38
Glu Propionic acid (39) 4.82 (39) −2.18 −0.36 (19) 4.84 −1.32
His 4-Methylimidazole (39) 6.95 (39) −0.30 8.77 (19) 5.66 −0.60
Lys N-Butylamine (39) 10.95 (39) −3.95 13.84 (19) 8.91 −2.77
Arg n-Propylguanidine (37) 13.6 (37) −6.60 18 (37) 10.95 −4.81

(expressed as mol/L in each phase) (6, 7) and the corresponding expected when water is released from close contact with the polar
values of their free energies, enthalpies, and entropies of transfer at groups of a solute when it passes from water into cyclohexane;
25 °C. Most of the values at 25 °C resemble values reported earlier positive ΔHw>c values (5–14 kcal/mol) are observed for the un-
(4), with minor discrepancies arising from new information about charged, but polar, side-chains of Arg, Asp, Asn, Glu, Lys, Gln, His,
the critically evaluated solubilities of the aliphatic hydrocarbon side- Ser, and Thr. Much smaller ΔH values (−0.25 to 3.1 kcal/mol) are
chains in water and in cyclohexane in the International Union of observed for the nonpolar side-chains of Ile, Leu, Val, Pro, Phe,
Pure and Applied Chemistry Solubility Data Series (15). Values of Ala, Met, Trp, Cys, and Gly, consistent with their inability to
Kw>c(uncharged) for the uncharged side-chains at 100 °C were esti- participate in strong H-bonding or other polar interactions with
mated by combining values of Kw>c(uncharged) at 25 °C with the water. The enthalpy of transfer of the Tyr side-chain occupies an
present values of ΔH for water-to-cyclohexane transfer. intermediate position (ΔHw>c = 4.3 kcal/mol), consistent with its
Table 2 shows the fractions (α) of molecules representing the intermediate position in the overall scale of hydrophobicities
ionizing side-chains of Asp, Glu, His, Lys, and Arg (acetic acid, pro- (Fig. 1).
pionic acid, 4-methylimidazole, butylamine, and N-propylguanidine)
that remain uncharged at pH 7 and 25 °C, based on pKa values from Entropies of Transfer of Uncharged Side-Chains. An increase in en-
the literature (21). Values for the heats of ionization of molecules tropy would be expected when structured water is released from
representing the ionizing side-chains of Asp, Glu, His, Lys, and Arg close contact with nonpolar groups around which it was orga-
were used to calculate their α values at 100 °C as described above at nized (Table 1). One would therefore anticipate an increase in
pH 6.14, the pH value of a neutral solution at 100 °C (22). Table 3 entropy when a nonpolar molecule leaves water and enters a
shows values of log Kw>c(total) at 25 °C for all species (charged plus nonpolar solvent like cyclohexane (23). However, because water
uncharged), obtained by dividing Kw>c(uncharged) by the value of α is also organized in the vicinity of polar substituents, a reduction
in the local organization of solvent water would also be expected
at 25 °C. A similar procedure was used to obtain values of Kw>c(total)
when a polar molecule leaves water and enters cyclohexane. It is
at 100 °C for all species (charged plus uncharged).
therefore not surprising that substantial positive values of TΔS
Discussion are observed for water-to-cyclohexane transfer of side-chains of
Ile, Leu, and Val, which are nonpolar, and for the side-chains of
Enthalpies of Transfer of Uncharged Side-Chains. Uncharged side-
Arg and Glu, which are strongly polar.
chains tend to become more hydrophobic with increasing temper-
ature, as indicated by their positive ΔH values for water-to-cyclo- Overall Equilibria of Transfer of Amino Acid Side-Chains (Including Both
hexane transfer (Table 1). Because H-bonds become weaker with Charged and Uncharged Species) from Water to Cyclohexane at pH 7.
increasing temperature (23), an increase in enthalpy would be Because amino acid side-chains enter cyclohexane only in their
uncharged forms, the effect of ionization of the side-chains of His,
Table 3. Logarithmic equilibrium constants [log10 Kw>c(total)] for
Lys, Arg, Asp, and Glu is to draw these molecules preferentially
transfer of molecules in both their charged and uncharged forms
into the aqueous phase [as indicated by Kw>c(total)], to an extent
from neutral aqueous solution to cyclohexane at 25 °C and 100 °C
that varies with the extent to which the pKa value of the ionizing
side-chain differs from 7 (Table 3). Values of the overall equilib-
Amino acid 25 °C 100 °C rium constant [Kw>c(total)] for transfer of each side-chain, taking
Ile 4.24 4.39
into account the sum of the concentrations of both the charged and
Leu 4.24 4.33
uncharged forms (Eqs. 1 and 2), span a range of 1015.2-fold at
Val 4.09 4.31
25 °C, with Arg occupying an extreme position (Table 3 and Fig. 1).
Pro 3.75 3.83
At elevated temperatures, that range of values becomes
Phe 2.64 2.60
smaller, for at least two reasons. First, the effect of temperature
Ala 2.11 2.49
in enhancing Kw>c(uncharged) is more pronounced for the more
Met 1.91 2.03
polar than for the less polar amino acids, so that the overall
Trp 1.83 1.81
range of Kw>c values for the uncharged side-chains shrinks with
Cys 1.53 1.99
increasing temperature, from 108.6-fold at 25 °C to 106.7-fold at
Gly 0.20 0.53
100 °C (Table 1 and Fig. 1). Second, the fraction (α) of the side-
Tyr −0.31 0.33
chains of His, Lys, Arg, Asp, and Glu that remains uncharged
Thr −1.83 −0.85
decreases with increasing temperature (Table 2).
Ser −2.82 −1.72
Relationship Between Side-Chain Transfer Equilibria and Protein
His −3.79 −2.38
Folding. Considerable effort has been devoted to the search for
Gln −4.07 −2.78
a general scale that might relate the physical properties of the
Lys −4.22 −2.26
side-chain of each the 20 amino acids to its solvent-accessible
Glu −4.44 −2.06
surface area in folded proteins (ASAfold), as determined using a
Asn −4.88 −3.83
probe that is often a water-sized sphere (24–32). Values of
Asp −5.53 −3.44
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ASAfold depend on the size and shape of the probe, the presence
Arg −10.92 −7.11
or absence of steric constraints on the approach of a probe that

7486 | www.pnas.org/cgi/doi/10.1073/pnas.1507565112 Wolfenden et al.

 25 °C Kw>c 100 °C (w>c,v>w = red; w>c,v>c = green; v>c,v>w = blue; Fig. 2B)
from the three-way vapor phase cycle (34), and e is a residual.
When each of these pairs was tested for its 2D ability to predict
105 105 log KSurf, correlations of those predictions with the observed
VAL LEU ILE VAL LEU ILE values averaged R2 = 0.90 (SI Appendix, Fig. S3). Using the
PRO PRO
Student t test to determine whether this improved correlation
PHE ALA PHE (Fig. 2C) exceeded that expected for an additional random pre-
ALA MET
CYS TRP MET TRP dictor, we obtained P < 0.0001 for each of the two transfer free
CYS
energies and P = 0.02 for their interaction. Using the results of a
GLY GLY TYR more recent model for predicting ASAfold, proposed by Tien
1 TYR
1 et al. (32), similar values of the three coefficients were obtained
THR
THR (SI Appendix, Table S2).
SER The one-dimensional correlations described in Fig. 2A in-
SER LYS
GLU GLN dicate the more prominent role for Kw>c values in both sets of
HIS
HIS ASN
correlations (SI Appendix, Fig. S3). The Kv>c branch of the
GLU GLN ASP phase-transfer cycle (Fig. 2B) is related to amino acid size as
ASN LYS represented by its surface area in a tripeptide (R2 = 0.86), its
10-5 10-5
ASP
volume (R2 = 0.83), or its mass (R2 = 0.89). It is uncorrelated
ARG
with Kw>c (R2 = 0.01; SI Appendix) and constitutes a linearly
independent source of information that was omitted from pre-
vious attempts to understand how the properties of amino acids
are related to protein folding. Its apparent helpfulness in con-
10-10 10-10 tributing to prediction of the locations of the common amino
acids in folded proteins agrees with the well-established close-
ARG
packing of amino acid side-chains in proteins, in which large and
small amino acids differ in their structural requirements (35).
second codeletter = U C G A
The finding that the core/surface distributions of the 20 amino
acids can be better approximated using two complementary types
Fig. 1. Hydrophobicities [Kw>c(total)] of amino acid side-chains at 25 °C and of solvent transfer free energies seems likely to be useful in
100 °C, colored according to the middle base of the corresponding codon. protein design and phylogenetics (36), by identifying suitable
Changes in the ordering of side-chain hydrophobicities at 25 °C and 100 °C, amino acid substitutions.
colored according to the second base of the corresponding codon as in-
dicated. Hydrophobic residues, near the top, tend to be associated with
Effects of Temperature on Protein Folding and the Genetic Code. As
pyrimidines (red and purple), whereas hydrophilic residues, near the bottom,
tend to be associated with purines (blue and black).
noted earlier, most side-chains become more hydrophobic with
rising temperature, and those increases are greater for the more
polar amino acids. For the uncharged forms of the side-chains,
may be imposed by flanking peptide bonds, and the rotameric the numerical range of ΔGw>c values (11.6 kcal/mol at 25 °C)
shrinks to 6.7 kcal/mol at 100 °C. When both the charged and

COMPUTATIONAL BIOLOGY
preferences of the larger side-chains, all of which can affect the
normalization of estimated ASA values. Despite those potential

BIOPHYSICS AND
pitfalls, we decided to explore the relationship, if any, between a
recent set of weighted average values of ASAfold reported by
Moelbert et al. (28) and the three branches of the vapor phase
thermodynamic cycle (Fig. 2B). Proline was omitted from con-
sideration because it occurs frequently in turns (26), and cysteine
was omitted because it tends to be buried by cross-linkage with
other cysteine residues or coordination by metal ions (33). After
conversion of each ASAfold value to a virtual equilibrium con-

CHEMISTRY
stant (Ksurf) (SI Appendix, Fig. S2), correlations between log Ksurf
and each of the three phase transfer equilibria were found to be
R2 = 0.62 for log Kw>c, R2 = 0.32 for log Kv>w, and R2 = 0.19 for
log Kv>c (Fig. 2A). Thus, the strongest correlation was with Kw>c,
confirming the results of an earlier study (4).
Agreement was found to improve (Fig. 2C) when a second
independent physical property was included in Eq. 3
Fig. 2. Correlations between amino acid properties and their behavior in
log  K surf   = β0 + βw>c ðΔGw>c Þ + βv>w ðΔGv>w Þ folded proteins. (A) Predictions of the logarithm of the distribution constant
+ βw>c*v>w ðΔGw>c ÞðΔGv>w Þ + e [3a] (KSurf), derived from the exposed surface area of amino acids in folded
proteins based on distribution coefficients of their side-chains. Rose-colored
log  K surf   = β0 + βw>c ðΔGw>c Þ + βv>c ðΔGv>c Þ ellipses denote 95% of the area necessary to include all points in the scat-
terplots. Squared correlation coefficients are given above the scattergrams.
+ βw>c*v>c ðΔGw>c ÞðΔGv>c Þ + e [3b] (B) Vapor phase analysis of amino acid transfer free energies (4). Colored
lines indicate pairs of values used to calculate log KSurf, according to the
log  Ksurf   = β0 + βv>c ðΔGv>c Þ + βv>w ðΔGv>w Þ background colors in C. (C) Scatterplots between observed values of log KSurf
and values calculated using the three independent pairs of experimental
+ βv>c*v>w ðΔGv>c ÞðΔGv>w Þ + e [3c] phase transfer free energies in B, two at a time, along with their synergistic
interaction (Eq. 3). These correlations average R2 = 0.9, whereas those in A
in which the β values were estimated by least-squares regression
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average R2 = 0.4. Correlations between estimates based on each pair of

analysis. Eqs. 3a–3c represent different pairs of transfer equilibria vapor phase cycle values (panels with white background), average 0.95.

Wolfenden et al. PNAS | June 16, 2015 | vol. 112 | no. 24 | 7487
 uncharged forms of all of the side-chains are taken into con-
sideration, the details change. The large heat of deprotonation of
the guanidinium side-chain of Arg (18 kcal/mol) (37) causes its
pKa value to decrease by 2.7 units when the temperature rises
from 25 °C to 100 °C, whereas the pKa values of the side-chains
of Lys (10.95) and His (6.95) decrease by 2.0 and 1.3 units, re-
spectively. In contrast, the heats of ionization of acetic and
propionic acids are −0.1 kcal/mol (19), so that the pKa values of
the side-chains of Asp and Glu do not change significantly when
the temperature increases from 25 °C to 100 °C. As a result, the
numerical range of ΔGw>c values for water-to-cyclohexane
transfer from neutral aqueous solution to cyclohexane shrinks
from 15.2 kcal/mol at 25 °C to 10.74 kcal/mol at 100 °C.
There is also a modest reshuffling of relative hydrophobicities,
so that the order His > Gln > Lys > Glu > Asn > Asp at 25 °C is
reordered to the series Glu > Lys > His > Gln > Asp > Asn at
100 °C. However, their Kw>c(total) values at 100 °C fall within a
factor of 60, an extremely narrow range compared with a range
of 1.5 × 1015-fold for all of the amino acid side-chains (SI Ap-
pendix, Table S1, last column). The limited magnitude of these
changes, for the more polar amino acids, would presumably have
tended to minimize the potentially disruptive effects of changes
in thermal environment on the structures of globular proteins
and also on the relative tendencies of amino acids to be found in
deeply buried sequences of transmembrane proteins. It seems
reasonable to infer that the rules of protein folding, insofar as
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ACKNOWLEDGMENTS. We thank Jan Hermans and Daniel Isom for helpful
discussions during the preparation of this manuscript. This work was
supported by National Institutes of Health Grants GM-18325 (to R.W.) and
GM-78227 (to C.W.C.).
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