From the Department of Biochemistry, Beckman Center, Stanford School of Medicine, Stanford, California 94305
Forming peptide hydrogen bonds was considered to be probably
the most important driving force for protein folding in 1951, when
Linus Pauling and Robert Corey proposed the hydrogen-bonded structures
of the The change in internal energy (
INTRODUCTION
-helix (1) and two
-sheets (2). Because the peptide CO and
NH groups form competing hydrogen bonds (H-bonds) to water when a
protein is unfolded it was evident, however, that the net contribution
of the peptide H-bond (CO···HN)
to protein stability might be small. In 1955 John Schellman (3, 4) made
a first analysis of the energetics of peptide H-bonds in protein
folding reactions. He listed the factors that should affect the
stability of a peptide
-helix in water, and he estimated the
strength of the peptide H-bond in water by attributing the unusual
thermodynamics of aqueous urea solutions (which had been measured
accurately) to H-bonded urea dimers. His analysis indicated that a
peptide helix in water should have at most marginal stability. Any
observable helix formation should be driven by the net enthalpy change
(
H) of forming the peptide H-bond in water, and the
aqueous urea data gave
H =
1.5 kcal/mol. The
proposal of Schellman that helix formation should be driven by the
enthalpy of the peptide H-bond was adopted by Zimm and Bragg (5) and by
Lifson and Roig (6) in their treatments of the statistical mechanics of helix formation. Klotz and Franzen (7) found, however, that the
dimerization of N-methylacetamide (NMA) in water was too
small to measure, and the energetic significance of peptide H-bonds for
protein folding lapsed into uncertainty. Interest in the problem diminished further as support grew for the bold proposal by Walter Kauzmann (8) in 1959 that the hydrophobic interaction provides the
major driving force for protein folding.
Hydrogen Bond Inventory
E) for forming a
hydrogen bond in the gas phase can be calculated by quantum mechanics,
and with steady improvement in methods of calculation, calculated values of H-bond energies are now believed to be comparable in accuracy
to good experimental values. A recent calculated value for the
E of dimerization of NMA in vacuum is
6.6 kcal/mol, which has been used as a model for the peptide H-bond (9). Some older
calculated values indicate that the H-bonds formed by amide CO and NH
groups to water (W), and also the
W···W and CO···NH H-bonds, have roughly
equal energies of about
6 ± 1 kcal/mol (10, 11). The conclusion
a decade ago was that a hydrogen bond inventory, discussed
by Alan Fersht (12, 13), is sufficient to describe the net enthalpy of
the peptide H-bond in water. In writing the inventory, the number of
H-bonds is assumed to be the same, whereas their types are similar on
both sides of the equation.
If the assumptions made in writing Equation 1 are valid, then
the net enthalpy of the peptide H-bond in water is 0 ± 1 kcal/mol to a first approximation. In making quantum mechanics calculations to
model the peptide H-bond and the
NH···W and
CO···W H-bonds, the energies
found by using different model compounds to estimate one type of H-bond
differ by as much as the energies found for different types of H-bond
(11). The enthalpy change
(Eq. 1)
H and the internal energy
change
E are considered here to be interchangeable in
aqueous solution because any difference between them should be negligible.
Note, however, that even a small net enthalpy change per peptide H-bond
could be important because a typical protein forms about 70% of the
possible peptide H-bonds. The patterns of peptide H-bonds found in
proteins are complex (14), and any average number must be considered as
a rough estimate. If H is as large as
1 kcal/mol per
H-bond, this projection would contribute
70 kcal/mol to the folding
reaction of a protein with 100 peptide groups. The net free energy
change
G for folding is in the range
5 to
10 kcal/mol
for typical small proteins, and a
70 kcal/mol contribution would be
highly significant. For comparison, the hydrophobic interaction is
estimated to contribute about
126 kcal/mol to the folding reaction of
a protein with 100 peptide bonds and 101 residues. The estimate of
1.25 kcal/mol residue is based on the average
amount of nonpolar surface area buried per residue in the folding
reaction of a typical protein (~50 Å2 (15, 16)) and on a
conversion factor between free energy and buried nonpolar area of 25 cal/Å2. If the energy of the H-bond is
6 kcal/mol, it
would be highly unfavorable to bury a peptide NH or CO group in the
interior of a protein without making its H-bond, and burial of a free
NH or CO group rarely occurs (14).
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Use of the Alanine Peptide Helix to Analyze H-bond Energetics |
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A new approach to understanding peptide H-bond energetics became possible with the discovery by Susan Marqusee (17) that the alanine peptide helix is stable by itself in water. Curiously only alanine, of the 20 naturally occurring amino acids, has this property (18). Thus, helices formed by alanine-based peptides can provide absolute helix propensities when the effect of a guest residue is studied, whereas other systems typically give only relative values because the helix must be stabilized by some other interaction(s), such as salt bridges, whose strength is difficult to measure independently. Amino acids with larger nonpolar side chains, such as leucine, form less stable helices than alanine (18), which suggests that alanine helix formation is not driven by the hydrophobic interaction. Because alanine has just a methyl side chain and only a small amount of nonpolar surface area is buried when an alanine peptide forms a helix (19), it is unlikely at first sight that the hydrophobic interaction could be responsible for helix formation. Instead, the peptide H-bond and solvation of the peptide group probably drive alanine helix formation. Recent calorimetric studies (see below) demonstrate that in fact the hydrophobic interaction is not responsible for alanine helix formation. Alanine helices are prone to be water-insoluble, but two charged ornithine residues at either end of a 13-residue alanine sequence are sufficient to solubilize the helix (20).
The stability of a peptide -helix formed from a single type of amino
acid depends on two different equilibrium constants. The first is a
nucleation constant for a reaction in which three adjacent peptide
groups assume a helical conformation but no peptide H-bonds are formed,
and the second is a helix propagation constant (the helix propensity)
for a reaction in which a single H-bonded helical residue is added (5).
A peptide H-bond is formed between the CO of peptide group i
and the NH of peptide group i + 3. To find the absolute
value of the helix propensity from measurements of helix stability, it
is necessary to determine the helix nucleation constant, which is found
from measurements of helix stability for a set of peptides of varying
chain lengths. The ratio of helical residues formed in the nucleation
reaction to those formed in the propagation reaction decreases as the
helix becomes longer and more H-bonded residues are added, and so
overall helix stability increases with chain length. The nucleation
constant for an alanine-based helix was initially determined by Marty
Scholtz (21), who used circular dichroism to measure the thermal
unfolding curves of a set of peptides of chain lengths varying from 14, 20, 26 ... to 50 residues. Later, Carol Rohl (22) confirmed the
value of the nucleation constant by a new method, based on NMR
measurement of the kinetics of hydrogen exchange, using a different
peptide series (22). When we analyzed these results by an adaptation (23) of the Lifson-Roig theory (6), which is particularly well suited
to analyzing helix formation by peptides with mixed sequences (24), the
helix nucleation constant was found to be 0.0013, more than 1000-fold
lower than the helix propagation constant for alanine, which is 1.70 at
0 °C (18).
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The Enthalpy of Helix Formation |
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In using the alanine peptide helix as a wedge to pry open the
energetics of peptide H-bonds, the first problem was to determine accurately the net enthalpy change for helix formation. Thermal unfolding curves (21) show that alanine helices unfold with increasing
temperature, which suggests that that helix formation is
enthalpy-driven. A standard method for measuring the enthalpy change on
protein unfolding is differential scanning calorimetry (16). Thermal
unfolding curves of peptide helices are quite broad, however, and the
calorimetrically measured unfolding curve of even a 50-residue
alanine-based helix (25) fails to show either 100% helix at 0 °C or
0% helix at 80 °C, the highest temperature reached in this
experiment. The broad thermal melting curve prevents fitting the base
line reliably, which limits the accuracy of measuring the enthalpy
change. Nevertheless, we were able to measure, in collaboration with
Wayne Bolen, an approximate value of the enthalpy change, 1.0
kcal/mol residue (25), which agrees with the value found by fitting
helix melting curves (21). Unfortunately the broad melting curve is
unsuitable for measuring
Cp, the change in heat capacity
on unfolding, which is the critical quantity for determining if the
hydrophobic interaction drives folding. If helix formation is driven by
the hydrophobic interaction, then a large
Cp dominates
the expression describing the thermal unfolding curve.
Recently, a way around the problem of the broad curves for thermal
unfolding was found. In a peptide system developed by Andrzej Bierzynski and his co-workers (26), based on a peptide sequence taken
from an EF-hand protein, helix formation occurs on adding La3+ so that H can be measured by titration
calorimetry. Independent measurements by us, in collaboration with
George Makhatadze (27), gave
H =
0.90 ± 0.1 kcal/mol residue, in good agreement with the value of
0.98 ± 0.1 kcal/mol residue measured by Bierzynski and
co-workers.1 Moreover,
measurements of
H made at two temperatures show that
Cp is zero within error (27), which confirms that folding
is not driven by the hydrophobic interaction. In the following, an average value of
H from these two studies is used,
0.95 ± 0.1 kcal/mol residue. Using this value as
the
H for the peptide H-bond in protein folding gives the
scenario mentioned above, in which (even though
H per
H-bond has only a modest value) the large number of peptide H-bonds
(~70 for a protein with 100 peptide bonds) should produce a large
enthalpic contribution,
66.5 kcal/mol, to the folding reaction if the
assumptions of the H-bond inventory are applicable.
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Application of H-bond Inventory to the Alanine Helix and to Amide Solvation |
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Does the H-bond inventory approach (Equation 1) give a correct
prediction of the measured value of H for alanine helix
formation, as it should if peptide H-bonds and solvation of the peptide
group provide the only important contributions to
H?
First an important question must be answered: does the free peptide
group form one or two H-bonds to water? As written above, Equation 1
implies that the peptide group makes only one H-bond to water, but both the peptide NH and CO groups should be able to form H-bonds to water.
According to the H-bond inventory, the answer can be found from the
enthalpy of solvation of simple amides, which must first be corrected
for the contributions to the solvation enthalpy from van der Waals
interactions and from making a cavity in water for the solute. (It is
also necessary to use the correct standard state for transfer of a
solute from the gas phase to liquid solution.) Calorimetric data for
the solvation enthalpy of some amides are available when the starting
material is the amide in gaseous form, and Peizhi Luo (29) analyzed the
results to give the enthalpy of interaction between water and the amide
polar groups. For four different amides,
H is close to
12 kcal/mol; the value for N-methylacetamide is
11.65
kcal/mol (29). Thus, following the rule discussed above in which
H for making one H-bond of this type is
6 ± 1 kcal/mol, we might at first conclude that the free peptide group makes
two H-bonds to water (however, see below).
In any case, if we use the solvation enthalpy found with amides to
predict the enthalpy of interaction between the free peptide group and
water, then a simple enthalpy balance combined with the H-bond
inventory gives H for forming the alanine peptide helix
as follows:
H(pred) = 12 (breaking H-bond(s) to
water)
6.6 (forming the peptide H-bond)
6 (forming a
W···W H-bond) =
0.6 ± 1, which agrees satisfactorily with the observed
H
value for the alanine helix of
0.95 ± 0.1 kcal/mol residue.
However, when we use the H-bond inventory approach to predict the
enthalpy of solvation of amides, we encounter a paradox, which I have
discussed elsewhere (30). The solvation reaction, starting with the
unsolvated amide (Am) in the gas phase (g), can be written as
follows.
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(Eq. 2) |
According to the H-bond inventory, the unsolvated amide breaks one
W···W H-bond as it dissolves in
liquid (l) water. Thus, the predicted enthalpy of solvating the two
amide polar groups is 12 (making two H-bonds to water) + 6 (breaking
one W···W H-bond) =
6
kcal/mol, but the observed value is
12 kcal/mol. If the assumptions
of the H-bond inventory are used to argue that the dry amide makes only
one H-bond when it dissolves in water, then the contradiction with the
observed value of
12 kcal/mol is even worse. The predicted value then
is
6 (making one H-bond to water) + 6 (breaking one
W···W H-bond) = 0 kcal/mol.
Because the H-bond inventory approach fails when applied to this simple
problem, its validity is doubtful. Probably the main reason for its
failure is that the role of water is not as simple as written in
Equations 1 and 2, in which one
W···W H-bond is broken per
peptide H-bond when a protein unfolds or when a dry amide molecule is
solvated (see the critique of the H-bond inventory by Ben-Naim (31)). A
different approach to understanding the solvation enthalpies of amides
is discussed next.
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Electrostatic Approach to Understanding Amide Solvation |
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The standard approach used by chemists to understand the
solvation of polar groups in model compounds is based on electrostatics (32, 33). The properties of the Born equation (1920) (34) provide a
background for explaining why electrostatics are all important.
Atom-splitting experiments were still a novelty in 1920, and Max Born
was interested in why Wilson's cloud chamber can be used to see the
tracks of charged particles in supersaturated water vapor. The path of
a charged particle is visualized by the trail of water droplets it
leaves behind. Born calculated the free energy G of
transferring an ion (with radius r, charge q) from vacuum to a continuum solvent (water) that has a dielectric constant D.
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(Eq. 3) |
The solvation enthalpy of NaCl (infinite dilution) has the large value
of 184 kcal/mol (35)! It is not surprising then that polar molecules
such as amides, which contain large partial charges on the amide oxygen
and nitrogen, have substantial solvation enthalpies associated with
their partial charges, nor is it surprising that chemists have devised
schemes for using electrostatics to compute the solvation free energies
associated with polar groups. The calculation scheme devised by Barry
Honig and his co-workers (32) has the attractive feature that the
partial charges and atomic radii of the PARSE parameter set are
calibrated from data for the solvation free energies of a large base of
model compounds, including amides. The electrostatic algorithm DelPhi
is used to compute the ESF. As discussed above, ESF values for the
solvation of amide polar groups agree within error with the
corresponding solvation enthalpies so long as the solvent is water.
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Electrostatic Approach to Analyzing the Role of Solvation in Forming the Alanine Helix |
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The use of experimental solvation free energies to calibrate
the parameters employed in calculating ESF values guarantees that the
calculated ESF values will fit the experimental solvation free energies
when applied to polar groups of the same type used for calibration.
Will electrostatic calculations also give meaningful ESF values when
applied to related but different polar groups such as the peptide NH
and CO groups in an alanine helix? The only groups that have
significant partial charges in an alanine peptide with blocked end
groups are the polar CO and NH groups. The ESF value of the central
peptide group in a solvent-exposed, 15-residue alanine helix was
computed by Franc Avbelj in collaboration with us (29) to be 2.5
kcal/mol, most of which (
2.0 kcal/mol) results from the interaction
with water of the peptide CO group. Avbelj and I later found a similar
result for peptide H-bonds in a
-structure, an alanine
-hairpin
(37), namely that the H-bonded peptide group is solvated and its ESF
value is about
2.5 kcal/mol. This calculated interaction between the
H-bonded peptide group and water is a basic contradiction of the H-bond inventory.
Solvation of the peptide H-bond should have major energetic
consequences for steps in the process of protein folding (i). There is
a desolvation penalty for burying an H-bonded peptide group because the
ESF value drops to zero when the peptide H-bond is buried out of
contact with water (37). Thus, for an alanine helix, the desolvation
penalty is exactly the opposite of the ESF of the peptide group in a
solvent-exposed helix or 2.5 kcal/mol. The peptide H-bond in an alanine
helix changes from being favorable for folding (H =
0.95 kcal/mol residue) when the H-bond is
solvent-exposed to being unfavorable (
H = 1.55 kcal/mol residue) when the helix is buried.
Consequently, solvent-exposed peptide H-bonds should stabilize but
buried H-bonds should destabilize (ii). The size of the calculated
desolvation penalty (2.5 kcal/mol) is remarkably large compared with
the numerically smaller free energy change per residue for burying the
nonpolar surface during folding (
1.4 kcal/mol for an average
residue). Consequently, the desolvation penalty ought to be a major
factor limiting domain size in globular proteins, because the ratio of
solvent-exposed H-bonds to buried H-bonds must then affect the overall
stability in a critical manner. The large size of the desolvation
penalty suggests that burial of peptide H-bonds must be coupled to a
process that provides a favorable free energy change, most likely
burial of nonpolar surface area. Coupling has been reported recently
(38) between formation of peptide H-bonds and burial of nonpolar
surface area when the folding transition state is formed (iii). Side
chains larger than alanine hinder the access of water to the helix
backbone and substantially reduce the ESF values of peptide groups in a helix (29). Thus, larger nonpolar side chains should give smaller desolvation penalties when H-bonds are buried through folding.
Do calculated ESF values yield the known value for the H
of alanine helix formation? The answer to this question is not yet known reliably because it depends critically on the ESF values of
peptide groups in the unfolded peptide, which are sensitive both to
backbone conformation and to the access of water. Neither is known
accurately at present. Until recently, unfolded peptides were assumed
to adopt the "random coil" conformation of polymer chemistry, in
which there is no preferred backbone conformation and the peptide chain
follows a random flight description. However, a recent NMR study of a
7-residue alanine sequence by Neville Kallenbach and co-workers (39)
showed surprisingly that the alanine sequence has predominantly the
polyproline II backbone conformation. Moreover, the tendency of the
unfolded peptide to bend back on itself is important in determining the
ESF values of the peptide groups, and little is known about this. I
recently compared predicted and observed values for the
H
of alanine helix formation, based on ESF values, and concluded that
they agree within error (30), but the uncertainty associated with ESF
values in the unfolded peptide is uncomfortably large.
Some ESF values of a peptide group in structures representative of characteristic stages of protein folding are given for an all alanine peptide in Table I. These ESF values and structures may also be used to consider the process of alanine helix formation. Note that the major uncertainty in representing alanine helix formation lies in the unknown structure of the "unfolded" peptide.
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A Paradigm Shift from the H-bond Inventory to Electrostatic Solvation |
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Adoption of the electrostatic approach to solvation by protein chemists will require a major change in their thinking about peptide H-bonds. The traditional view has been governed by the assumptions of the H-bond inventory in which the H-bonded peptide group does not interact with water, and the net energy of the peptide H-bond depends only on its geometry, not on its exposure to water.
Pressure for change in this traditional view has been building for some time. In 1979 Shneior Lifson and co-workers in their pioneering development of a molecular force field found that the peptide H-bond is represented to a good first approximation by placing dipoles on the peptide NH and CO groups (40) (see Ref. 41 for a recent treatment). If the peptide H-bond can be represented by peptide dipoles, then it is logical to use an electrostatic approach to analyze the solvation free energy associated with the dipoles. In his 1990 review of "dominant forces in protein folding," Ken Dill observed that "transferring a hydrogen bond into a nonpolar medium is generally disfavored" (42), and he gives references to earlier work on the subject. In a 1991 critique of the H-bond inventory, Ben-Naim (31) also discusses the interaction of water with H-bonded polar groups. The desolvation penalty for burying a peptide H-bond was reviewed in 1995 (43) and again later (9) by Barry Honig and co-workers. Modern work on the electrostatic approach to peptide solvation relies particularly on Honig's framework for calculating electrostatic solvation free energy (32) by an algorithm applicable to peptides and proteins.
My own interest in the electrostatic solvation approach was aroused in
1999 with the observation by Peizhi Luo (44) that helix melting curves
of peptides differing by only one nonpolar amino acid cross each other
at high temperatures. His results indicate that differences in helix
propensity among the nonpolar amino acids must be chiefly enthalpic,
rather than being entropic as believed. Luo's observation can be
explained if nonpolar amino acids with bulky side chains reduce the
access of water to the helix backbone (29), as argued earlier by Franc
Avbelj and John Moult (45) and elaborated on later by Franc Avbelj
(46). A change in solvation of the peptide CO group, when leucine is
substituted by alanine, cannot be detected by Fourier-transform
infrared measurements (47), however. Avbelj and I found that
-structure propensities, like helix propensities, depend on backbone
solvation as judged by a strong correlation between ESF values (37) and
unfolding free energies (48) for mutants of a zinc finger protein.
Scientists who make electrostatic calculations on proteins generally
agree there is a desolvation penalty when polar groups are wholly or partly buried in proteins. Time will tell whether protein chemists will
accept that there is a desolvation penalty for burial of peptide
H-bonds.
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Historical Footnote |
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I have been asked how I first became interested in this
problem. My interest goes back a long time. John Schellman and I became close friends in the 1950s when I was at the University of Wisconsin and he was at the University of Minnesota. As I studied John's classic
paper (3) predicting the factors that should control the stability of a
peptide helix in water, I often pondered the question: is the -helix
stable in water or not? When our laboratory took up the study of the
mechanism of protein folding in 1970, a central question was whether
the peptide H-bond is sufficient to stabilize an isolated secondary
structure such as an
-helix. In 1970 it was commonly believed that
the answer was known, namely that the peptide H-bond cannot by itself
stabilize a helix in water because studies of peptides from helical
segments of myoglobin (49) and staphylococcal nuclease (50) failed to
detect any helix formation. Moreover, helix propensities and helix
nucleation constants measured in a host-guest system (51), using a
non-natural amino acid as the host (hydroxypropyl- or
hydroxybutyl-L-glutamine), gave results indicating that the
peptide H-bond will not stabilize a short peptide helix in water. There
was, however, a different result reported in 1971 by James Brown and
Werner Klee (52), who found marginal helix formation (but only near
0 °C) for the N-terminal helix of ribonuclease A studied in the
"C-peptide," which contains the first 13 residues of RNase A. That
the C-peptide does indeed form some helix near 0 °C was confirmed in
1982 by Andrzej Bierzynski and Peter Kim in our laboratory (53), and we
then began a search for the factors stabilizing this anomalous C-peptide helix. Today many peptides from helical segments of proteins
are known to show some helix formation in water.
The search for the origin of helix stability in C-peptide, which was
begun both in our laboratory and that of Professor Manuel Rico in
Madrid, uncovered one interaction that had been seen in the structure
of RNase S (the
Glu-2···Arg-10+
salt bridge) and another interaction that was visible in the RNase S
structure but not recognized (the
Phe-8···His 12+
amino-aromatic interaction). The search also found the interaction between the helix dipole and a charged group at either end of the
helix, which can be either stabilizing or destabilizing. Finally in
1989 the search led to the discovery by Susan Marqusee (17) that
alanine by itself forms a stable peptide helix, although no other
natural amino acid has this ability. Therefore, the peptide H-bond must
be sufficient to stabilize a helix in water, because very little
nonpolar surface area is buried in an alanine helix. The stabilizing
effect of the peptide H-bond is still disputed, and it has been
proposed (28) that the charged residues needed to solubilize an alanine
helix are instead responsible for its stability, although uninterrupted
alanine sequences as long as 9 residues (44) or even 13 residues (20)
are found to have the high helix propensity characteristic of alanine.
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ACKNOWLEDGEMENTS |
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I thank Franc Avbelj, John Brauman, Bob Lehman, and an anonymous reviewer for discussion.
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FOOTNOTES |
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Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.X200009200
Address correspondence to: bbaldwin{at}cmgm.stanford.edu.
1 G. Goch, M. Maciejczyk, M. Oleszczuk, D. Stachowiak, J. Malicka, and A. Bierzynski, submitted for publication.
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