Peptides adsorbed on hydrophobic surfaces - A sum frequency generation vibrational spectroscopy and modeling study

Article abstract of DOI:10.1560/IJC.47.1.51

Sum frequency generation (SFG) vibrational spectroscopy has been used to characterize the interfacial structure of a series of model peptides at the hydrophobic polystyrene-buffer interface. The peptides contain two types of amino acids, one hydrophobic (X) and one hydrophilic (Y). Their sequences are Ac-XYYXXYXXYYXXYX-NH₂ (XY₁₄) and Ac-XYXYXYX-NH₂ (XY₇), respectively, where the X and Y combinations are: leucine (L) and lysine (K); alanine (A) and lysine (K); alanine (A) and arginine (R); and phenylalanine (F) and arginine (R), respectively. One additional peptide was synthesized and characterized, Ac-LKKLLKL-NH₂, referred to as LK ₇ α. The XY₁₄ peptides showed SFG spectra that were characteristic of the hydrophobic (X) amino acid of the peptide. Comparison with the 7-amino acid peptides shows that the molecular orientation of alanine is more sensitive to changes in sequence and chain length than leucine or phenyl-alanine. The hydrophilic amino acids are not observed in the SFG spectra of these peptides at the hydrophobic polystyrene interface (with the possible exception of the AR₇ peptide), suggesting the hydrophilic amino acids studied here have a random orientation at this interface. The results of these studies are put into the context of recent SFG studies of proteins adsorbed onto hydrophobic surfaces. Furthermore, our approach to theoretical understanding of interfacial peptide structure is outlined. The results of a molecular dynamics simulation of the LK₁₄ peptide on a hydrophobic interface are presented and discussed.

Full text of DOI:10.1560/IJC.47.1.51

Peptides Adsorbed on Hydrophobic Surfaces—A Sum Frequency Generation
Vibrational Spectroscopy and Modeling Study
RogeR L. YoRk, WiLLiam k. BRoWne, PhiLLiP L. geissLeR, and gaBoR a. somoRjai*
Department of Chemistry, University of California, Berkeley and Material Science Division,
Lawrence Berkeley National Laboratories, Berkeley, California 94720, USA
(Received 19 December 2006)
Abstract. Sum frequency generation (SFG) vibrational spectroscopy has been
used to characterize the interfacial structure of a series of model peptides at the
hydrophobic polystyrene–buffer interface. The peptides contain two types of
amino acids, one hydrophobic (X) and one hydrophilic (Y). Their sequences are
Ac-XYYXXYXXYYXXYX-NH2 (XY14) and Ac-XYXYXYX-NH2 (XY7), respec-
tively, where the X and Y combinations are: leucine (L) and lysine (K); alanine (A)
and lysine (K); alanine (A) and arginine (R); and phenylalanine (F) and arginine
(R), respectively. One additional peptide was synthesized and characterized, Ac-
LKKLLKL-NH2, referred to as LK7 α. The XY14 peptides showed SFG spectra that
were characteristic of the hydrophobic (X) amino acid of the peptide. Comparison
with the 7-amino acid peptides shows that the molecular orientation of alanine is
more sensitive to changes in sequence and chain length than leucine or phenyl-
alanine. The hydrophilic amino acids are not observed in the SFG spectra of these
peptides at the hydrophobic polystyrene interface (with the possible exception of the
AR7 peptide), suggesting the hydrophilic amino acids studied here have a random
orientation at this interface. The results of these studies are put into the context of
recent SFG studies of proteins adsorbed onto hydrophobic surfaces. Furthermore,
our approach to theoretical understanding of interfacial peptide structure is outlined.
The results of a molecular dynamics simulation of the LK14 peptide on a hydropho-
bic interface are presented and discussed.
IntroductIon
ExpErImEntAl
The molecular understanding of protein adsorption
on biopolymer surfaces provides the opportunity to de-
velop biocompatible implant systems or prevent unfa-
vorable protein adsorption from the blood onto medical
devices, which alters their biological and immunologi-
Peptide Synthesis
Peptides were synthesized using Rink amide 4-methyl-
benz-hydrylamine (MBHA) resin (Novabiochem) and stan-
dard 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry on an
ABI 431A synthesizer (Applied Biosystems). An excess of
cal responses.1,2 Unfortunately, adsorbed proteins are reagents (9 equivalents) was used in each step, beginning with
approximately 200 mg of MBHA.Acetic anhydride was added
to the resin-bound peptide prior to peptide cleavage off the
resin to acetylate the N-terminus of the peptide. The purity and
composition of the peptides were characterized using Liquid
Chromatography-Electrospray Ionization-Mass Spectrometry
(LC-ESI-MS). Peptides were precipitated in cold methyl-tert-
butylether and lyophilized.4
Two different sequences of peptides were synthesized.6
One had sequence Ac-XYYXXYXXYYXXYX-NH2 and is
*Author to whom correspondence should be addressed.
E-mail: somorjai@berkeley.edu
difficult molecules to study with vibrational spectros-
copy on a molecular level due to their size and structural
complexity.3 Therefore, we have turned to a class of
small, model peptides that contain two types of amino
acids, one hydrophilic and the other hydrophobic.4–7
In this paper we will review their adsorption onto hy-
drophobic surfaces studied experimentally by sum fre-
quency generation (SFG) vibrational spectroscopy and
outline recent theoretical advances in the understanding
of these experiments.
Israel Journal of Chemistry
Vol. 47
2007
pp. 51–58

52
referred to as XY14. The other sequence was Ac-XYXYXYX- are measured in a sum frequency experiment: a mode must be
NH2 and is referred to as XY7 β or simply XY7. There were IR and Raman active, and it must be ordered (i.e., a mode that
four XY combinations synthesized in this study: leucine (L) has a random geometrical distribution does not produce a reso-
and lysine (K), alanine (A) and lysine (K), alanine (A) and nant response, as seen by the integral in eq 2). Thus the physi-
arginine (R), and phenylalanine (F) and arginine (R). One cal interpretation of the SFG spectra presented here becomes
additional peptide was synthesized: Ac-LKKLLKL-NH2, clear: due to specific interactions between the peptide and
referred to as LK7 α. The LK, AK, AR, and FR peptides had surface (i.e., hydrophobic or electrostatic) certain vibrational
different solution solubilities, which decreased in the order modes will become ordered at an interface, and those modes
AR~AK>LK>FR. The concentrations used in this study are are seen in the SFG spectra.4,6,7
given in Table 1.
Sum Frequency Generation Vibrational Spectroscopy
Our experimental laser setup, substrate preparation, buf-
fer salt concentration, and experimental geometry have been
described in detail elsewhere.4,8 Deuterated d8-polystyrene is
used in this study to avoid spectral confustion between the
substrate and adsorbates. The theory of sum frequency gen-
eration has been presented elsewhere.4,6,9,10 Our spectra are fit
to the equation:
rEsults
The SFG spectra of the 14-amino acid peptides adsorbed
on deuterated d8-polystyrene are shown in Figs. 1–4.6
The solid lines in each figure are the results of fitting
the spectra to eq 1. Peptide modes are visible for all of
the peptides adsorbed on hydrophobic polystyrene sur-
faces. As we have previously demonstrated, LK14 shows
three distinct modes on PS-d8: 2870 cm–1, assigned to
a methyl symmetric stretch; 2895 cm–1, assigned to a
CH stretch or CH2 Fermi resonance; and 2935 cm–1, as-
signed to a Fermi resonance of a methyl mode (Fig. 1).4
The FR14 peptide shows a strong resonance at 3050 cm–1,
assigned to the phenyl stretch of the phenylalanine side
chains (Fig. 2).6 The AR14 shows similar, yet slightly
different results from the LK14 peptide: 2870 cm–1, as-
signed to symmetric stretch of the methyl side chain of
alanine and the 2925 cm–1 mode, assigned to a Fermi
resonance of the methyl side chain of alanine (Fig. 3).6
The SFG spectrum of the AK14 peptide is similar to
AR14, with the two modes observed at 2870 cm–1 and
2930 cm–1 (Fig. 4).6
2
Aq
2 = |]N2Rg +/
]
|
g
2
(1)
~
IRh
^
~IR - ~q + iCq
q
]
g
2
|
where
is the non-resonant part of the surface nonlinear
NR
susceptibility, χ(2); ωIR is the frequency of the infrared beam;
and ωq and Γq are the frequency and damping constant of the
qth vibrational mode, respectively. The strength of a vibra-
tional mode, Aq, is given by:
(2)
] g
q f X dX
#
=
Aq Ns
a
where Ns is the surface density of molecules, aq is the ampli-
tude of a molecular vibration, f( ) is an orientation distribu-
X
tion function over , a set of orientational angles that de-
X
scribes a transformation between the laboratory and molecular
coordinate system.11 aq can be understood as:
The SFG spectra of the 7-amino acid (β-strand
sequences) peptides adsorbed on d8-polystyrene are
shown in Figs. 5–8.6 The SFG spectra of LK7 show
three resonances: 2870 cm–1, again a symmetric stretch
of a methyl group; 2910 cm–1, CH stretch or CH2 Fermi
resonance; and 2935 cm–1, a methyl Fermi resonance
(Fig. 5). A peak at 3050 cm–1 in the SFG spectra in FR7
g
2a]1
2n
aq ∝
(3)
7
2Qq 2Qq
2a]1 /2Qq
g
where
tive and Raman polarizability derivative with respect to Qq,
and
are the infrared dipole deriva-
2n/2Qq
the classical normal coordinate of the qth vibrational mode,
respectively. The previous equations demonstrate what modes is attributed to the phenyl ring of phenylalanine (Fig. 6).
Table 1. Peptide sequences and concentrations used in this study
peptide
sequence
solution
concentration
LK14
AK14
AR14
FR14
LK7 β
AK7
Ac-LKKLLKLLKKLLKL-NH2
Ac-AKKAAKAAKKAAKA-NH2
Ac-ARRAARAARRAARA-NH2
Ac-FRRFFRFFRRFFRF-NH2
Ac-LKLKLKL-NH2
0.1 mg/mL
1 mg/mL
1.5 mg/mL
0.05 mg/mL
0.05 mg/mL
1 mg/mL
Ac-AKAKAKA-NH2
AR7
FR7
LK7 a
Ac-ARARARA-NH2
Ac-FRFRFRF-NH2
Ac-LKKLLKL-NH2
1 mg/mL
0.05 mg/mL
0.05 mg/mL
Israel Journal of Chemistry
47
2007

53
Fig. 1. SFG spectra of LK14 adsorbed on hydrophobic deu- Fig. 3. SFG spectra of AR14 adsorbed on hydrophobic deu-
terated polystyrene. The spectra show three modes from the terated polystyrene. The spectra show two modes from the
hydrophobic leucine side chains: 2870 cm–1, a symmetric CH3 hydrophobic alanine side chains: 2870 cm–1, a symmetric CH3
stretch; 2895 cm–1, a CH stretch or CH2 Fermi resonance; and stretch, and 2930 cm–1, a CH3 Fermi resonance.
2935 cm–1, a CH3 Fermi resonance.
Fig. 2. SFG spectra of FR14 adsorbed on hydrophobic deuter- Fig. 4. SFG spectra of AK14 adsorbed on hydrophobic deu-
ated polystyrene. The spectra show one mode from the hydro- terated polystyrene. The spectra show two modes from the
phobic phenylalanine side chains. This mode at 3050 cm–1 is hydrophobic alanine side chains: 2870 cm–1, a symmetric CH3
assigned to the υ2 mode of the phenyl ring.
stretch, and 2925 cm–1, a CH3 Fermi resonance.
York et al. / SFG of Adsorbed Peptides

54
Fig. 5. SFG spectra of LK7 β adsorbed on hydrophobic deu- Fig. 7. SFG spectra of AR7 adsorbed on hydrophobic deuter-
terated polystyrene. The spectra show three modes from the ated polystyrene. The spectra show one mode from the peptide
hydrophobic leucine side chains: 2870 cm–1, a symmetric CH3 at 3030 cm–1. The origin of this mode is not certain, but could
stretch; 2910 cm–1, a CH stretch or CH2 Fermi resonance; and be assigned to the arginine side chain (see text for details).
2935 cm–1, a CH3 Fermi resonance.
Fig. 6. SFG spectra of FR7 adsorbed on hydrophobic deuter- Fig. 8. SFG spectra ofAK7 adsorbed on hydrophobic deuterat-
ated polystyrene. The spectra show one mode from the peptide ed polystyrene. The spectra show no modes from the adsorbed
side chains at 3050 cm–1. This mode at 3050 cm–1 is assigned to peptide itself; rather, interfacial water is observed. The spectra
the υ2 mode of the phenyl ring of phenylalanine.
here is fit with two modes at ~3200 cm–1 and ~ 3400 cm–1.
Israel Journal of Chemistry
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55
The SFG spectra of AR7 do not show modes associ-
ated with alanine, but a strong CH stretching mode at
3030 cm–1 (Fig. 7). Previous work of Larsson has as-
signed a CH mode at 3030 cm–1 to the arginine side
chain.12,13 In contrast, there are no peptide modes visible
in the SFG spectra of AK7 (Fig. 8). Figure 9 shows the
SFG spectra of the 7-amino acid LK a peptide, with
sequence Ac-LKKLLKL-NH2. The two LK7 spectra
have three similar modes: 2870 cm–1, assigned to the
CH3 symmetric stretch; a mode around 2900 cm–1, as-
signed to a CH resonance or CH2 Fermi resonance; and
2935 cm–1, assigned to a Fermi resonance of a methyl
group.6
dIscussIon
In this section, we will briefly discuss the historical back-
ground of SFG studies on biomolecules at interfaces,
then put the results outlined above into the context of
these studies. We will subsequently explain how theoret-
ical studies can aid in the interpretation of these spectra.
Fig. 9. SFG spectra of LK7 a adsorbed on hydrophobic deu-
terated polystyrene. The spectra show three modes from the
hydrophobic leucine side chains: 2870 cm–1, a symmetric CH3
stretch; 2900 cm–1, a CH stretch or CH2 Fermi resonance; and
2935 cm–1, a CH3 Fermi resonance.
SFG Studies on Biomolecules: From Proteins to
Peptides
SFG was first demonstrated as a surface-specific
vibrational spectroscopy in 1987 by Shen and cowork- of various proteins adsorbing onto various surfaces and
ers.14,15 There are a number of recent reviews of this have demonstrated how a number of parameters (e.g.,
technique and the many interfaces studied by it in the surface hydrophobicity) influence protein interfacial
literature,16–18 and we will not attempt to give a com- structure, with much of the work done by the Chen
prehensive review here. The first study of proteins at group.20,21 Reviews are now in the literature concerning
interfaces was performed by the Cremer group, which the application of SFG to biological molecules at inter-
examined bovine serum albumin adsorption on silica.19 faces.22–24 However, determining the orientation of indi-
Since then, a number of groups have performed studies vidual amino acids has proven difficult due to the large
Fig. 10. An LK14 peptide adsorbed at the surface of a SiO2 surface with no partial charge separation. The peptide’s initial con-
figuration was alpha helical, persisting for several picoseconds of simulation time. This conformation remains for simulation
times up to 1 ns.
York et al. / SFG of Adsorbed Peptides

56
number and types of amino acids in a protein.3 Clearly, from the AR and AK experiments reveal that the aver-
a study of individual amino acids at hydrophobic and age orientation of alanine is sensitive to the sequence
hydrophilic surfaces would be an ideal method to eluci- and chain length of a peptide.
date the interfacial structure of amino acids. However,
The use of the model peptides described here allows
this has proven somewhat difficult at the solid/liquid for a molecular-level understanding of how individual
interface,5 although amino acids have been well char- amino acid residues order at hydrophobic interfaces.
acterized at the carbon tetrachloride/water interface.25 The results presented here demonstrate that the orienta-
Therefore, we have turned to a series of short, model tions of the most hydrophobic amino acids studied here
peptides that contain two types of amino acids in order (phenylalanine and leucine) are insensitive to peptide
to elucidate how individual amino acids change orienta- chain length and sequence. However, the more hydro-
tion at hydrophobic surfaces as a function of the peptide philic alanine residues’ orientation is much more sensi-
chain length and sequence. Our studies demonstrate that tive to the sequence and chain length of a peptide. The
the molecular orientation of alanine is more sensitive most hydrophilic side chains are not observed in the
to peptide chain length and sequence than the more hy- SFG spectra presented here (with the possible excep-
drophic leucine or phenylalanine amino acids.6
tion of the AR7 peptide), suggesting that the hydrophilic
The 14 amino acid peptides adsorbed on hydrophobic residues of a peptide have random orientation at a hy-
polystyrene studied here provide characteristic spectro- drophobic surfaces.
scopic signatures of the hydrophobic amino acid con-
tained in the peptide. The LK14 (Fig. 1) and FR14 (Fig. 2) Theoretical Models of Peptide Adsorption on
SFG spectra are nearly identical to the spectra observed Hydrophobic and Hydrophilic Surfaces
for leucine and phenylalanine at the carbon tetrachloride/
Although SFG reports directly on orientational sym-
water interface, respectively.25 The LK14 SFG spectra are metry-breaking near interfaces, inferring microscopic
also very similar to the SFG spectra of leucine at the air/ structure from its frequency dependence is not straight-
water interface.26 A comparison with the work of Kim et forward. Indeed, for solutes that are sensitive to rear-
al.,27 who studied polyalanine at the air/water interface, rangements in water’s hydrogen bond network, even
shows that the SFG spectra of AR14 (Fig. 3) and AK14 linear spectra can be difficult to interpret. One need look
(Fig. 4) from this study are characteristic of the alanine no farther than Raman scattering by HOD in D2O to find
side chains. This is further supported by the observation long-standing debates over the molecular implications
that the AR14 and AK14 spectra are nearly identical.
of shifts in frequency and intensity.28 Recent theoretical
The 7-amino acid peptides (β sequence) show some calculations have offered new insight into those trends
interesting trends.6 The LK7 peptide (Fig. 5) shows and have provided a microscopic basis for understand-
the three characteristic peaks associated with leucine. ing their physical origins.28–30 Computer simulations
The observation that intensity ratios between the CH3 promise to become a similarly powerful counterpart to
symmetric mode and CH3 Fermi resonance mode are SFG, but theoretical developments necessary to realize
slightly different for the LK7 (Fig. 5) than for the LK14 this potential have not been fully developed.
(Fig. 1) suggests that the leucine side chains in each
Practical strategies for calculating the response
peptide adopt slightly different orientations at the poly- functions that determine SFG have been devised and
styrene interface. The LK7 α (Fig. 9) also shows these demonstrated.31 They require not only a scheme for
three peaks, again with the intensity ratio between the determining instantaneous oscillator frequencies but
CH3 symmetric mode and CH3 Fermi resonance being also numerical integrations weighted by complicated
slightly different than the LK7 β. The SFG spectra of the orientational factors. These convolutions, together with
FR7 peptide (Fig. 6) are nearly identical to FR14 peptide interference of resonant and non-resonant contribu-
(Fig. 2), indicating that the orientation of phenylalanine tions, obscure any simple connection between spectral
is not sensitive to changes in peptide sequence or chain intensities and the distribution of microscopic environ-
length. The peptides containing alanine show the op- ments. Understanding of bulk vibrational spectra has
posite effect. The AK7 peptide (Fig. 8) shows no modes benefited greatly from such connections, which draw
in SFG spectra from the peptide, and the AR7 spectra special attention to electric fields generated by aqueous
(Fig. 7) are completely different than the AR14 (Fig. 3). surroundings.32–34 Lacking a direct correspondence, one
The two characteristic modes of alanine observed in can nonetheless calculate SFG spectra for complex sys-
AR14 are not present in the SFG spectra ofAR7; instead a tems, provided a representative ensemble of molecular
mode at 3030 cm–1 is observed. This mode is postulated configurations. Agreement with experiment provides a
to originate from the side chains of arginine.6 Regard- measure of confidence in the intermolecular structures
less of the exact origin of this mode, the results obtained generated by simulation.
Israel Journal of Chemistry
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2007

57
Competing requirements of physical realism and single LK14 peptide situated above a hydrophobic sub-
computational tractability heavily constrain the choice strate. SFG data suggest that helical ordering should not
of microscopic models useful for describing polypep- be observed in this case,7 and our simulations concur.
tide adsorption. An empirical force field must account Initial conditions in which the peptide adopts perfect
for conformational variability, fluctuations in solvent helical structure rapidly evolve toward disordered con-
density that underlie the hydrophobic effect, and in- formations that associate strongly with the substrate.
trinsic interfacial affinity of individual amino acids. Secondary structure all but disappears within tens of
Two distinct approaches can be pursued toward this picoseconds and does not reappear within the nanosec-
end. A minimalist view would depict the polypeptide ond timescale of our calculation. This preliminary result
as a connected chain with varying degrees of hydrogen does not, however, unambiguously implicate the sub-
bonding potential along its backbone. Solvent would be strate in loss of helical order, since LK14 does not exhibit
represented by a smoothly varying density field capable strong helicity in bulk solution at low concentrations
of drying at hydrophobic surfaces. The substrate itself and low ionic strength.7,35 In order to clarify the role of
would be caricatured as uniformly hydrophobic or hy- surface, we are currently investigating effects of salt, in
drophilic, or as a statistically patterned mixture of the the form of a phosphate buffer known to stabilize sec-
two. This kind of model is ideal for posing general ques- ondary structure in bulk, as well as interactions among
tions about the role of hydrophobicity in polymer ad- multiple peptide solutes at the surface.
sorption, for investigating correlations among multiple
solutes, and for exploring broad ranges of parameters
conclusIon
and molecular compositions. Making contact with ex-
SFG has been used to characterize the adsorbed struc-
periment, however, is made difficult by coarse-graining
ture of a class of model peptides. The results show
over local structures that might influence spectroscopic
that on hydrophobic polystyrene surfaces, side chain
probes. Furthermore, mimicking experimental condi-
vibrational modes are observed in the SFG spectra of
tions requires simultaneous parameterization of many
these peptides. The modes observed for the 14-amino
effective interaction strengths.
acid peptides are characteristic of the hydrophobic side
As a first step we have pursued an alternate route,
chains of the peptide. With the possible exception of
which attempts a more detailed representation of molec-
AR7, no hydrophilic amino acid side chains are ob-
ular arrangements. Specifically, we model the motions
served in the SFG spectra. Additionally, the molecular
of all atoms in the peptide and solvent, as determined
orientations of phenylalanine and leucine are less sensi-
by Newton’s equations of motion and the empirical
tive to peptide sequence and chain length than alanine.
AMBER 94 force field. The substrate appears as a fixed
Our approach to achieving a molecular level theoretical
external field, which we constructed from the atomic
understanding of the experiments presented here has
structure of a quartz sheet with partial charges assigned
been outlined. Finally, our initial results of the interfa-
to each Si and O atom. By varying these partial charges,
cial structure of the LK14 peptide have been presented.
we can vary the substrate continuously between hydro-
Acknowledgments. Professor Levine is the experimentalist’s
dream of a theorist; he listens to experimental details and his
involvement in extracting the key concepts of an experiment
and then developing models to describe the phenomena permit
the creation of new science. It has been my privilege to interact
with him in the field of surface science throughout the years.
This work was supported by the Director, Office of Sci-
ence, Office of Advanced Scientific Computing Research,
Office of Basic Energy Sciences, Materials Science and En-
gineering Division, of the U.S. Department of Energy under
Contract No. DE-AC02-05CH11231. The authors would like
phobic and hydrophilic extremes.
Proper treatment of the hydrophobic effect near ex-
tended interfaces requires a careful choice of simula-
tion geometry. In particular, boundary conditions that
suppress fluctuations in net solvent density can inhibit
formation of a vapor-like layer above hydrophobic sub-
strates. This kind of bias can be avoided in several ways,
for example by sampling an open ensemble that permits
exchange of molecules and/or volume with an external
bath. Because the dynamics of such exchange is artifi-
cial in most protocols, we chose instead to enforce con- to thank Dr. David King and the HHMI Mass Spectrometry
Facility (UC Berkeley) for assistance with peptide synthesis.
ditions of liquid–vapor coexistence by placing an ideal
hydrophobic sheet and a thin layer of vapor on the top
surface of our simulation cell (opposite the substrate).
Integrating molecular equations of motion for this sys-
tem does not curtail the natural range of local density
variations, even though its total volume is fixed.
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