Structure and Orientation of Interfacial Proteins Determined by Sum Frequency Generation Vibrational Spectroscopy

CHAPTER SEVEN

Structure and Orientation of Interfacial Proteins Determined by Sum Frequency Generation Vibrational Spectroscopy: Method and Application Shuji Ye*,†,1, Feng Wei*,†, Hongchun Li*,†, Kangzhen Tian*,†, Yi Luo*,† *Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, PR China † Department of Chemical Physics, University of Science and Technology of China, Hefei, PR China 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Theoretical Background of SFG-VS 3. Orientation Determination of Interfacial Protein Structures 3.1 Calculation method for Raman polarizability tensor 3.2 Calculation method for IR dipole moments 3.3 Orientation analysis methods for the interfacial proteins using SFG amide I signal 4. Recent Progresses on the Determination of Protein Molecular Structure and Orientation at Different Interfaces 4.1 The study of a-helix 4.2 The study of 310-helix 4.3 The study of antiparallel b-sheet 4.4 The study of parallel b-sheet 5. Summary Acknowledgments Appendix A: Euler Transformation Between Different Coordinate Systems Appendix B: Macroscopic (x, y, z) Coordinates of Different Secondary Structures Appendix C: The Full Name of Lipid Molecules References

214 217 219 221 227 233 238 239 243 245 247 248 249 249 250 251 251

Abstract In situ and real-time characterization of molecular structures and orientation of proteins at interfaces is essential to understand the nature of interfacial protein interaction. Such work will undoubtedly provide important clues to control biointerface in a desired

Advances in Protein Chemistry and Structural Biology, Volume 93 ISSN 1876-1623 http://dx.doi.org/10.1016/B978-0-12-416596-0.00007-5

#

2013 Elsevier Inc. All rights reserved.

213

214

Shuji Ye et al.

manner. Sum frequency generation vibrational spectroscopy (SFG-VS) has been demonstrated to be a powerful technique to study the interfacial structures and interactions at the molecular level. This paper first systematically introduced the methods for the calculation of the Raman polarizability tensor, infrared transition dipole moment, and SFG molecular hyperpolarizability tensor elements of proteins/peptides with the secondary structures of a-helix, 310-helix, antiparallel b-sheet, and parallel b-sheet, as well as the methodology to determine the orientation of interfacial protein secondary structures using SFG amide I spectra. After that, recent progresses on the determination of protein structure and orientation at different interfaces by SFG-VS were then reviewed, which provides a molecular-level understanding of the structures and interactions of interfacial proteins, specially understanding the nature of driving force behind such interactions. Although this review has focused on analysis of amide I spectra, it will be expected to offer a basic idea for the spectral analysis of amide III SFG signals and other complicated molecular systems such as RNA and DNA.

1. INTRODUCTION When protein molecules touch a surface, they will accumulate at interfaces, which can be both a practical asset and a problem (Garland, Shen, & Zhu, 2012; Szott & Horbett, 2011). Thus, effective and molecular-level control of interfacial protein structures has the potential to advance the development of novel synthetic methods for biomedical coatings and implant devices, biosensors, immunological tests, and drugdelivery schemes with improved performance (Hlady & Buijs, 1996; Ye, Nguyen, Boughton, Mello, & Chen, 2010). Yet, the performance of a biosensor or biochip is greatly affected by the structure and activity of the interfacial proteins used for biological recognition (Ye, Nguyen, Boughton, et al., 2010; Zhu & Snyder, 2003). If the protein-binding domains are randomly oriented or deformed, the selectivity and sensitivity of the sensor will be significantly reduced; uniformly orienting the molecules for optimal binding is thus expected to improve sensor performance (Ye, Nguyen, Boughton, et al., 2010; Zhu & Snyder, 2003). On the other hand, structures of membrane-associated proteins govern their biological functions, such as signal transduction, cellular regulation, immune recognition, and molecular anchoring. The aggregation of these membrane proteins in cell membranes may induce many “protein deposition diseases,” including Alzheimer’s disease, Parkinson’s disease, and prion disease (Chiti & Dobson, 2006; Outeiro, 2011). Therefore, in situ and real-time characterization of molecular structures and orientation of proteins at interfaces is essential to understand the

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

215

nature of interfacial protein interaction, the properties of the interfacial biodevices, and disease mechanisms. Such work will undoubtedly provide important clues to control biointerface in a desired manner. Extensive researches have been performed to gain insight into interfacial protein structures and properties using a number of techniques, such as surface plasmon resonance (Yano, 2012), nuclear magnetic resonance (NMR) (Judge & Watts, 2011), atomic force microscopy (Fotiadis, 2012), and so on. The applications of these techniques (and others) to the study of the interactions between proteins and surface can be found in previous review articles (Fotiadis, 2012; Judge & Watts, 2011; Yano, 2012). Even though some molecular-level information can be acquired by probing the interactions between proteins and surface using these analytical tools, further details regarding such molecular structure and orientation at interfaces need to be elucidated in situ and real time. To this end, sum frequency generation vibrational spectroscopy (SFGVS) has been developed into a powerful and versatile technique to study interfacial molecular structures at the molecular-level in situ in different chemical environments. As a polarized vibrational spectroscopy, SFG-VS permits the identification of interfacial molecular species (or chemical groups) and also provides information about the interfacial structures, such as the orientation and the orientation distribution of functional groups on a surface or at an interface in situ in real time (Castellana & Cremer, 2006; Gopalakrishnan, Liu, Allen, Kuo, & Shultz, 2006; Lambert, Davies, & Neivandt, 2005; Shen, 1984; Tian, Li, & Ye, 2013; Wang, Gan, Lu, Rao, & Wu, 2005; Wei & Ye, 2012). SFG-VS has been applied to study the structure and orientation of various biomolecules (including peptides and proteins) in modeling membrane or other interfacial environments (Chen, 2012; Chen & Chen, 2006; Chen, Clarke, Wang, & Chen, 2005; Fu, Wang, & Yan, 2011; Liu, Jasensky, & Chen, 2012; Liu, Monson, Yang, Pace, & Cremer, 2011; Ye, Nguyen, Clair, & Chen, 2009). For example, Chen group has successfully determined the interfacial structures of membrane-bounded peptides and proteins such as magainin 2, melittin, cecropin P1 (CP1c), and G protein (Chen, 2012; Chen & Chen, 2006; Chen, Clarke, et al., 2005; Liu et al., 2012; Ye et al., 2009). Yan group used SFG-VS to characterize the secondary structures of a-helix, b-sheet, and random-coil at interfaces (Fu, Liu, & Yan, 2011). Bonn group investigated the inhibition efficiency of amyloid formation at phospholipid interfaces by polyphenol EGCG inhibitors (Engel et al., 2012). Castner group probed the orientation and conformation of a-helix and b-strand model peptides on

216

Shuji Ye et al.

different surfaces using SFG, NEXAFS, and NMR spectroscopy (Weidner, Apte, Gamble, & Castner, 2010; Weidner, Breen, Li, Drobny, & Castner, 2010). Our group monitored the molecular action of the peptides such as alamethicin, prion, and mastoparan (MP) at lipid bilayers (Li, Ye, Wei, Ma, & Luo, 2012; Wei, Ye, Li, & Luo, 2013; Ye et al., 2012). Since several review papers have been published to summarize details about SFG studies on elucidating molecular-level information of interfacial peptides/proteins involved in different biological environments (Chen, 2012; Chen & Chen, 2006; Chen, Clarke, et al., 2005; Fu, Wang, et al., 2011; Liu et al., 2012; Ye et al., 2009), in this paper, we will focus on the method for determining molecular structure and orientation of interfacial protein using SFG-VS, as well as introduce some examples for the application of this technique. SFG signal from interfacial molecules depends on a property known as the SFG susceptibility tensor elements w(2) ijk (i, j, k ¼ x, y, z) (Castellana & Cremer, 2006; Gopalakrishnan et al., 2006; Lambert et al., 2005; Shen, 1984; Wang, Gan, et al., 2005). Molecular orientation information can be obtained by relating w(2) ijk (i, j, k ¼ x, y, z) to the numerical density of interfacial molecules (Ns) and molecular hyperpolarizability tensor elements blmn(l, m, n ¼ a, b, c) via an Euler transformation (Eq. (7.1)) (Moad & Simpson, 2004; Shen, 1984). blmn(l, m, n ¼ a, b, c) is a tensor product of the
 @mn and the Raman polarizability infrared (IR) transition dipole moment @Q q
 lm tensor @a @Qq (Eq. (7.2), marked as alm below). If either the change in polarizability or the change in dipole moment of a vibrational mode is equal to zero, then no SFG signal will be observed, which requires the vibrational modes observed in SFG are both IR and Raman active. Therefore, to determine the orientation of interfacial protein and accurately analyze the SFG spectra, we need to obtain the information of hyperpolarizability of blmn(l, m, n ¼ a, b, c). X   ð2Þ Ns Ril Rjm Rkn blmn,q ð7:1Þ wijk,q ¼ l,m,n @alm @mn ð7:2Þ blmn,q ∝ @Qq @Qq In this paper, we will first present a brief introduction of the theoretical background needed to understand SFG, and then summarize recent studies on the calculation methods of hyperpolarizability as well as the orientation

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

217

Figure 7.1 Molecular structures of (A) right-handed a-helix, (B) antiparallel b-sheet, and (C) parallel b-sheet. The molecular axes used in the below analysis were marked. Antiparallel b-sheet and parallel b-sheet adopt different axes.

analysis methods for the proteins with different secondary structures (a-helix, 310-helix, antiparallel b-sheet, and parallel b-sheet, shown in Fig. 7.1). Finally, we will introduce recent progresses for the application of this technique on characterizing molecular structures and orientation of various interfacial proteins.

2. THEORETICAL BACKGROUND OF SFG-VS SFG-VS is a second-order nonlinear optical laser technique which provides vibrational spectra of surfaces and interfaces. Details regarding SFG theories and instruments have been reported previously (Castellana & Cremer, 2006; Gopalakrishnan et al., 2006; Lambert et al., 2005; Shen, 1984; Wang, Gan, et al., 2005). Currently, two different kinds of SFG systems are used to measure SFG spectra: frequency scanning system or broad-band system (Smith & Hinson-Smith, 2004). Frequency scanning system involves two picosecond pulse laser beams (visible and infrared), while broad-band system involves a picosecond narrow-band visible beam and a femtosecond broad-band IR beam. No matter which system is used, in all the SFG experiments, the pulsed visible beam (with a fixed frequency in the visible frequency range (oVis)) and the IR beam (with a tunable or broad-band frequency in the IR frequency range (oIR)) must be overlapped spatially and temporally on the samples. The SFG signal is generated at the sum frequency of the two input beams by the nonlinear process, oSFG ¼ oVis þ oIR. Therefore, the SFG process can be simply viewed as a

218

Shuji Ye et al.

combination of IR absorbance and Raman scattering. Under the dipole approximation, the intensity of the sum frequency signal is proportional to the intensity of incoming visible laser beam I1(oVis), IR laser beam I2(oIR), and the square of the vibration’s second-order nonlinear susceptibility, which vanished when a material has inversion symmetry (Castellana & Cremer, 2006; Gopalakrishnan et al., 2006; Lambert et al., 2005; Shen, 1984; Wang, Gan, et al., 2005).  2  ð2Þ  I ðoSFG Þ∝weff  I1 ðoVis ÞI2 ðoIR Þ

ð7:3Þ

Therefore, bulk materials that possess inversion symmetry do not generate a sum frequency signal, but surfaces and interfaces, where the symmetry is broken, do generate a sum frequency signal. As the IR beam frequency is tuned over the vibrational resonance of surface/interface molecules, the effective surface nonlinear susceptibility w(2) R can be enhanced. The fre(2) quency dependence of weff is described by ð2Þ

ð2Þ

weff ðoÞ ¼ wNR þ

X q

Aq o  oq þ iGq

ð7:4Þ

where Aq, oq, and Gq are the strength, resonant frequency, and damping coefficient of the vibrational mode q, respectively, and w(2) NR is the nonresonant background (Castellana & Cremer, 2006; Gopalakrishnan et al., 2006; Lambert et al., 2005; Shen, 1984; Wang, Gan, et al., 2005). The plot of SFG signal versus the IR input frequency shows a polarized vibrational spectrum of the surface or interface. The components of w(2) eff can be probed by using different polarization combinations of the input and output laser beams, such as ssp (s-polarized SFG signal, s-polarized visible beam, p-polarized IR beam), sps, spp, psp, and ppp. Each measured polarization combination is related to the different components of w(2) ijk (i, j, k ¼ x, y, z) defined in the lab coordinate system and is given in Eqs. (7.5)–(7.9) (Castellana & Cremer, 2006; Gopalakrishnan et al., 2006; Lambert et al., 2005; Shen, 1984; Wang, Gan, et al., 2005). Here, the lab coordinate is defined as the z-axis being along the surface normal and the x-axis being in the incident plane. ð2Þ

ð7:5Þ

ð2Þ

ð7:6Þ

2Þ weff ,ssp ¼ Lyy ðoSF ÞLyy ðoVis ÞLzz ðoIR Þ sinbIR wðyyz 2Þ weff ,sps ¼ Lyy ðoSF ÞLzz ðoVis ÞLyy ðoIR Þsin bVis wðyzy

219

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

ð2Þ

2Þ weff ,spp ¼ Lyy ðoSF ÞLxx ðoVis ÞLzz ðoIR Þ cosbVis sinbIR wðyxz 2Þ þ Lyy ðoSF ÞLzz ðoVis ÞLxx ðoIR Þsin bVis cosbIR wðyzx ð2Þ

ð7:7Þ

2Þ weff ,psp ¼ Lxx ðoSF ÞLyy ðoVis ÞLzz ðoIR Þ cos bSF sinbIR wðxyz 2Þ þ Lzz ðoSF ÞLyy ðoVis ÞLxx ðoIR Þ sinbSF cos bIR wðzyx

ð7:8Þ

2Þ weff ,ppp ¼ Lxx ðoSF ÞLxx ðoVis ÞLzz ðoIR Þ cos bSF cos bVis sinbIR wðxxz ð2Þ  Lxx ðoSF ÞLzz ðoVis ÞLxx ðoIR Þcos bSF sinbVis cosbIR wxzx 2Þ þ Lzz ðoSF ÞLxx ðoVis ÞLxx ðoIR Þsin bSF cosbVis cosbIR wðzxx ð2Þ þ Lzz ðoSF ÞLzz ðoVis ÞLzz ðoIR Þ sin bSF sin bVis sin bIR wzzz

ð7:9Þ

ð2Þ

where bSF, bVis, and bIR are the angles between the surface normal and the sum frequency beam, the input visible beam, and the input IR beam, respectively. Lii(i ¼ x, y, or z) denotes the Fresnel coefficients and its detailed calculation can be found elsewhere (Castellana & Cremer, 2006; Gopalakrishnan et al., 2006; Lambert et al., 2005; Shen, 1984; Wang, Gan, et al., 2005).

3. ORIENTATION DETERMINATION OF INTERFACIAL PROTEIN STRUCTURES Earlier SFG studies on interfacial proteins structure mainly focused on the CdH stretching frequency region. For example, Cremer et al. investigated the orientation of gramicidin A in a DMPC monolayer using the CdH SFG stretching signals generated from the side chains of gramicidin A (Kim, Gurau, Lim, & Cremer, 2003). However, SFG signals acquired in the CdH stretching frequency region are comprised of signals from protein side chains and other molecules at surface. In addition to the side-chain vibrations, proteins give rise to backbone vibrations, known as the amide vibration (Barth & Zscherp, 2002; Tamm & Tatulian, 1997). All amide frequencies are conformation-sensitive, but the amide I vibration depends on the secondary structure of the backbone and is hardly affected by the nature of the side chains. Different types of secondary structures show different peak centers (Table 7.1). For example, a-helical structure has a peak center at about 1655 cm1, while antiparallel b-sheet structure shows the characteristic peaks at about 1630 and 1685 cm1 (Barth & Zscherp, 2002; Tamm & Tatulian, 1997). Therefore, the amide I vibration is most commonly used for secondary-structure analysis. Chen and his coworkers pioneered to demonstrate the feasibility of detecting SFG amide I signals from interfacial proteins/peptides in 2005

220

Shuji Ye et al.

Table 7.1 Properties of different types of secondary structures of proteins (Nguyen et al., 2009a; Nguyen, King, et al., 2010; Xiao et al., 2012; Tamm & Tatulian, 1997) Secondary Repeating SFG-active Peak center structure residues Symmetry modes (cm1) psp

a-Helix

3.6

C18/5

A, E1

1655

No

310-Helix

3

C3v

A, E1

1635

No

Antiparallel b-sheet

4

D2

B1 B2 B3

1685 1630 1720

Yes Yes Yes

Parallel b-sheet

2

C2

A B

1625 1670

Yes Yes

(Wang, Chen, Clarke, & Chen, 2005). Adopting a near total reflection experimental geometry, they can obtain very strong SFG amide I signals of interfacial proteins. Currently a growing number of research groups apply SFG for interfacial protein studies. As indicated in Eq. (7.2), the SFG hyperpolarizability tensor of a vibrational mode can be deduced if both Raman polarizability tensor and IR transition dipole moment are known. Therefore, it is necessary to introduce the calculation methods of Raman polarizability tensor and IR transition dipole moment before we go further to review the method to determine the orientation of interfacial proteins. Raman polarizability tensor and IR transition dipole moment can be approached by three methods: polarized IR and Raman spectroscopic techniques, bond additivity model, and ab initio calculation. For example, Yan group used tripeptide segments as modeling peptides to deduce Raman polarizability tensor and IR transition dipole moment using ab initio calculation (Xiao, Fu, Liu, Batista, & Yan, 2012). However, proteins are very large molecules, which makes the calculation become complicated and difficult. In contrast, Raman polarizability tensor and IR transition dipole moment for a secondary structure (helix or b-sheet) can be calculated from the Raman polarizability tensor and IR transition dipole moment of a peptide unit using the bond additivity model according to the symmetry and structure. Recently, Chen group has confirmed that the calculated quantities deduced by bond additivity model are compared to and validated by the experimentally measured quantities acquired by polarized Raman and IR spectra measurements (Nguyen, Le Clair, Ye, & Chen, 2009; Nguyen, King, & Chen, 2010). Therefore, the bond additivity model shows more advantages when dealing with the large molecules such as polymers and proteins.

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

221

3.1. Calculation method for Raman polarizability tensor 3.1.1 Raman polarizability tensor of peptide bone structure Before we go further to apply the bond additivity model to calculate the susceptibility tensors of peptides with different secondary structures, we need to know the Raman polarizability tensor and IR transition dipole moment of peptide unit. The a-helix and b-sheet structures are the most common protein secondary structures, which were first predicted by Linus Pauling based on the peptide bonds that connect each amide acid residue (Pauling & Corey, 1953). Other helices such as the 310-helix and p-helix are two variations from the a-helix. Details of the introduction of Pauling’s a-helix, 310-helix, and b-sheet have been introduced by Nguyen et al. (2009a) and Nguyen, King, et al. (2010), who have developed a methodology to determine the orientation of a-helical and antiparallel b-sheet structure using SFG amide I spectra collected with different polarization combinations by treating a-helical structure as having C18/5 symmetry and antiparallel b-sheet structure as having D2 symmetry. All of the proteins are built up by peptide unit on which every backbone carbonyl C]O and NdH group is hydrogen bonded to another NdH and C]O. Using polarized Raman spectroscopy, Tsuboi et al. have successfully measured the Raman tensors of vibrational modes of the functional groups such as the ester C]O stretch, the amide I and amide III modes, and the CdCphenyl stretch (Tsuboi, Ikeda, & Ueda, 1991). Figure 7.2 shows the molecular coordinates of peptide bond structure described in Tsuboi’s study (Tsuboi et al., 1991). The direction of C]O bond and NdH bond in each peptide bond structure is opposite to each other, which enables the backbone of peptide chain to form a hydrogen bonding network within the molecular frame. Its principal axis x is perpendicular to the peptide plane, z and y axes are in the plane, and z-axis makes an angle d from the C]O bond. The angle d is determined to be about 34 for a-helical structures z O d

y C x

N H

Figure 7.2 The molecular coordinates of peptide bond structure described in Tsuboi’s study (Tsuboi et al., 1991, 2006).

222

Shuji Ye et al.

and 22 for b-sheet structures with the excitation laser at 488 nm in Raman experiments (Tsuboi, Kubo, Akahane, Benevides, & Thomas, 2006). The Raman tensor for the amide I mode of peptide unit (Fig. 7.2) has the following form:    0:05 0 0    aunit ¼  0 0:2 0  ð7:10Þ  0 0 1 3.1.2 Raman polarizability tensor of secondary structures As mentioned above, the a-helical and b-sheet structures with respect to the laboratory reference frames (Fig. 7.1) were first proposed by Pauling and Corey (1953). In order to apply Tsuboi’s Raman tensor to the a-helical and b-sheet structures, we need to transform Tsuboi’s Raman tensor to the first peptide unit (an isolated amide linage) of the a-helical and b-sheet structures by applying Euler transformation (Eq. (7.11)) (Kip, van Gurp, van Heel, & Meier, 1993; Rintoul, Carter, Stewart, & Fredericks, 2000). alink ¼ xaunit xT

ð7:11Þ

The rotation x matrix can be calculated based on the coordinates of atoms N, C, and O of the amide bond in the lab coordinate (x, y, z) and the molecular coordinate (a, b, c) (Overman, Tsuboi, & Thomas, 1996). Assuming that the coordinates of atoms N, C, and O are marked as N(xN, yN, zN); C(xC, yC, zC); O(xO, yO, zO) in (x, y, z) frame and N(aN, bN, cN); C(aC, bC, cC); O(aO, bO, cO) in (a, b, c) frame, the direction-cosine matrix for the selected bond in (x, y, z) and (a, b, c) coordinate system is then calculated by Eqs. (7.12) and (7.13), respectively.   lNC mNC nNC A¼ ð7:12Þ lC¼O mC¼O nC¼O  0  lNC m0NC n0NC ð7:13Þ B¼ 0 lC¼O m0C¼O n0C¼O where

Dxi ffi; li ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 2 Dxi þDyi þDzi

Dyi ffi; mi ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 2 Dxi þDyi þDzi

Dzi ffi; ni ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 2 Dxi þDyi þDzi

Dai Dbi Dci ffi; m0i ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi; n0i ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi; Dxi, Dyi, Dzi, li0 ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 2 2 2 2 2 2 2 Dai þDbi þDci

Dai þDbi þDci

Dai þDbi þDci

Dai, Dbi, and Dci are the differences of the two atoms in the given bond in experimental coordinates and molecular coordinates (Overman et al., 1996). And then the x matrix can be calculated using the relationship of

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

223

  1 0 0   AT ¼ x  BT and xxT ¼  0 1 0 . With the knowledge of the rotation x 0 0 1 matrix, the Raman tensor of the amide link in a-helical and b-sheet structures is ready to be calculated using Eq. (7.11). The resulting x matrices and alink of different secondary structures (a-helix, 310-helix, antiparallel b-sheet, and parallel b-sheet) are given in Table 7.2. A super polarizability tensor for the amide I vibration was then calculated using the polarizability tensor of an isolated amide linkage as a starting point and bond additivity model. 3.1.2.1 Raman polarizability tensor of a-helical structures

By using the Raman tensor for the amide I mode of peptide unit given by Tsuboi et al. (1991, 2006), the super polarizability tensor for the amide I vibration of a-helix has been calculated by Rintoul et al. in terms of bond additivity model (Rintoul et al., 2000). The Pauling’s a-helix has a repeat unit comprising 18 amino acid residues in five turns (Pauling & Corey, 1951). Each consecutive residue linkage is effectively symmetry related to the preceding one by a 100 rotation. The tensors of each of the remaining 17 linkages can be calculated by successive Euler rotation of 100 . The super polarizability tensor is then given by summing over all the residues in the unit cell (Nguyen et al., 2009a).    cos ð100l Þ sin ð100lÞ 0     aahelix ¼  sin ð100lÞ cos ð100lÞ 0 aalink    l¼0  0 0 1    cos ð100lÞ sin ð100lÞ 0 T      sin ð100lÞ cos ð100lÞ 0  eilphase    0 0 1 n  X

ð7:14Þ

For an ideal a-helix, n ¼ 17. The ideal a-helix could be considered having C18/5 symmetry, resulting in three vibrational modes A, E1, and E2 (Nguyen et al., 2009a). Only A and E1 are both IR and Raman active. The relative phases between each peptide structure can be extracted from the character table of corresponding point group. For A mode, phase ¼ 0 and for E1 mode, phase ¼ 100 and 100 . Resulting Raman polarizability tensors of A mode and E1 mode are given by

Table 7.2 Rotation x matrices and Raman polarizability tensors of peptide unit (alink) of different secondary structures Secondary structure j matrices alink (aa-link, a310-link, aiAPb-link, aiPb-link)

a-Helix

310-Helix

Antiparallel b-sheet

Parallel b-sheet

   0 0:6858 0:7278      1 0 0    0 0:7278 0:6858     0:4598 0:4113 0:7870     0:8670 0:3990 0:2985     0:1897 0:8204 0:5394     0:9275 0:3433 0:1475     0:005 0:4068 0:9135      0:3731 0:8468 0:3791   0:9275 0:3433 0:1475     0:005 0:4068 0:9135       0:3731 0:8468 0:3791  0:9275 0:3433 0:1475     0:005 0:4068 0:9135     0:3731 0:8468 0:3791     0:9275 0:3433 0:1475     0:005 0:4068 0:9135     0:3731 0:8468 0:3791     0:856 0:4879 0:1709     0:5144 0:8285 0:2211     0:0369 0:2779 0:96     0:856 0:4879 0:1709     0:5144 0:8285 0:2211     0:0369 0:2779 0:96 

   0:6238 0 0:3993     0 0:05 0    0:3993 0 0:5763     0:6638 0:2478 0:3527     0:2478 0:1585 0:1038     0:3527 0:1038 0:4274     0:0883 0:1070 0:0968     0:1070 0:8676 0:2773     0:0968 0:2773 0:2941     0:0883 0:1070 0:0968     0:1070 0:8676 0:2773     0:0968 0:2773 0:2941     0:0883 0:1070 0:0968     0:1070 0:8676 0:2773     0:0968 0:2773 0:2941     0:0883 0:1070 0:0968     0:1070 0:8676 0:2773     0:0968 0:2773 0:2941     0:1135 0:0966 0:1354     0:0966 0:1994 0:1672     0:1354 0:1672 0:9371     0:1135 0:0966 0:1354     0:0966 0:1994 0:1672     0:1354 0:1672 0:9371 

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

   6:064 0 0   aAahelix ¼  0 6:064 0   0 0 10:373     0 0 3:594   1 ¼  0 0 3:594i  aEahelix  3:594 3:594i 0 

225

ð7:15Þ

ð7:16Þ

3.1.2.2 Raman polarizability tensor of 310-helical structures

310-helix is a variation from a-helix. The 310-helical structure has a repeat unit comprising three amino acid residues in one turn. Each consecutive residue linkage is effectively symmetry related to the preceding one by a 120 rotation. Similar to the case of a-helix, the super polarizability tensor can be given by summing over all the residues in the unit cell.    cos ð120l Þ sin ð120lÞ 0   n  X   a310 helix ¼  sin ð120lÞ cos ð120lÞ 0 a310 link    l¼0  0 0 1 ð7:17Þ    cos ð120l Þ sin ð120lÞ 0 T      sin ð120lÞ cos ð120l Þ 0  eilphase    0 0 1 For an ideal 310-helix, n ¼ 2. The 310-helix has C3v symmetry. 310-Helix also shows three vibrational modes A, E1, and E2 (Nguyen et al., 2009a). Only A and E1 are both IR and Raman active. The phase of A mode is equal to 0 while the phase of E1 mode is equal to 120 and 120 . Correspondingly, Raman polarizability tensors of A mode and E1 mode are given by    1:2335  0 0   A  1:2335 0  a310 helix ¼  0 ð7:18Þ  0 0 1:2824     0:3789  0:3717i 0:3717  0:3789i 0:5291 þ 0:1557i    aE3101 helix ¼  0:3717  0:3789i 0:3789 þ 0:3717i 0:1557 þ 0:5291i   0:5291 þ 0:1557i 0:1557 þ 0:5291i  0 ð7:19Þ 3.1.2.3 Raman polarizability tensor of antiparallel b-sheet structures

Antiparallel b-sheet structure is a fundamental structural component of native protein. The major structural element is antiparallel b-sheet in many native proteins such as the silk of silkworms and spiders. The defined abc

226

Shuji Ye et al.

system in Fig. 7.1 is described as below: c is directed along the polypeptide chain in the plane of the sheet, b is in the same plane, b is perpendicular to c and approximately parallel to the peptide carbonyls, and a is perpendicular to bc plane. Tsuboi et al. have developed a method to calculate the super polarizability tensor for the amide I vibration of antiparallel b-sheet (Tsuboi et al., 2006). The antiparallel b-sheet has a repeat unit comprising four peptide bond structures from two b-sheet chains. Two of these four peptide bond structures are from the same b-sheet chain and maintain the opposite directions to form a strong hydrogen bonding network with the other two peptide bond structures. Because antiparallel b-sheet has D2 symmetry, four amide I vibrations are expected with the D2 symmetry: A, B1, B2, and B3 modes. The Raman polarizability tensor of A, B1, B2, and B3 modes of the repeating unit can be calculated with the consideration of the phase of the amide I mode for each peptide unit (Nguyen, King, et al., 2010). A mode:    0:3534 0 0  4 X  aAantiparallel ¼ aiAPblink ¼  0 3:4703 0  ð7:20Þ  i¼1 0 0 1:1764  B1 mode: 1 aBantiparallel ¼ cos ðpÞa1APblink þ cos ðpÞa2APblink þ cos ð0Þa3APblink    0 0:4282 0   ð7:21Þ þcos ð0Þa4APblink ¼  0:4282 0 0   0 0 0 B2 mode: 2 aBantiparallel ¼ cos ð0Þa1APblink þ cos ðpÞa2APblink þ cos ðpÞa3APblink    0 0 0:3870   ð7:22Þ þcos ð0Þa4APblink ¼  0 0 0   0:3870 0 0  B3 mode: 3 aBantiparallel ¼ cos ðpÞa1APblink þ cos ð0Þa2APblink þ cos ðpÞa3APblink   0  0 0   ð7:23Þ 4  þcos ð0ÞaAPblink ¼  0 0 1:1093   0 1:1093  0 3.1.2.4 Raman polarizability tensor of parallel b-sheet structures

Parallel b-sheet is characterized by two peptide strands running in the same direction held together by hydrogen bonding between the strands. The

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

227

repeat unit has C2 symmetry, thus two amide I vibrations are expected: A and B modes. Different from the abc system of antiparallel b-sheet, the abc system of the parallel b-sheet in Fig. 7.1 is defined as that c is along the rotation C2-axis in the plane of the sheet and approximately parallel to the peptide carbonyls, while b is directed along the polypeptide chain in the plane of the sheet and perpendicular to c, and a is perpendicular to bc plane. Following the methodology developed by Chen lab (Nguyen, King, et al., 2010), we deduced the Raman polarizability tensor based on the Raman tensor for the amide I mode in the molecular frame as defined in Fig. 7.2 proposed by Tsuboi et al. (1991, 2006) and molecular coordinate system of two peptide units of the Pauling–Corey parallel b-sheet (Pauling & Corey, 1953). The pleating angle of the parallel b-sheet is 31 , deduced from the Pauling–Corey’s coordinates. The resulting Raman polarizability tensors of A mode and B mode are given by following matrices. A mode:    0:2269 0 0:2707   aAparallel ¼ aiPblink ¼  0 0:3988 0   i¼1 0:2707 0 1:8742  2 X

ð7:24Þ

B mode: aBparallel ¼ cos ð0Þa1Pblink þ cos ðpÞa2Pblink    0 0:1932 0   ¼  0:1932 0 0:3343   0 0:3343 0 

ð7:25Þ

3.2. Calculation method for IR dipole moments It has been indicated that the biaxial orientation of a transition dipole moment (the absolute magnitude is equal to M0) can be described with respect to the lab coordinate system (x, y, z) by using the Euler angles as shown in Fig. 7.3 (Pelletier, Laurin, Buffeteau, & Pe´zolet, 2004). The relationship between the components of the transition dipole moment along each axis in the (x, y, z) coordinate system (Mx, My, Mz) and the components of the transition dipole moment along the axis of the molecular coordinate system (Ma, Mb, Mc) has been given by Zbinden in half century years ago (Zbinden, 1964).

228

Shuji Ye et al.

z

A

c b a

J g

Q M

y

f x

B

C

a

a j

c

b

b

j

YM YM

M

c

M

Figure 7.3 (A) Molecular axes (a, b, c) of the peptide chain and transition moment (M) relative to the axes in the lab coordinate system (x, y, z). (B) Molecular coordinate of antiparallel b-sheet structure. (C) Molecular coordinate of parallel b-sheet structure. Antiparallel b-sheet and parallel b-sheet adopt different axes. 1 Mx B C @ My A ¼ 0

0

Mz cos ð#Þ cos ðgÞ cos ðfÞ  sin ð#Þ sin ðfÞ

cos ðfÞ cos ðgÞ sin ð#Þ þ sin ðfÞ cos ð#Þ  cos ðfÞ sin ðgÞ

B @  sin ðfÞ cos ðgÞ cos ð#Þ  cos ðfÞsin ð#Þ  sin ðfÞ cos ðgÞ sin ð#Þ þ cos ðfÞcos ð#Þ sin ðgÞ cos ð#Þ

10

Ma

1

CB C sin ðfÞ sin ðgÞ A@ Mb A

sin ðgÞsin ð#Þ

cos ðgÞ

Mc

ð7:26Þ Using the axis system defined in Fig. 7.3, the components of the transition dipole moment along the axes of the molecular coordinate system (Ma, Mb, Mc) are given by Ma ¼ M0 sin ðYÞ, Mb ¼ 0, Mc ¼ M0 cos ðYÞ

ð7:27Þ

Here Y is the orientation of the transition moment relative to the long molecular axis. Inserting the relations of Eq. (7.27) into Eq. (7.26), the components of the transition dipole moment along each axis in the (x, y, z) coordinate system (Mx, My, Mz) can be written as

229

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

Mx ¼ ð cos ðfÞ cos ðgÞ cos ð#Þ  sin ðfÞ sin ð#ÞÞM0 sin ðYÞ  cos ðfÞ sin ðgÞM0 cos ðYÞ

ð7:28Þ

My ¼ ðsin ðfÞcos ðgÞ cos ð#Þ  cos ðfÞ sin ð#ÞÞM0 sin ðYÞ þ sin ðfÞ sin ðgÞM0 cos ðYÞ

ð7:29Þ

Mz ¼ ð sin ðgÞ cos ð#ÞÞM0 sin ðYÞ þ cos ðgÞM0 cos ðYÞ

ð7:30Þ

3.2.1 IR dipole moments of a-helix and 310-helix By using Eqs. (7.28)–(7.30), the IR transition dipole moment of the individual peptide unit comprising one repeat unit of a-helical and antiparallel b-sheet structures has been calculated by Marsh (1997), Marsh, Mu¨ller, and Schmitt (2000), and Marsh (2004). For the a-helix and 310-helix, when the axis of the helix is aligned along the molecular c-axis, at the same time, the (x, y, z) coordinate system is overlapped with the molecular (a, b, c) coordinate system, the angles (#, g, f) in Eq. (7.26) will all be equal to 0. Then the component of the transition dipole moment of the amide linage is given by Eq. (7.27). The magnitudes of the parallel and perpendicular transition dipole moment can be calculated using the bond additivity model by integrating the projections of the dipole moment of each peptide unit in the helix onto the parallel and perpendicular directions in the helix’s molecular frame. The calculated IR transition dipole moments for the two IR active modes are given by Nguyen et al. (2009a). The A mode (the parallel mode, phase ¼ 0 ):

0 1  0 @mA ∘ A ð0 Þ ¼ @ X 0 @Qq M0 cos ðYÞ

ð7:31Þ

The E1 mode of a-helix (the perpendicular mode, phase ¼ 100 ) 0

1

n X

M0 cos ð100l Þsin ðYÞ cos ðl  phaseÞ C B  B l¼1 C @maE1 B C n ð100∘ Þ ¼ B X C B i M0 sin ð100lÞ sin ðYÞsin ðl  phaseÞ C @Qq @ A l¼1

0

ð7:32Þ

230

Shuji Ye et al.

The E1 mode of 310-helix (the perpendicular mode, phase ¼ 120 ) 0 n 1 X M0 cos ð120lÞ sin ðYÞ cos ðl  phaseÞ C B " # B l¼1 C @m3E101 B C n ð120∘ Þ ¼ B X C ð7:33Þ B i M0 sin ð120lÞ sin ðYÞ sin ðl  phaseÞ C @Qq @ A l¼1

0 Extensive studies have been performed to deduce the angle of Y using polarized FTIR spectra. The value of a-helix (Y ¼ 38 ) was reported by Marsh according to the studies on poly(g-methyl-L-glutamate)x-co-(g-noctodecyl-L-glutamate) (Marsh, 1997, 2004; Marsh et al., 2000). Considering the fact that the amide I signal can be affected by the hydrogen bonding and the dipole–dipole coupling among the neighboring groups, Wang et al. corrected Y angle to be 42 (Wang, Lee, & Chen, 2008). The Y angles of a and 310-helix (Ya ¼ 42 and Y310 ¼ 45.6 ) have been adopted by Khoi and Ye in previous studies (Nguyen et al., 2009a; Ye, Nguyen, & Chen, 2010). Inserting these Y angles into Eqs. (7.31)–(7.33), and assuming M0 ¼ 1, the calculated IR transition dipole moments for A and E1 modes of ideal a and 310-helix are given by 0 1 0 1 0 1 0 6:02 0

a

a

310  @mA B C @mE1 B C @mA B C ¼ @ 0 A; ¼ @ 6:02i A; ¼ @ 0 A; @Qq @Qq @Qq 13:38 0 2:099 0 1 " # 1:0717 @m3E101 B C ¼ @ 1:0717i A @Qq 0 ð7:34Þ 3.2.2 IR dipole moments of b-sheet structures Compared to helical structure, b-sheet structure is more complicated because the angles (#, g, f) do not correspond directly with its geometry. To obtain the IR dipole moments of the amide linage, we first need to calculate the angles of (#, g, f) and Y. The calculation details have been given by Marsh et al. (2000) and Marsh (2004). In Marsh’s calculation, the inclination angle (Y) of the transition dipole moment (M) to the strand c-axis and the angle (#) of transition dipole moment relative to the a-axis are given by cos ðYÞ ¼ cos ð’Þsin ðcM Þ

ð7:35Þ

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

cos ðcM Þ ffi cos ð#Þ ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1  cos 2 ð’Þsin 2 ðcM Þ

231

ð7:36Þ

where ’ is the inclination angle of the peptide plane with respect to the strand c-axis, cM is the tilt angle of the individual transition dipole moment to the perpendicular a-axis within the plane of the sheet. ’ can be calculated using the molecular coordinates. For the coordinates in Appendix B, ’ is equal to 22 and 31 for antiparallel b-sheet and parallel b-sheet, respectively. The value of ’ ¼ 30 was assumed for antiparallel b-sheet by Khoi et al. in their calculation (Nguyen, King, et al., 2010). cM can be determined from the relative intensities of the n?(p,0) and n//(0,p) amide I modes using Eq. (7.37). By combining the intensities of the amide I components for Bombyx mori silk fibroin with the value of ’ ¼ 22 obtained from the original X-ray pseudostructure, a value of cM ¼ 19 for antiparallel b-sheet was determined by Suzuki (1967). As for parallel b-sheet, no IR data are available; therefore, no experimental results were reported by now. However, a value of 23 has been adopted by Schweitzer-Stenner and Cho in their theoretical simulation (Lee & Cho, 2004; SchweitzerStenner, 2012).

I n== ð0,pÞ tan ðcM Þ ¼ cos 2 ð’ÞI ½n? ðp,0Þ 2

ð7:37Þ

With the knowledge of the value of ’ and cM, the angles of Y and # can be calculated using Eqs. (7.35) and (7.36). Then the components of IR dipole moments of the amide linage are obtained, given in Table 7.3. From the transition dipole moments of individual peptide units, the overall transition dipole moment of the IR active vibrational modes can be calculated (Li et al., 2012; Nguyen, King, et al., 2010): For the antiparallel b-sheet B1 mode: mBnð10,pÞ ¼

" B1 # @mAPb @Qq

" 1 # " 2 # " 3 # @mAPb @mAPb @mAPb ¼ cos ðpÞ þ cos ðpÞ þ cos ð0Þ @Qq @Qq @Qq 0 1 " 4 # 0 @mAPb @ ð7:38Þ þ cos ð0Þ ¼ 0 A @Qq 1:208

Table 7.3 The components of IR dipole moments of the individual peptide units ∂ miAPb/∂ Qq or ∂ miPb/∂ Qq Structure

w ( )

c M ( )

Q ( )

q ( )

Antiparallel b-sheet (APb)

22

19

72.43

7.35

APb (Nguyen, King, et al., 2010)

30

19

73.62

9.77

Parallel b-sheet

31

23

70.43

12.33

g¼0 ’ ¼ 270∘

0

1 0:122 @ 0:946 A 0:302 0 1 0:163 @ 0:946 A 0:282 0 1 0:201 @ 0:335 A 0:921

g¼0 ’ ¼ 90∘

0

1 0:122 @ 0:946 A 0:302 0 1 0:163 @ 0:946 A 0:282 0 1 0:201 @ 0:335 A 0:921

g ¼ 180∘ ’ ¼270∘

0

1 0:122 @ 0:946 A 0:302 0 1 0:163 @ 0:946 A 0:282

g ¼ 180∘ ’ ¼ 90∘

0

1 0:122 @ 0:946 A 0:302 0 1 0:163 @ 0:946 A 0:282

233

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

B2 mode: " B # " # " # " # 2 @mAPb @m3APb @m1APb @m2APb B2 ¼ cos ð0Þ þ cos ðpÞ þ cos ðpÞ mnðp,0Þ ¼ @Qq @Qq @Qq @Qq 1 " # 0 0 @m4APb @ ¼ 3:784 A þcos ð0Þ ð7:39Þ @Qq 0 B3 mode: " B # " # " # " # 3 3 1 2 @m @m @m @m APb APb APb APb ¼ cos ðpÞ þ cos ð0Þ þ cos ðpÞ mBnð3p,pÞ ¼ @Qq @Qq @Qq @Qq 1 " # 0 0:804 @m4APb ¼@ 0 A þ cos ð0Þ ð7:40Þ @Qq 0 For the parallel b-sheet A mode: 1 " # " # " # 0 0 A 1 2 @m @m @m Pb Pb Pb mAnð0,0Þ ¼ ¼ cos ð0Þ þ cos ð0Þ ¼ @ 0:670 A ð7:41Þ @Qq @Qq @Qq 0 B mode:

"

@mBPb

#

" # @m1Pb

"

@m2Pb

¼ cos ð0Þ þ cos ðpÞ @Qq @Qq @Qq 0 1 0:402 ¼@ 0 A 1:842

mBnðp,0Þ ¼

#

ð7:42Þ

3.3. Orientation analysis methods for the interfacial proteins using SFG amide I signal With the knowledge of Raman polarizability tensor and IR transition dipole moment, the molecular hyperpolarizability tensor elements blmn(l, m, n ¼ a, b, c) of a vibrational mode can be calculated using Eq. (7.2). The ratio of different blmn(l, m, n ¼ a, b, c) as a function of amino acid residue number of each a-helix (N) was plotted in Fig. 7.4. The Euler transformation in Eq. (7.1) follows the z–y–z convention and is given in Eq. (7.43). By integrating

234

Shuji Ye et al.

the Euler angles (f, or both f and c) from 0 to 2p in Eq. (7.1), SFG susceptibility tensor elements of w(2) ijk (i, j, k ¼ x, y, z) as a function of orientational tilt angle (y) and twist angle (c) are deduced (Li et al., 2012; Nguyen et al., 2009a; Nguyen, King, et al., 2010). 

 Ril Rjm Rkn ¼ 0 1 cos ðfÞcos ðyÞcos ðcÞ  sin ðfÞsin ðcÞ sin ðfÞcos ðyÞ cos ðcÞ  cos ðfÞsin ðcÞ sin ðyÞcos ðcÞ @ cos ðfÞcos ðyÞsin ðcÞ þ sin ðfÞcos ðcÞ sin ðfÞcos ðyÞ sin ðcÞ þ cos ðfÞcos ðcÞ sin ðyÞsin ðcÞ A cos ðfÞsin ðyÞ sin ðfÞsin ðyÞ cos ðyÞ

ð7:43Þ where the y, f, c are tilt angle, rotational angle, and twist angle which are defined by the angular differences between the experimental axis and molecular axis, shown in Fig. A1 in Appendix A. 3.3.1 The case of a and 310-helix The SFG hyperpolarizability tensor is a third rank tensor with 27 elements. Because of the symmetry of the vibrational modes, some of the 27 elements can be equal to 0. Therefore, the relations between w(2) ijk (i, j, k ¼ x, y, z) and the angles of y and c will be quite different for the vibrational modes of different symmetries (Li et al., 2012; Nguyen et al., 2009a; Nguyen, King, et al., 2010).

0.75 baac /bccc

0.70

baca /bccc

0.65 0.60 Ratio

0.55 0.50 0.45 0.40 0.35 0.30 0.25 0

10 20 30 40 50 100 200 Amino acid residue number of each helix (N)

300

Figure 7.4 The ratio of different blmn(l, m, n ¼ a, b, c) as a function of amino acid residue number of each a-helix (N).

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

235

In terms of the symmetries of the a-helix and 310-helix, the relationships of A mode and E1 mode can be expressed by Nguyen et al. (2009a). For the A mode:   1 ð2Þ ð2Þ wA,xxz ¼ wA,yyz ¼ Ns ð1 þ r Þh cos yi  ð1  r Þ cos 3 y bccc ð7:44Þ 2   1 ð2Þ ð2Þ ð2Þ ð2Þ wA,xzx ¼ wA,yzy ¼ wA,zxx ¼ wA,zyy ¼ Ns h cosyi  cos 3 y ð1  r Þbccc 2 ð7:45Þ  

ð2Þ wA,zzz ¼ Ns r h cos yi þ ð1  r Þ cos 3 y bccc ð7:46Þ For the E1 mode:   ð2Þ ð2Þ wE1 ,xxz ¼ wE1 ,yyz ¼ Ns h cos yi  cos 3 y baca   ð2Þ ð2Þ ð2Þ ð2Þ wE1 ,xzx ¼ wE1 ,yzy ¼ wE1 ,zxx ¼ wE1 ,zyy ¼ Ns cos 3 y baca   ð2Þ wE1 ,zzz ¼ 2Ns h cos yi  cos 3 y baca

ð7:47Þ ð7:48Þ ð7:49Þ

where r ¼ baac/bccc and baac, baca, and bccc are the molecular hyperpolarizability elements. Previous IR studies indicated that the frequency difference of A mode and E1 mode is only several cm1 (Tamm & Tatulian, 1997), the A mode and E1 mode cannot be readily resolved in the frequency domain due to the limited resolution of many SFG spectrometers (5 cm1 or more). Chen group assumed the total susceptibility to be the sum of the susceptibilities from these two modes (Chen, Wang, Boughton, Kristalyn, & Chen, 2007): ð2Þ

ð2Þ

ð7:50Þ

ð2Þ

ð2Þ

ð7:51Þ

2Þ wðyyz ¼ wA,yyz þ wE1 ,yyz 2Þ wðzzz ¼ wA,zzz þ wE1 ,zzz

According to Eqs. (7.46)–(7.53), the orientation angle (y) can be obtained by measuring the ppp and ssp spectral intensity ratio of peptide amide I signals (Eqs. (7.52) and (7.53)). 2Þ wðssp

  

  1 þ r baca 1  r baca  3  cos y bccc ð7:52Þ ¼ Fyyz Ns   h cos yi  bccc bccc 2 2

236

Shuji Ye et al.

! ! # 1 þ r b 1  r b aca aca 2Þ h cos yi  wðppp ¼ Fxxz Ns h cos 3 yi bccc   bccc bccc 2 2 ! ! " # 2baca 2baca 3 þ Fzzz Ns r þ h cosyi þ 1  r  h cos yi bccc bccc bccc "

ð7:53Þ 3.3.2 The case of antiparallel b-sheet Compared to helical structure, antiparallel b-sheet structure is somewhat more complicated when integrating the Euler angles to calculate the surface susceptibility because the molecule cannot be assumed to be axially symmetric, the result being that the orientation of a b-sheet structure is characterized by two angles: a tilt angle (y) and a twist angle (c) (Wang, Chen, et al., 2005). Among the four vibration modes, only B1, B2, and B3 modes are SFG active. Chen group has deduced the relations between w(2) ijk (i, j, k ¼ x, y, z) and the angles of y and c for these three vibrational modes in the laboratory coordinate system (Wang, Chen, et al., 2005). B1 mode:    2Þ wðyyz ¼ Ns h cos ysin c cos ci  cos 3 y sin ccosc babc ð7:54Þ   3  ð2Þ ð7:55Þ wzzz ¼ 2Ns h cos ysin c cos ci  cos y sin ccosc babc    1  2Þ ¼  Ns sin 2 y sin 2 c  sin 2 y cos 2 c babc ð7:56Þ wðyzx 2 B2 mode:

   2Þ wðyyz ¼ Ns h cosy sin c cosci  cos 3 y sin ccosc bacb    2Þ ¼ 2Ns h cosy sin c cosci  cos 3 y sin ccosc bacb wðzzz    1  2Þ wðyzx ¼ Ns cos 2 y  sin 2 ycos 2 c bacb 2

ð7:57Þ ð7:58Þ ð7:59Þ

B3 mode:

   2Þ wðyyz ¼ Ns h cosy sin c cosci  cos 3 y sin ccosc bbca    2Þ ¼ 2Ns h cosy sin c cosci  cos 3 y sin ccosc bbca wðzzz    1  2Þ wðyzx ¼  Ns cos 2 y  sin 2 y sin 2 c bbca 2

ð7:60Þ ð7:61Þ ð7:62Þ

237

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

where Ns is the surface number density of the repeating units of the antiparallel b-sheet. The standard (or achiral) susceptibility components (2) (2) (2) (2) (2) (2) w(2) yyz ¼ wxxz ¼ wxzx ¼ wyzy ¼ wzxx ¼ wzyy and wzzz can be obtained by fitting (2) (2) (2) achiral SFG spectra, and chiral tensors wxzy ¼  w(2) yzx ¼ wzxy ¼  wzyx can be deduced from chiral SFG spectra. The achiral susceptibility tensor elements for the B2 and B3 modes have the same form as the B1 mode, except that babc should be replaced by bacb and bbca, respectively. According to the orientation analysis of interfacial b-sheet structures reported by Nguyen et al., the intensity of the B2 vibrational mode (the 1625 cm1 peak) is roughly ninefold stronger than the intensity of the B1 and B3 modes in SFG-VS spectra (Nguyen, King, et al., 2010). 3.3.3 The case of parallel b-sheet The method of the orientation determination of interfacial parallel b-sheet structure has been introduced by Yan et al. recently (Xiao et al., 2012). With the consideration of C2 symmetry, the parallel b-sheet has two SFG-active modes: A mode and B mode. The nonzero tensor elements of blmn,q are baab, bacb ¼ bcab, bbbb, and bccb for the A mode and baba ¼ bbaa, babc ¼ bbac, bbca ¼ bcba, and bbcc ¼ bcbc for the B mode (Li et al., 2012; Xiao et al., 2012). Yan et al. deduced the hyperpolarizability tensor elements using ab initio quantum chemistry calculation and determined the orientation by using the relative psp SFG intensity of the B mode to the A mode (Xiao et al., 2012). The (2) (2) (2) susceptibility elements of w(2) yyz, wzzz, wzyx, and wxyz can be obtained by an integration of the in-plane-rotation angle f from 0 to 2p. With the knowing of nonzero tensor elements, the relationship between the SFG susceptibility (2) (2) w(2) ssp, wppp, and wpsp and the tilt (y) and twist angle (c) of the parallel b-sheet is deduced. A mode:   1 h siny sin ciðbaab þ bbbb Þ þ h sin 3 y sinciðbccb  baab Þ ð2Þ wyyz ¼ Ns þh sin 3 y sin 3 ciðbaab  bbbb Þ þ 2h sin 2 y cosy sin ccoscibacb 2 ð7:63Þ

2Þ wðzzz ¼ Ns



h siny sincibccb  h sin 3 y sin ciðbccb  baab Þ þh sin 3 y sin 3 ciðbbbb  baab Þ  2h cos ysin 2 y sinccos cibacb



ð7:64Þ

238

Shuji Ye et al.

    1  2Þ 2Þ wðzyx ¼ wðzxy ¼  Ns cos 2 y bcab  sin 2 y cos 2 c bacb þ h sinycos y cosciðbccb  baab Þ 2

ð7:65Þ 2Þ wðxyz ¼0

ð7:66Þ

B mode:   h sin 2 y cosy sinc cosciðbabc þ bbca Þ  h siny sincibbcc ð2Þ ð7:67Þ wyyz ¼ Ns þh sin 3 y sin ciðbbcc  baba Þ þ h sin 3 y sin 3 cibaba 

2Þ wðzzz

¼ 2Ns

2Þ wðzyx

2Þ ¼ wðzxy

h sin 2 y cos ysin c cos ciðbabc þ bbca Þ  h sinysin cibbcc þh sin 3 y sinciðbbcc  baba Þ þ h sin 3 y sin 3 cibaba



ð7:68Þ   1 h cos 2 yibcba þ h sin 2 y sin 2 ciðbbca  bbac Þ ¼  Ns þh sin 2 y cos 2 cibabc þ h sin y cosycos ciðbaba  bcbc Þ 2 ð7:69Þ 2Þ wðxyz ¼0

ð7:70Þ

4. RECENT PROGRESSES ON THE DETERMINATION OF PROTEIN MOLECULAR STRUCTURE AND ORIENTATION AT DIFFERENT INTERFACES According to the analysis in above sections, the orientation angles of interfacial protein can be deduced by measuring the intensity ratio between SFG spectra collected with different polarization combinations of input and output laser beams. By measuring the SFG intensity ratio of ssp, and ppp or other polarization combinations, the orientation of a variety of peptides and proteins such as melittin, G protein b subunit, magainin 2, MSI-78, cytochrome b5, Pep-1, CP1c, fibrinogen, alamethicin, tachyplesin I has been determined (Chen, 2012; Chen & Chen, 2006; Chen, Clarke, et al., 2005; Liu et al., 2012; Ye et al., 2009; Ye, Nguyen, Boughton, et al., 2010). In this section, we will summarize recent progresses on the determination of protein molecular structure and orientation at different interfaces.

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

239

4.1. The study of a-helix The a-helical structure is a common secondary structure in peptides/ proteins and has been widely used to investigate the interaction between proteins and cell membrane, the adsorption at solid surfaces, and the immobilization on solid surfaces (Ye, Nguyen, Boughton, et al., 2010). Normally, the soluble or membrane-inserted a-helical structures are dominated by the peak at 1655 cm1 (Tamm & Tatulian, 1997). The orientation information of helical structures only includes one tilt angle. Thus, it is enough to determine the orientation angle by measuring the SFG intensity ratio from the ssp and ppp (or sps) spectra. The earlier studies mainly focused on determining the static orientation of proteins in pure DI water or buffer solution environments. For example, Chen et al. used melittin as a model peptide to develop the methodology to measure multiple orientation distribution of peptide molecules by combining SFG and ATR-FTIR measurements (Chen, Wang, et al., 2007) and used G protein b subunit as an example to show the possibility to determine the orientation of global proteins with several helical segments (Chen, Boughton, Tesmer, & Chen, 2007). However, the interactions between protein and solid surfaces or cell membranes are quite complex, which are driven by different peptide–membrane forces such as van der Waals, hydrophobic, hydrogen bonding, and electrostatic forces. Important parameters for interaction include pH, temperature, the ionic strength, the properties of protein and surface, the nature of solvent. Thus the researches on how these parameters mediate the interaction become important topics in recent studies. Temperature effect. The interaction between protein and cell membrane is strongly influenced by the nature of cell membrane. With temperature increases, the thermal energy can increase the fluidity of the lipid bilayer as well as the mobility of protein molecules, thus favors the protein insertion into membranes. Recently, Nguyen et al. evaluated the temperature effect on the interaction between mutant Cytochrome b5 and d-DMPC/ d-DMPC lipid bilayer (Nguyen, Soong, et al., 2010). While elevating the temperature from 25 to 40  C, a dramatic increase of ppp SFG amide I signal was observed (Fig. 7.5A). From the orientation analysis of the helical structure using SFG amide I band, it was found that the SFG signal intensity increases when the tilt angle decreases (Fig. 7.5B). This observed SFG signal intensity increase was caused by a reorientation process in the bilayer at higher temperatures, rather than by more proteins adsorbed onto the bilayer at a higher temperature. However, when the temperature is even higher,

Shuji Ye et al.

2000 1800 1600 1400 1200 1000 800 600 400 200 0

80

A

B

25 ⬚C 30 ⬚C 35 ⬚C 40 ⬚C 45 ⬚C

Helical tilt angle (⬚)

Intensity (a.u.)

240

70 60 50 40 30 20 10

1550

1600

1650

1700

1750 –1

Wavenumber (cm )

1800

1850

20

25

30 35 40 Temperature (⬚C)

45

50

Figure 7.5 (A) ppp polarized SFG amide I band of an 8-deletion mutant-Cyt-b5 in a d-DMPC/d-DMPC lipid bilayer as a function of temperature. (B) Tilt angle of an 8-deletion mutant-Cyt-b5 as a function of temperature determined using SFG ppp/ssp signal strength ratio. Reproduced with permission from Nguyen, Soong, et al. (2010). Copyright 2010, American Chemical Society.

besides the protein’s reorientation process, an induced change in the membrane protein coverage may have also occurred. Ionic strength effect. Ions are important components in human body. The human body contains about 70% water on average. But this body fluid is not pure water. It is actually a salt solution containing 1% salts in ionic form. Phosphate ion is one of the most important anions present in the intracellular and extracellular fluid. It can form strongly hydrogen-bonded and saltbridged complexes with arginine and lysine to activate the voltage-gated channel protein. A molecular-level insight into how the phosphate anions mediate the interaction between peptides and cell membrane is critical to understand membrane-bound peptide actions. Recently, Wei et al. used MP, a G-protein-activating peptide, as modeling peptide to investigate how the phosphate anions mediate the interaction between peptides and different lipid bilayers and thus affect the peptide actions. It is found that phosphate ions can greatly promote the association of MP with lipid bilayers and accelerate the conformation transition of membrane-bound MP from aggregation into a-helical structure (Fig. 7.6). In phosphate buffer (PB) solution, MP can insert not only into negatively and neutrally charged lipid bilayers, but also into positively charged lipid bilayer. In neutrally and negatively charged lipid bilayers, the tilt angle of a-helical structure becomes smaller with the increasing buffer concentration, while MP adopts a multiple orientation distribution in positively charged lipid bilayer. MP interacts with lipid bilayers in salt solution environment most likely by formation of toroidal pores inside the bilayer matrix (Wei et al., 2013).

241

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

A

B

0.2

0.2

0 mM

0.1

0 mM

0.1

0.2 0.1 0.0 0.2 0.1 0.0 0.2 0.1 0.0 0.2 0.1 0.0 0.2 0.1 0.0 1500 1550 1600 1650 1700

2 mM

SFG intensity (a.u.)

SFG intensity (a.u.)

0.0

5 mM

10 mM

20 mM

50 mM

1750 1800

0.0 0.5 mM 0.3 0.2 0.1 0.0 4 mM 0.3 0.2 0.1 0.0 8 mM 0.3 0.2 0.1 0.0 20 mM 0.3 0.2 0.1 0.0 1500 1550 1600 1650 1700 1750 1800

Wavenumber (cm-1)

SFG intensity (a.u.)

C

0.6 0.4 0.2 0.0 0.4 0.2 0.0 0.4 0.2 0.0 0.4 0.2 0.0 0.4 0.2 0.0

Wavenumber (cm-1)

0 mM

5 mM

10 mM

20 mM

40 mM

100 mM 0.4 0.2 0.0 1500 1550 1600 1650 1700 1750 1800

Wavenumber (cm-1)

Figure 7.6 The amide I ssp spectra of MP molecules when interacting with lipid bilayer in the presence of different phosphate buffer (PB) concentrations. (A) DMPG; (B) DMPC; (C) DMEPC. Reproduced with permission from Wei et al. (2013). Copyright 2013, American Chemical Society.

242

Shuji Ye et al.

Protein concentration effect. The protein concentration is one of the most important parameters for protein–surface interaction. The protein concentration not only affects the interaction kinetics and mechanism, but also influences the orientation distribution of proteins on membranes. Recently, Nguyen et al. investigated the interactions of magainin 2 with symmetric POPG bilayers. It was observed that magainin 2 orients relatively parallel to the POPG lipid bilayer surface at low solution concentrations, around 200 nM. When increasing the magainin 2 concentration to 800 nM, magainin 2 molecules insert into the POPG bilayer and adopt a transmembrane orientation with an angle of about 20 from the POPG bilayer normal (Nguyen, Le Clair, Ye, & Chen, 2009). Ding et al. examined peptide concentration dependence of the orientation distribution of the a-helical Pep-1 segment associated with the fluid-phase bilayers. It was found that Pep-1 molecules adopted an orientation nearly perpendicular to the plane of the bilayer for peptide concentrations of 0.28 and 1.4 mM. When Pep-1 concentration was increased to 7.0 mM, Pep-1 molecules were associated with the bilayer with a broad orientation distribution (Ding & Chen, 2012). Yang et al. determined the orientation of different concentrations of MSI-78 in various model membranes, indicating that the a-helical MSI-78 molecules are associated with the bilayer surface with 70 deviation from the bilayer normal in the negatively charged gel-phase DPPG bilayer at 400 nM peptide concentration. However, when the concentration was increased to 600 nM, MSI-78 molecules changed their orientation to make a 25 tilt from the lipid bilayer normal whereas multiple orientations were observed for an even higher concentration (Fig. 7.7) (Yang, Ramamoorthy, & Chen, 2011). The solvent effect. Immobilization of biomolecules such as peptides/proteins on the solid surface is a necessary and critical step in the design of biosensors. Yet the activity of the surface-immobilized peptides depends on the structure and orientation of the immobilized peptides/proteins. Recently,

Low concentration

Middle concentration

High concentration

Figure 7.7 Schematics showing the orientation of MSI-78 in a DPPG/DPPG bilayer at different peptide concentrations. Reproduced with permission from Yang et al. (2011). Copyright 2011, American Chemical Society.

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

243

Ye et al. chemically immobilized CP1c, modified with a cysteine amino acid at the C-terminus, onto a polystyrene maleimide (PS-MA) surface by forming a thioether bond between the cysteine group in CP1c and the maleimide group on the PS-MA surface (Ye, Nguyen, Boughton, et al., 2010). It was found that chemically immobilized peptides on polymers adopt a more ordered orientation than less tightly bound physically adsorbed peptides. Later, Han et al. investigated the effect of solvents on this immobilization process by using solvents with different ratios between 2,2,2trifluoroethanol (TFE) and PB. They found that TFE induced the formation of a-helical structure in solution and made the immobilization simpler than in PB solution alone. CP1 adopt in a preferential orientation rather than multiple orientations in PB–TFE solvent (Han, Soblosky, Slutsky, Mello, & Chen, 2011).

4.2. The study of 310-helix The 310-helical structure represents an important secondary structure in protein. It is found that 33% of them are adjacent to the terminus of an a-helix or b-strand and account for about 10% of helical residues in crystal structures (Barlow & Thornton, 1988; Pal, Chakrabarti, & Basu, 2003; Pal, Dasgupta, & Chakrabarti, 2005; Schweitzer-Stenner, Gonzales, Bourne, Feng, & Marshall, 2007). The peptides containing AIB (a-aminoisobutyric acid) residues prefer to form the 310 rather than a-helical conformation. Alamethicin is a 20-residue hydrophobic antibiotic peptide that contains eight aminoisobutyric acid units. Its crystal structure contains an a-helical domain and a 310-helical domain (Fox & Richards, 1982). Alamethicin has been used frequently as a model for larger channel proteins since it can form voltage-gated ion channels in membranes (Tamm & Tatulian, 1997). Ye et al. first applied SFG to characterize interactions between alamethicin and different lipid bilayers in the absence of membrane potential (Ye, Nguyen, & Chen, 2010). It was found that the SFG spectra are dominated by two peaks at 1635 and 1670 cm1 in fluid-phase lipid bilayers (shown in Fig. 7.8). The 1670 cm1 peak is contributed by a helical structure dominated by a-helix but with a 310-helix part. The 1635 cm1 peak arises from the 310-helix. The frequency of the 1670 cm1 peak in a-helix is higher than those normally found for soluble or membrane-inserted a-helices (1655 cm1). The higher frequency attributed to the coupling between the 310-helix and a-helix (Tamm & Tatulian, 1997). Ye et al. determined the orientation of alamethicin in lipid bilayers using SFG amide

244

Shuji Ye et al.

2400

250

A

200

1600

E

150

800

Normalized SFG intensity

Normalized SFG intensity

100 2400

B

1600 800 3200 2400 1600 800

C

2400

D

200

F

150 100 50 200

G

150

1600

100

800 0 1550

50

50 1600

1650

1700

1750

Wavenumber (cm−1)

1800

0 1550

1600

1650

1700

1750

1800

Wavenumber (cm−1)

Figure 7.8 SFG ppp spectra of alamethicin in different bilayers at pH 6.7. (A) POPC/ POPC; (B) POPC/POPG; (C) d-DMPC/DMPC; (D) d-DMPC/d-DMPC; (E) d-DPPC/DPPC; (F) d-DPPG/DPPG; (G) d-DSPC/DSPC. Reproduced with permission from Ye, Nguyen, and Chen (2010). Copyright 2010, American Chemical Society.

I spectra detected with different polarization combinations and found that the helix (mainly a-helix) at the N-terminus tilts at about 63 versus the surface normal in a fluid-phase d-DMPC/DMPC bilayer. The 310-helix at the C-terminus (beyond the Pro14 residue) tilts at about 43 versus the surface normal. However, when alamethicin interacts with a gel-phase lipid bilayer, only two weak SFG peaks at 1685 and 1720 cm1 were observed. The absence of the strong alamethicin helical signal on gel-phase lipid bilayer indicates that both the helical structures lie down on the lipid bilayer surface and/or may be changed into other secondary structures, for example, antiparallel b-sheet or aggregated strand. Recently, Ye et al. further characterized the interactions between alamethicin (a model for larger channel proteins) and POPC lipid bilayers in the presence of an electric potential across the membrane (Ye et al., 2012). In Ye et al.’s study, the membrane potential difference was controlled by adding PB into the solution in contact with the bilayer and measured using fluorescence spectroscopy. There is evidence in the literatures suggesting that phosphate forms strongly hydrogen-bonded and salt-bridged complexes with arginine to activate the voltage-gated channel protein (Freites, Tobias, von Heijne, & White, 2005). The orientation angle of alamethicin in POPC lipid bilayers was then determined using polarized

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

A

245

B q2 q1

f K3PO4

Figure 7.9 (A) The definition of tilt angle and bend angle of alamethicin in POPC/POPC bilayer. (B) A schematic to show the PB-dependent channel gating action of alamethicin. Reproduced with permission from Ye et al. (2012). Copyright 2012, American Chemical Society.

SFG amide I spectra before and after adding PB. It was observed that the a-helix at the N-terminus and the 310-helix at the C-terminus tilt at about 72 (y1) and 50 (y2) versus the surface normal in pure DI water environment, respectively. When adding 100 mL K3PO4 (in 1 M solution) into the subphase (1.6 mL DI water), y1 and y2 decrease to 56.5 and 45 , respectively. The localized change of phosphate ion in proximity to the bilayer can modulate the membrane potential and thus induces a decrease in both the tilt and bend angles of the two helices in alamethicin (Fig. 7.9): (i) The orientation angle becomes narrower. (ii) The bend angle between the two helical components becomes smaller. This is the first reported application of SFG to the study of model ion channel gating mechanisms in model cell membranes. More recently, Chen group determined the relationship between the solution concentration of alamethicin and its orientation in POPC lipid bilayers (Yang, Wu, & Chen, 2013). Their SFG results indicated that the alamethicin molecules more or less lay down on the surface of POPC lipid bilayers at a low peptide concentration of 0.84 mM (y1 ¼ 88 , y2 ¼ 58 ). However, when the peptide concentration was increased to 15.6 mM, it was observed that the alamethicin molecules further inserted into the lipid bilayers (y1 ¼ 37 , y2 ¼ 50 ). They found that the membrane orientation of the alamethicin a-helical component changed substantially with the increase of the alamethicin concentration, while the membrane orientation of the 310-helical component remained more or less the same (Fig. 7.10) (Yang et al., 2013).

4.3. The study of antiparallel b-sheet The b-sheet structure is another common secondary structure in peptides/ proteins. The major structural element of many native proteins is b-sheet.

246

Shuji Ye et al.

R1

4-2

0

R1-13

R1

4-

20

-13 R1

Low concentration

High concentration

Figure 7.10 Schematics showing the orientation of alamethicin in a POPC/POPC bilayer at 0.84 mM (left) or 15.6 mM (right) peptide solution concentration. Reproduced with permission from Yang et al. (2013). Copyright 2013, American Chemical Society.

Different from a-helical structures, the SFG spectra of antiparallel b-sheet structure are centered at 1625, 1690, and 1730 cm1, which correspond to B2, B1, and B3 modes, respectively. Besides this difference, orientation information of antiparallel b-sheet structure includes both the tilt (y) and the twist (c) angles. Therefore, only one SFG intensity ratio obtained from the ppp and ssp spectra is not enough to determine the orientation. In addition, due to the D2 symmetry, b-sheet structures exhibit apparent chiral SFG signal with the polarization combination of spp, psp, and pps. Although there have been many researches on helical structures using SFG, only several SFG applications on antiparallel b-sheet structure were reported since Chen group first detected amide I signal from tachyplesin I (which forms antiparallel b-sheet structure) on polystyrene (PS) surface. Previous studies mainly focus on characterizing the formation of b-sheet structure in terms of the SFG special spectral features (Chen, Wang, Sniadecki, Even, & Chen, 2005; Wang, Chen, et al., 2005). For example, Castner group probed the orientation of electrostatically immobilized protein G B1 onto both amine (NH3þ) and carboxyl (COO) functionalized gold (Baio et al., 2012). Yan group investigated proton exchange in antiparallel b-sheets at interfaces (Fu, Xiao, Wang, Batista, & Yan, 2013). As mentioned above, Chen group developed a methodology to determine the orientation of antiparallel b-sheet structure using SFG amide I spectra collected with different polarization combinations by treating antiparallel b-sheet structure as having D2 symmetry (Nguyen, King, et al., 2010). They estimated orientations of antiparallel b-sheet structures (tachyplesin I) in DPPG/d-DPPG lipid bilayers by using the relative SFG signal intensities of the B1 and B2 modes and found that the tilt angle (y) has a range of 75–90 and the twist angle (c) has a range of 75–90 . It is evident to show that tachyplesin I adopts quite a different

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

247

twist angle on the DPPG/d-DPPG lipid bilayer than it does on PS or deuterated-PS polymer surface (Nguyen, King, et al., 2010).

4.4. The study of parallel b-sheet Parallel b-sheet structure is not as common as antiparallel b-sheet structure. However, recent studies indicated that the proteins such as prion and amyloid proteins mainly form parallel b-sheet structures (Cobb, Sonnichsen, McHaourab, & Surewicz, 2007; Helmus, Surewicz, Apostol, Surewicz, & Jaroniec, 2011; Shewmaker, Wickner, & Tycko, 2006; Wickner, Dyda, & Tycko, 2008). Yet such protein aggregation is associated with many “protein deposition diseases” such as Alzheimer’s disease and Parkinson’s disease. Distinguished from antiparallel b-sheet, parallel b-sheet lacks the amide I peaks at high frequency (>1680 cm1). Human islet amyloid polypeptide (hIAPP) is known to misfold into the b-sheet structure upon interaction with membranes. It has been widely studied using IR and Raman spectroscopy (Oladepo et al., 2012; Wang et al., 2011). Recently, Yan group used hIAPP as an example to monitor the changes in the amide I spectra at the air/water interface after addition of DPPG lipid molecules. They used the chiral-sensitive psp polarization combination to obtain amide I spectra and observed a gradual buildup of the chiral structures that display the vibrational characteristics of parallel b-sheets (1622 cm1), indicating that DPPG induces the misfolding of hIAPP from a-helical and random-coil structures to the parallel b-sheet structure at the air/water interface (Fu, Ma, & Yan, 2010). Later then, Yan group combined SFG-VS and ab initio quantum chemistry calculations to characterize the orientation of hIAPPs at lipid/aqueous interfaces and found that the aggregates bind with b-strands oriented at 48 relative to the interface (Xiao et al., 2012). More recently, Li et al. used prion protein fragment [118–135] (PrP118–135) as a model to characterize interactions between PrP118–135 and POPG lipid bilayer (Li et al., 2012). They determined the conformation change and orientation of PrP118–135 in lipid bilayer using SFG spectra with different polarization combinations. It is found that low-concentration PrP118–135 adopts predominantly a-helical structure but with tiny b-sheet structure. With PrP118–135 increasing concentration, the signals from the 1657 cm1 peak at the concentration of >0.03 mg/mL, which is characteristic of an a-helical structure, are much weaker than those at the concentration of 0.03 mg/mL. In addition, the intensity of psp signals at 1625 cm1 increases with the concentration (Fig. 7.11). By analyzing

248

Shuji Ye et al.

A

B 0.2

ssp

0.10 mg/mL

0.1

Normallized SFG intensity

0.05 mg/mL 0.1 0.0 0.6 0.03 mg/mL 0.4 0.2 0.0 0.02 mg/mL 0.6 0.4 0.2 0.0 0.6 0.01 mg/mL 0.4 0.2 0.0 0 mg/mL 0.15 0.10 0.05 0.00 1500 1550 1600 1650 1700 1750 1800

Wavenumber (cm-1)

Normallized SFG intensity

0.2

0.15 0.10 mg/mL psp 0.10 0.05 0.00 0.09 0.05 mg/mL 0.06 0.03 0.00 0.03 mg/mL 0.015 0.010 0.005 0.000 0.02 mg/mL 0.015 0.010 0.005 0.000 0.01 mg/mL 0.015 0.010 0.005 0.000 0 mg/mL 0.015 0.010 0.005 0.000 1500 1550 1600 1650 1700 1750 1800

Wavenumber (cm-1)

Figure 7.11 SFG spectra of PrP118–135 in POPG/POPG bilayer at different concentrations in amide I region. (A) ssp; (B) psp. Solid line represents the fitting profile. Reproduced with permission from Li et al. (2012). Copyright 2012, American Chemical Society.

the SFG spectra at different polarization combinations, they concluded that the molecular number ratio of parallel b-sheet structure on POPG bilayer increases with prion concentration and reaches 44% at the prion concentration of 0.10 mg/mL. The a-helical structure inserts into lipid bilayer with a tilt angle of 32 versus the surface normal, while the b-sheet structure lies down on the lipid bilayer with the tilt and twist angle both of 90 (Li et al., 2012).

5. SUMMARY In this review, we have systematically summarized the methods for the calculation of the Raman polarizability tensor, IR transition dipole moment, and SFG molecular hyperpolarizability tensor elements of proteins/peptides with the secondary structures of a-helix, 310-helix, antiparallel b-sheet, and parallel b-sheet, as well as the methodology to determine the orientation of protein secondary structures using SFG amide I spectra collected with

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

249

different polarization combinations. Recent progresses on the determination of protein molecular structure and orientation at different interfaces have demonstrated that SFG-VS is a powerful technique to provide a molecular-level understanding of the structures and interactions of interfacial proteins, specially understanding the nature of driving force behind such interaction. Although this review focused on analysis of amide I spectra, it will be expected to offer a basic idea for the spectral analysis of amide III SFG signals. Amide III spectral region (1200–1350 cm1) not only can avoid the interference from the water vibration (1640 cm1) in the amide I region, but also presents a more characterized spectral feature, which is easily resolved and better defined bands for quantitative analysis.

ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (grant nos 2010CB923300), the National Natural Science Foundation of China (grant nos 21273217, 91127042), the Fundamental Research Funds for the Central Universities (WK2070000007), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.

APPENDIX A: EULER TRANSFORMATION BETWEEN DIFFERENT COORDINATE SYSTEMS The Euler transformation used here follows the z–y–z convention, which has a matrix in the form shown in Eq. (7.43). The Euler angles y (tilt angle), c (twist angle), f (in-plane rotation angle) are defined in Fig. A1. z

c b Y

a

q y f

x

Figure A1 Euler angles (y, c, f) relating the molecular (a, b, c) and macroscopic (x, y, z) coordinate systems.

250

Shuji Ye et al.

APPENDIX B: MACROSCOPIC (X, Y, Z) COORDINATES OF DIFFERENT SECONDARY STRUCTURES Atom

x

y

z

a-helix N

1.16

0

0.89

C

2.42

0

0.44

O

2.69

0

0.76

310-helix N

0.07

1.12

0.957

C

1.12

0.54

1.189

O

1.64

0.51

2.304

0.285

1.211

Antiparallel b-sheet N1

0.2

C1

0.4845

2.24

0.21

O1

1.71

2.282

0.22

N2

0.285

4.711

0.2

C2

0.4845

5.74

0.21

O2

1.71

5.782

0.22

N3

5.035

1.211

0.2

C3

4.2655

2.24

0.21

O3

3.04

2.282

0.22

N4

4.465

2.289

0.2

C4

5.2345

1.26

0.21

O4

6.46

1.218

0.22

N1

0.3201

1.209

0.26

C1

0.5723

2.0475

0.28

O1

1.79935

1.9175

0.25

N2

0.3201

4.459

0.26

C2

0.5723

5.2975

0.28

O2

1.79935

5.1675

0.25

Parallel b-sheet

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

251

APPENDIX C: THE FULL NAME OF LIPID MOLECULES POPC 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPG 1-Palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] DMPC 1,2-Dimyristoyl-sn-glycero-3-phosphocholine d-DMPC Fully deuterated DMPC DMPG 1,2-Dimyristoyl-sn-glycero-3-phospho-(10 -rac-glycerol) (sodium salt) DMEPC 1,2-Dimyristoyl-sn-glycero-3-ethylphosphocholine (chloride salt) DPPC 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine DPPG 1,2-Dipalmitoyl-sn-glycero-3-phospho-(10 -rac-glycerol) (sodium salt) DSPC 1,2-Distearoyl-sn-glycero-3-phosphocholine d-DSPC Fully deuterated DSPC

REFERENCES Baio, J. E., Weidner, T., Baugh, L., Gamble, L. J., Stayton, P. S., & Castner, D. G. (2012). Probing the orientation of electrostatically immobilized protein G B1 by time-of-flight secondary ion spectrometry, sum frequency generation, and near-edge X-ray adsorption fine structure spectroscopy. Langmuir, 28, 2107–2112. Barlow, D. J., & Thornton, J. M. (1988). Helix geometry in proteins. Journal of Molecular Biology, 201, 601–619. Barth, A., & Zscherp, C. (2002). What vibrations tell about proteins. Quarterly Reviews of Biophysics, 35, 369–430. Castellana, E. T., & Cremer, P. S. (2006). Solid supported lipid bilayers: From biophysical studies to sensor design. Surface Science Reports, 61, 429–444. Chen, Z. (2012). Molecular structures of buried polymer interfaces and biological interfaces detected by sum frequency generation vibrational spectroscopy. Acta Physico-Chimica Sinica, 28, 504–521. Chen, X., Boughton, A. P., Tesmer, J. J. G., & Chen, Z. (2007). In situ investigation of heterotrimeric G protein bg subunit binding and orientation on membrane bilayers. Journal of the American Chemical Society, 129, 12658–12659. Chen, X., & Chen, Z. (2006). SFG studies on interactions between antimicrobial peptides and supported lipid bilayers. Biochimica et Biophysica Acta – Biomembranes, 1758, 1257–1273. Chen, X., Clarke, M. L., Wang, J., & Chen, Z. (2005). Sum frequency generation vibrational spectroscopy studies on molecular conformation and orientation of biological molecules at interfaces. International Journal of Modern Physics B, 19, 691–713. Chen, X., Wang, J., Boughton, A. P., Kristalyn, C. B., & Chen, Z. (2007). Multiple orientation of melittin inside a single lipid bilayer determined by combined vibrational spectroscopic studies. Journal of the American Chemical Society, 129, 1420–1427. Chen, X., Wang, J., Sniadecki, J. J., Even, M. A., & Chen, Z. (2005). Probing a-helical and b-sheet structures of peptides at solid/liquid interfaces with SFG. Langmuir, 21, 2662–2664. Chiti, F., & Dobson, C. M. (2006). Protein misfolding, functional amyloid, and human disease. Annual Review of Biochemistry, 75, 333–366. Cobb, N. J., Sonnichsen, F. D., McHaourab, H., & Surewicz, W. K. (2007). Molecular architecture of human prion protein amyloid: A parallel, in-register beta-structure. Proceedings of the National Academy of Sciences of the United States of America, 104, 18946–18951.

252

Shuji Ye et al.

Ding, B., & Chen, Z. (2012). Molecular interactions between cell penetrating peptide Pep-1 and model cell membranes. The Journal of Physical Chemistry B, 116, 2545–2552. Engel, M. F. M., vandenAkker, C. C., Schleeger, M., Velikov, K. P., Koenderink, G. H., & Bonn, M. (2012). The polyphenol EGCG inhibits amyloid formation less efficiently at phospholipid interfaces than in bulk solution. Journal of the American Chemical Society, 134, 14781–14788. Fotiadis, D. (2012). Atomic force microscopy for the study of membrane proteins. Current Opinion in Biotechnology, 23, 510–515. Fox, R. O., Jr., & Richards, F. M. (1982). A voltage-gated ion channel model inferred from ˚ resolution. Nature, 300, 325–330. the crystal structure of alamethicin at 1.5-A Freites, J. A., Tobias, D. J., von Heijne, G., & White, S. H. (2005). Interface connections of a transmembrane voltage sensor. Proceedings of the National Academy of Sciences of the United States of America, 102, 15059–15064. Fu, L., Liu, J., & Yan, E. C. Y. (2011). Chiral sum frequency generation spectroscopy for characterizing protein secondary structures at interfaces. Journal of the American Chemical Society, 133, 8094–8097. Fu, L., Ma, G., & Yan, E. C. Y. (2010). In situ misfolding of human islet amyloid polypeptide at interfaces probed by vibrational sum frequency generation. Journal of the American Chemical Society, 132, 5405–5412. Fu, L., Wang, Z., & Yan, E. C. (2011). Chiral vibrational structures of proteins at interfaces probed by sum frequency generation spectroscopy. International Journal of Molecular Sciences, 12, 9404–9425. Fu, L., Xiao, D., Wang, Z., Batista, V. S., & Yan, E. C. Y. (2013). Chiral sum frequency generation for in situ probing proton exchange in anti-parallel b-sheets at interfaces. Journal of the American Chemical Society, 135, 3592–3598. Garland, A., Shen, L., & Zhu, X. (2012). Mobile precursor mediated protein adsorption on solid surfaces. Progress in Surface Science, 87, 1–22. Gopalakrishnan, S., Liu, D., Allen, H. C., Kuo, M., & Shultz, M. J. (2006). Vibrational spectroscopic studies of aqueous interfaces: Salts, acids, bases, and nanodrops. Chemical Reviews, 106, 1155–1175. Han, X., Soblosky, L., Slutsky, M., Mello, C. M., & Chen, Z. (2011). Solvent effect and time-dependent behavior of c-terminus-cysteine-modified cecropin P1 chemically immobilized on a polymer surface. Langmuir, 27, 7042–7051. Helmus, J. J., Surewicz, K., Apostol, M. I., Surewicz, W. K., & Jaroniec, C. P. (2011). Intermolecular alignment in Y145Stop human prion protein amyloid fibrils probed by solid-state NMR spectroscopy. Journal of the American Chemical Society, 133, 13934–13937. Hlady, V., & Buijs, J. (1996). Protein adsorption on solid surfaces. Current Opinion in Biotechnology, 7, 72–77. Judge, P. J., & Watts, A. (2011). Recent contributions from solid-state NMR to the understanding of membrane protein structure and function. Current Opinion in Biotechnology, 15, 690–695. Kim, G., Gurau, M. C., Lim, S.-M., & Cremer, P. S. (2003). Investigations of the orientation of a membrane peptide by sum frequency spectroscopy. The Journal of Physical Chemistry B, 107, 1403–1409. Kip, B. J., van Gurp, M., van Heel, S. P. C., & Meier, R. J. (1993). Orientational order in polyethylene foils: A polarized Raman spectroscopic study. Journal of Raman Spectroscopy, 24, 501–510. Lambert, A. G., Davies, P. B., & Neivandt, D. J. (2005). Implementing the theory of sum frequency generation vibrational spectroscopy: A tutorial review. Applied Spectroscopy Reviews, 40, 103–145.

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

253

Lee, C., & Cho, M. (2004). Local amide i mode frequencies and coupling constants in multiple-stranded anti-parallel b-sheet polypeptides. The Journal of Physical Chemistry B, 108, 20397–20407. Li, H. C., Ye, S. J., Wei, F., Ma, S. L., & Luo, Y. (2012). In situ molecular-level insights into the interfacial structure changes of membrane-associated prion protein fragment [118–135] investigated by sum frequency generation vibrational spectroscopy. Langmuir, 28, 16979–16988. Liu, Y., Jasensky, J., & Chen, Z. (2012). Molecular interactions of proteins and peptides at interfaces studied by sum frequency generation vibrational spectroscopy. Langmuir, 28, 2113–2121. Liu, C., Monson, C. F., Yang, T., Pace, H., & Cremer, P. S. (2011). Protein separation by electrophoretic–electroosmotic focusing on supported lipid bilayers. Analytical Chemistry, 83, 7876–7880. Marsh, D. (1997). Dichroic ratios in polarized Fourier transform infrared for nonaxial symmetry of b-sheet structures. Biophysical Journal, 72, 2710–2718. Marsh, D. (2004). Infrared dichroism of isotope-edited a-helices and b-sheets. Journal of Molecular Biology, 338, 353–367. Marsh, D., Mu¨ller, M., & Schmitt, F.-J. (2000). Orientation of the infrared transition moments for an a-helix. Biophysical Journal, 78, 2499–2510. Moad, A. J., & Simpson, G. J. (2004). A unified treatment of selection rules and symmetry relations for sum-frequency and second harmonic spectroscopies. The Journal of Physical Chemistry B, 108, 3548–3562. Nguyen, K. T., King, J. T., & Chen, Z. (2010). Orientation determination of interfacial b-sheet structures in situ. The Journal of Physical Chemistry B, 114, 8291–8300. Nguyen, K. T., Le Clair, S., Ye, S. J., & Chen, Z. (2009a). Orientation determination of protein helical secondary structures using linear and nonlinear vibrational spectroscopy. The Journal of Physical Chemistry B, 113, 12169–12180. Nguyen, K. T., Le Clair, S., Ye, S. J., & Chen, Z. (2009b). Molecular interactions between magainin 2 and model membranes in situ. The Journal of Physical Chemistry B, 113, 12358–12363. Nguyen, K. T., Soong, R., lm, S.-C., Waskell, L., Ramamoorthy, A., & Chen, Z. (2010). Probing the spontaneous membrane insertion of a tail-anchored membrane protein by sum frequency generation spectroscopy. Journal of the American Chemical Society, 132, 15112–15115. Oladepo, S. A., Xiong, K., Hong, Z., Asher, S. A., Handen, J., & Lednev, I. K. (2012). UV resonance Raman investigations of peptide and protein structure and dynamics. Chemical Reviews, 112, 2604–2628. Outeiro, T. F. (2011). Amyloidogenesis: FlAsH illuminates Ab aggregation. Nature Chemical Biology, 7, 581–582. Overman, S. A., Tsuboi, M., & Thomas, G. J., Jr. (1996). Subunit orientation in the filamentous virus Ff(fd, f1, M13). Journal of Molecular Biology, 259, 331–336. Pal, L., Chakrabarti, P., & Basu, G. (2003). Sequence and structure patterns in proteins from an analysis of the shortest helices: Implications for helix nucleation. Journal of Molecular Biology, 326, 273–291. Pal, L., Dasgupta, B., & Chakrabarti, P. (2005). 310-Helix adjoining a-helix and b-strand: Sequence and structural features and their conservation. Biopolymers, 78, 147–162. Pauling, L., & Corey, R. B. (1951). Atomic coordinates and structure factors for two helical configurations of polypeptide chains. Proceedings of the National Academy of Sciences of the United States of America, 37, 235–240. Pauling, L., & Corey, R. B. (1953). Two rippled-sheet configurations of polypeptide chains, and a note about the pleated sheets. Proceedings of the National Academy of Sciences of the United States of America, 39, 253–256.

254

Shuji Ye et al.

Pelletier, I., Laurin, I., Buffeteau, T., & Pe´zolet, M. (2004). Determination of molecular orientation in biaxially oriented ultrathin films. The Journal of Physical Chemistry B, 108, 7162–7169. Rintoul, L., Carter, E. A., Stewart, S. D., & Fredericks, P. M. (2000). Keratin orientation in wool and feathers by polarized Raman spectroscopy. Biopolymers, 57, 19–28. Schweitzer-Stenner, R. (2012). Simulated IR, isotropic and anisotropic Raman, and vibrational circular dichroism amide I band profiles of stacked b-sheets. The Journal of Physical Chemistry B, 116, 4141–4153. Schweitzer-Stenner, R., Gonzales, W., Bourne, G. T., Feng, J. A., & Marshall, G. R. (2007). Conformational manifold of a-aminoisobutyric acid (Aib) containing alanine-based tripeptides in aqueous solution explored by vibrational spectroscopy, electronic circular dichroism spectroscopy, and molecular dynamics simulations. Journal of the American Chemical Society, 129, 13095–13109. Shen, Y. R. (1984). The principles of nonlinear optics. Wiley: New York. Shewmaker, F., Wickner, R. B., & Tycko, R. (2006). Amyloid of the prion domain of Sup35p has an in-register parallel b-sheet structure. Proceedings of the National Academy of Sciences of the United States of America, 103, 19754–19759. Smith, J. P., & Hinson-Smith, V. (2004). Product review: SFG coming of age. Analytical Chemistry, 76, 287A–290A. Suzuki, E. (1967). A quantitative study of the amide vibrations in the infra-red spectrum of silk fibroin. Spectrochimica Acta, Part A, 23, 2303–2308. Szott, L. M., & Horbett, T. A. (2011). Protein interactions with surfaces: Cellular responses, complement activation, and newer methods. Current Opinion in Biotechnology, 15, 677–682. Tamm, L., & Tatulian, S. A. (1997). Infrared spectroscopy of proteins and peptides in lipid bilayers. Quarterly Reviews of Biophysics, 30, 365–429. Tian, K. Z., Li, H. C., & Ye, S. J. (2013). Methanol perturbing modeling cell membranes investigated using linear and nonlinear vibrational spectroscopy. Chinese Journal of Chemical Physics, 26, 27–34. Tsuboi, M., Ikeda, T., & Ueda, T. (1991). Raman microscopy of a small uniaxial crystal: tetragonal aspartame. Journal of Raman Spectroscopy, 22, 619–626. Tsuboi, M., Kubo, Y., Akahane, K., Benevides, J. M., & Thomas, G. J. (2006). Determination of the amide I Raman tensor for the anti-parallel b-sheet: Application to silkworm and spider silks. Journal of Raman Spectroscopy, 37, 240–247. Wang, J., Chen, X., Clarke, M. L., & Chen, Z. (2005). Detection of chiral sum frequency generation vibrational spectra of proteins and peptides at interfaces in situ. Proceedings of the National Academy of Sciences of the United States of America, 102, 4978–4983. Wang, H. F., Gan, W., Lu, R., Rao, Y., & Wu, B. H. (2005). Quantitative spectral and orientational analysis in surface sum frequency generation vibrational spectroscopy (SFGVS). International Reviews in Physical Chemistry, 24, 191–256. Wang, J., Lee, S.-H., & Chen, Z. (2008). Quantifying the ordering of adsorbed proteins in situ. The Journal of Physical Chemistry B, 112, 2281–2290. Wang, L., Middleton, C. T., Singh, S., Reddy, A. S., Woys, A. M., Strasfeld, D. B., et al. (2011). 2DIR spectroscopy of human amylin fibrils reflects stable b-sheet structure. Journal of the American Chemical Society, 133, 16062–16071. Wei, F., & Ye, S. J. (2012). Molecular-level insights into N–N pi-bond rotation in the pH-induced interfacial isomerization of PARC18 monolayer investigated by sum frequency generation vibrational spectroscopy. Journal of Physical Chemistry C, 116, 16553–16560. Wei, F., Ye, S. J., Li, H. C., & Luo, Y. (2013). Phosphate ions promoting association between peptide and modeling cell membrane revealed by sum frequency generation vibrational spectroscopy. Journal of Physical Chemistry C, 117, 11095–11103.

Structure and Orientation of Interfacial Proteins Determined by SFG-VS

255

Weidner, T., Apte, J. S., Gamble, L. J., & Castner, D. G. (2010). Probing the orientation and conformation of a-helix and b-strand model peptides on self-assembled monolayers using sum frequency generation and NEXAFS spectroscopy. Langmuir, 26, 3433–3440. Weidner, T., Breen, N. F., Li, K., Drobny, G. P., & Castner, D. G. (2010). Sum frequency generation and solid-state NMR study of the structure, orientation, and dynamics of polystyrene-adsorbed peptides. Proceedings of the National Academy of Sciences of the United States of America, 107, 13288–13293. Wickner, R. B., Dyda, F., & Tycko, R. (2008). Amyloid of Rnq1p, the basis of the [PINþ] prion, has a parallel in-register b-sheet structure. Proceedings of the National Academy of Sciences of the United States of America, 105, 2403–2408. Xiao, D., Fu, L., Liu, J., Batista, V. S., & Yan, E. C. Y. (2012). Amphiphilic adsorption of human islet amyloid polypeptide aggregates to lipid/aqueous interfaces. Journal of Molecular Biology, 421, 537–547. Yang, P., Ramamoorthy, A., & Chen, Z. (2011). Membrane orientation of MSI-78 measured by sum frequency generation vibrational spectroscopy. Langmuir, 27, 7760–7767. Yang, P., Wu, F.-G., & Chen, Z. (2013). Dependence of alamethicin membrane orientation on the solution concentration. Journal of Physical Chemistry C, 117, 3358–3365. Yano, Y. F. (2012). Kinetics of protein unfolding at interfaces. Journal of Physics: Condensed Matter, 24, 503101. Ye, S. J., Li, H. C., Wei, F., Jasensky, J., Boughton, A. P., Yang, P., et al. (2012). Observing a model ion channel gating action in model cell membranes in real time in situ: Membrane potential change induced alamethicin orientation change. Journal of the American Chemical Society, 134, 6237–6243. Ye, S. J., Nguyen, K. T., Boughton, A. P., Mello, C. M., & Chen, Z. (2010). Orientation difference of chemically immobilized and physically adsorbed biological molecules on polymers detected at the solid/liquid interfaces in situ. Langmuir, 26, 6471–6477. Ye, S. J., Nguyen, K. T., & Chen, Z. (2010). Interactions of alamethicin with model cell membranes investigated using sum frequency generation vibrational spectroscopy in real time in situ. The Journal of Physical Chemistry B, 114, 3334–3340. Ye, S. J., Nguyen, K. T., Clair, S. V. L., & Chen, Z. (2009). In situ molecular level studies on membrane related peptides and proteins in real time using sum frequency generation vibrational spectroscopy. Journal of Structural Biology, 168, 61–77. Zbinden, R. (1964). Infrared spectroscopy of high polymers. New York: Academic Press. Zhu, H., & Snyder, M. (2003). Protein chip technology. Current Opinion in Chemical Biology, 7, 55–63.