Heterodyne-Detected Achiral and Chiral Vibrational Sum Frequency Generation of Proteins at Air/Water Interface

Abstract

We present complex achiral and chiral vibrational sum frequency generation (VSFG) spectra at the air/ water interface of protein solutions by using heterodynedetected VSFG. Bovine serum albumin, pepsin, concanavalin A, and α-chymotrypsin were measured as model proteins. The obtained achiral Im[χ (2) ] spectra gave us insights into the molecular orientation of protein molecules and water at the interface. From the chiral Im[χ (2) ] spectra in the NH stretching and amide I regions, the secondary structures of the interfacial proteins were deduced. We attributed the chiral signals in the amide I and NH stretching regions to the interface on the basis of the phase of the signals. All the achiral and chiral spectra in each region showed the same sign despite different secondarystructure contents of the examined proteins. Real-time observation of the spectral change of α-chymotrypsin was also performed by heterodyne-detected chiral VSFG. The signal intensity of the chiral Im[χ (2) ] spectra in the NH stretching and amide I regions decreased on the scale of 10 min, originating from the decrease of the portion of antiparallel β-sheet conformation in the molecule. The conformational change occurred not in the bulk but at the interface. Heterodyne-detected achiral and chiral VSFG are capable of addressing the molecular orientation and conformation of proteins at air/water interfaces. ■ INTRODUCTIONIn living organisms, most of the intracellular enzymic processes occur at phase boundaries, that is, the membrane−water interface. At interfaces, protein molecules change their conformation and molecular orientation from those in bulk and then function properly. Understanding the molecular functionality of proteins in living systems requires gaining information not only on bulk but also on interfacial properties. In technological applications, the characterization of interfacial proteins at the molecular level is essential for developing functioning biomaterials such as biocatalysis and biosensors. Thus, determination of secondary structure and molecular orientation of proteins at interfaces is highly important to understand and control the function of proteins. However, there are few methods that investigate interfacial proteins in situ without staining, 1−4 while the tools for studying their properties in bulk have been well established.Vibrational sum frequency generation (VSFG) spectroscopy is suited for studying molecules adsorbed at interfaces. VSFG is a second-order nonlinear spectroscopy, which provides us vibrational spectra reflecting molecular structure with high surface specificity. 5 So far, many studies of proteins at various interfaces have been performed by using homodyne-detected VSFG, and molecular level insight has been obtained. 6−12 Recently, VSFG has been extended to the heterodyne detection scheme, which enables us to determine the phase of the second-order nonlinear susceptibility, χ (2) . 13−15 The phase carries the information on the polar molecular orientation. Contrastingly, in the conventional homod...

Sum frequency generation vibrational spectroscopy (SFG-VS) for complex molecular surfaces and interfaces: Spectral lineshape measurement and analysis plus some controversial issues

“…There have been many developments in measuring phase-resolved spectra of the much narrower spectral features in the C-H stretching vibration region and protein amide band region, etc. [79,[177][178][179][180]. However, as discussed in Section 3.2, when there are non-negligible bulk contributions, such as the (3) χ mechanism from charges surfaces/interfaces, the notion that the imaginary SFG-VS spectrum contains only the absorptive term of the susceptibility can no longer be taken for granted, and the interpretation of the SFG-VS imaginary spectra based only on this notion needs to be cautioned and carefully examined [29], particularly for charged aqueous surfaces/interfaces [92,181] and aqueous surfaces/interfaces of accumulated ions [93,182].…”

Misfolding of a Human Islet Amyloid Polypeptide at the Lipid Membrane Populates through β-Sheet Conformers without Involving α-Helical Intermediates

“…SFG-VS, a second-order nonlinear optical technique, is a powerful tool for the identification of interfacial molecular structures and dynamics in situ and in real time. , It has been applied to determine the structures of peptides and proteins at different interfaces by probing amide I vibrations. − ,− Specially, the β-sheet structure is active in the amide I spectra with chiral-sensitive polarization measurements (such as psp and spp), whereas the α-helical or unordered structures are both silent. , SFG-VS is therefore an ideal method to characterize the transition between an α-helix and a β-sheet at the interface . Unfortunately, different β-sheet structural types (including β-hairpin-like monomers, β-sheet oligomers, and fibrils) all contribute to chiral amide I signals.…”

“…Because SFG signals rely on a tensor product of the IR transition dipole moment and the Raman polarizability tensor, − the amide II SFG signals are too weak to be detected in the disordered, α-helical structure and β-hairpin-like structure. Indeed, with the exception of one example of an amide II signal reported in an intermolecular β-sheet structure by Yan et al, the amide II SFG spectra have not been applied to address the issue of amyloid formation when the SFG technique was applied to investigate the interfacial proteins. − ,− However, some recent studies have shown that upon formation of multistranded β-sheet structures, long-range vibrational coupling from intermolecular β-sheet contacts can largely enhance the amide II signals − and make the amide II Raman spectra become visible; − see Table S1. For example, the amide II Raman signals are absent in the disordered α-synuclein and appear when α-synuclein starts to aggregate to form oligomeric β-sheet structures. , It can be naturally anticipated that amide II SFG signals are active in β-sheet oligomers and fibrils.…”

Impact of intermolecular vibrational coupling effects on the sum-frequency generation spectra of the water/air interface

“…In recent years, this technique has been very successful in providing vibrational spectra for various interfacial systems [13][14][15][16][17][18]. Among those, the water/air interface has been the most intensively studied system, both experimentally [18][19][20][21][22][23][24][25][26] and theoretically [11,18,[25][26][27][28][29][30][31][32][33][34][35][36].…”