Accurate Determination of Interfacial Protein Secondary Structure by Combining Interfacial-Sensitive Amide I and Amide III Spectral Signals

Abstract

Accurate determination of protein structures at the interface is essential to understand the nature of interfacial protein interactions, but it can only be done with a few, very limited experimental methods. Here, we demonstrate for the first time that sum frequency generation vibrational spectroscopy can unambiguously differentiate the interfacial protein secondary structures by combining surface-sensitive amide I and amide III spectral signals. This combination offers a powerful tool to directly distinguish random-coil (disordered) and α-helical structures in proteins. From a systematic study on the interactions between several antimicrobial peptides (including LKα14, mastoparan X, cecropin P1, melittin, and pardaxin) and lipid bilayers, it is found that the spectral profiles of the random-coil and α-helical structures are well separated in the amide III spectra, appearing below and above 1260 cm(-1), respectively. For the peptides with a straight backbone chain, the strength ratio for the peaks of the random-coil and α-helical structures shows a distinct linear relationship with the fraction of the disordered structure deduced from independent NMR experiments reported in the literature. It is revealed that increasing the fraction of negatively charged lipids can induce a conformational change of pardaxin from random-coil to α-helical structures. This experimental protocol can be employed for determining the interfacial protein secondary structures and dynamics in situ and in real time without extraneous labels.

Facet-Specific Adsorption of Tripeptides at Aqueous Au Interfaces: Open Questions in Reconciling Experiment and Simulation

“…A few recent studies have also reported use of techniques such as nuclear magnetic resonance (NMR) and sum frequency generation vibration spectroscopy to provide information about the structure of biomolecules adsorbed at aqueous interfaces. [21][22][23][24] While valuable, such studies are not yet part of standard analyses, and therefore obtaining unambiguous information on the structures of peptides adsorbed at aqueous interfaces remains challenging. The difficultly in obtaining structural information regarding adsorbed peptides is compounded by the fact that many materials-binding peptides are intrinsically disordered peptides (IDPs), 25,26 implying that these biomolecules cannot be adequately characterized by a single representative structure (or a few such structures) in the adsorbed state; on the contrary, these systems are better captured as an ensemble of structures.…”

Amide III SFG Signals as a Sensitive Probe of Protein Folding at Cell Membrane Surface

“…Recently we employed several technical procedures to enhance the SFG signals in the fingerprint region and successfully probed the weak amide III signals. 29 We have demonstrated that the complicated 3D structures of proteins could effectively be deduced by probing the vibrational modes in the fingerprint region (such as amide III vibrations) combined with the functional groups of the amide I backbone vibrations. 28,29 Here, the amide I vibrations arise mainly from the C = O stretching vibration, whereas the amide III vibrations come from the in-phase combinations of C-N stretching and N-H bending.…”

“…29 We have demonstrated that the complicated 3D structures of proteins could effectively be deduced by probing the vibrational modes in the fingerprint region (such as amide III vibrations) combined with the functional groups of the amide I backbone vibrations. 28,29 Here, the amide I vibrations arise mainly from the C = O stretching vibration, whereas the amide III vibrations come from the in-phase combinations of C-N stretching and N-H bending. It was particularly notable that with these spectral fingerprints, SFG-VS is able to unambiguously differentiate the interfacial random-coil (or loop) and α-helical structures in proteins.…”

“…It was particularly notable that with these spectral fingerprints, SFG-VS is able to unambiguously differentiate the interfacial random-coil (or loop) and α-helical structures in proteins. 28,29 One can thus naturally anticipate that such ability could enable the direct observation of the early steps of folding and the dynamics of α-helical formation within 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 5 membrane environments. It is known that the membrane protein folding process can be regulated by the membrane parameters such as lipid chain length, fluidity, and head group charge.…”

Phase behavior of a binary lipid system containing long- and short-chain phosphatidylcholines

“…The planar bilayer region enriched in the long-chain lipids provides a favorable environment to study membrane proteins using various techniques, such as nuclear magnetic resonance (NMR), [4][5][6][7][8][9][10][11] X-ray crystallography, [12][13][14][15][16] and spectroscopy techniques. [17][18][19][20][21] Bicelles can also be used as delivery vehicles for membrane proteins or drugs, 22,23 or as templates for the synthesis of platinum nanowheels.…”