Sum frequency generation vibrational spectroscopy(SFG-VS)has been demonstrated to be a powerful technique to study the interfacial structures and interactions of biomolecules at the molecular level.Yet most previous studies mainly collected the SFG spectra in the frequency range of 1500–4000 cm-1,which is not always sufficient to describe the detailed interactions at surface and interface.Thorough knowledge of the complex biophysicochemical interactions between biomolecules and surface requires new ideas and advanced experimental methods for collecting SFG vibrational spectra.We introduced some advanced methods recently exploited by our group and others,including(1)detection of vibration modes in the fingerprint region;(2)combination of chiral and achiral polarization measurements;(3)SFG coupled with surface plasmon polaritons(SPPs);(4)imaging and microscopy approaches;and(5)ultrafast time-resolved SFG measurements.The technique that we integrated with these advanced methods may help to give a detailed and high-spatial-resolution 3D picture of interfacial biomolecules.
The amide A band of protein is sensitive to the hydrogen bands of amide groups of proteins. However, it is hard to distinguish the amide A band of aqueous protein in situ directly, since it overlaps with O-H stretching vibration of water. In this work, we presented a new analytical method of Raman ratio spectrum, which can extract the amide A band of proteins in water. To obtain the Raman ratio spectrum, the Raman spectrum of aqueous protein was divided by that of pure water. A mathematical simulation was employed to examine whether Raman ratio spectrum is effective. Two kinds of protein, lysozyme and （^-chymotrypsin were employed. The amide A bands of them in water were extracted from Raman ratio spectra. Additionally, the process of thermal denaturation of lysozyme was detected from Raman ratio spectrum. These results demonstrated the Raman ratio spectra could be employed to study the amide A modes of proteins in water.
Dehydration of a surface is the first step for the interaction between biomolecules and the surface. In this study, we systemati- cally investigated the influence of cholesterol analog 6-ketocholestanol （6-KC） on the dehydration of model cell membrane, using sum frequency generation vibrational spectroscopy. In pure DI water environment, two separate dehydration dynamic components were observed in neutrally charged and isotopically labeled 1,2-dimyristoyl-sn-glycero-3-phosphocholine （DMPC） and positively charged 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine（chloride salt） （DMEPC） bilayer： a large-amplitude fast component and a small-amplitude slow component, which originated from the water molecules with a weak and a strong water-membrane bound strengths, respectively. Dehydration of a negatively charged mixed DMPC/DMPG bilayer lead to the membrane-bound water being reorganized to ordered structures quickly. It is evident that the water-membrane bound strengths depend largely on the charge status of the lipid and has an order of neutrally charged membrane〈〈positively charged mem- brane〈〈negatively charged membrane. In an ionic environment, KC1 solution can not only dehydrate DMPC bilayer, but also prevent the 6-KC fiom further dehydrating this model cell membrane. We observed that the dehydration dynamics behavior of DMPC bilayer in the presence of the chaotropic anions is similar to that of the negatively charged DMPG bilayer because of the penetration of chaotropic anions into the DMPC bilayer. The degree of dehydration difficulty in kosmotropic anions fol- lows a Hofmeister series and linearly correlates with the hydration Gibbs free energy of the anions. Our results provide a molecular basis for the interpretation of the Hofmeister effect of kosmotropic anions on ion transport proteins.
