The Roles Of Aromatic Amino Acid Residues In Membrane Protein Investigated By FTIR Spectroscopy

Biomembrane is the foundation of life, membrane protein is the major undertaker of biomembrane function and the important drug target, and the aromatic residues play vital roles in the dynamics of membrane and membrane protein. So the study of the influence of membrane dynamics by interacting with aromatic residues containing peptides/protein and the effect of aromatic residues on the membrane protein/ligand dynamics are very important. Recently, fourier transform infrared spectroscopy (FTIR) combined with advanced accessory techniques, such as attenuated total reflection (ATR), low temperature transmission and time-resolved spectroscopy (TRS), etc. It has been become one of the important methods in biophysics field to investigate the dynamics of lipid membrane, membrane protein and ligand extensively with many significant advantages. In this thesis, we studied the influence of unsaturated lipid bilayers dynamics and structure by aromatic amino acid methyl esters and aromatic residues containing peptides via polarised ATR-FTIR technique, quantum mechanical (QM) calculations, and fluorescene spectroscopy. Moreover, we investigated the dynamics and structural changes of [10,11,14,15-13C4]-all-trans-retinal reconsituted wild type bacteriorhodopsin (WT-BR) and its mutation (Y185F-BR) in photocycle process by using low temperature transmission and isotopic labelling. Some new proposals were suggested. The main points and details of this thesis are as follows:In Chapter three, we investigated the effect of unsaturated 2-oleoyl-l-pamlitoyl-sn-glyecro-3-phosphocholine (POPC) acyl chains packing by a series of hydrophobic and aromatic amino acid methyl esters using polarised ATR-FTIR technique combined with the QM calculations. We suggested that the =C-H and C=C stretching vibrational modes of the C=C bond in middle of POPC acyl chain were used to probe the dynamics and structural changes of POPC membrane bilayers at the first time. ATR-FTIR measurements show that the=C-H and C=C stretching vibrational bands of POPC present evident red-shifts when aromatic amino acid methyl esters are mixed into membrane bilayers. The QM calculations further demonstrate that the red shifts of the =C-H and C=C stretching modes for POPC acyl chains are not through a direct interaction between the side chain groups and the HC=CH motif but are mainly caused by the conformational changes of POPC acyl chains and the decrease of acyl chains order, in which the HC=CH motif is packed closed to the other one of adjacent acyl chains with the dispersion interactions enhancing.In Chapter four, we describe the changes of acyl chains order and membrane dynamics of binary unsaturated lipid bilayers by studying the pure POPC bilayers, 2-oleoyl-1-pamlitoyl-sn-glyecro-3-glycerol (POPG) bilayers and POPC/POPG bilayers using polarised ATR-FTIR technique. The interactions between the POPC/POPG membrane and three model peptides containing aromatic and basic residues were employed to investigate the effects of the peptide structure on the membrane dynamics and the acyl chains order of POPC/POPG bilayers. ATR-FTIR spectroscopic studies present that the acyl chains order of POPC/POPG bilayers decreases and the conformation changes compared with that of pure POPC or POPG bilayers, evidenced by the obvious red shifts of v(=C-H) and v(C=C) frequencies. Besides, the chains order of POPC/POPG bilayers decreases significantly when Gravin was added compared with the adding of Vamp2 or Antp. Fluorescence spectroscopic measurements further show that the tryptophan of three peptides may interact with the hydrophobic region of POPC/POPG bilayers, thus resulting in the conformational order change of POPC/POPG acyl chains.In Chapter five, the synthesis of [10,11,14,15-13C4]-labeled all-trans-retinal was started from β-ionone as starting material and [1,2-13C2]-acetonitrile as isotope labeling unit. We perform the analysis of the low-temperature FTIR difference spectroscopy of [10,11,14,15-13C4]-all-trans-retinal reconsituted WT-BR and Y185F-BR in K, L, M, N intermediate states compared with WT-BR and Y185F-BR. The 13C isotope-edited effect can be used to make a plenty of IR spectroscopic information for v(C-C) region of retinylidene. The mutation of Y185 makes a significant difference between Y185F-BR and WT-BR in M intermediate state, it further demonstrates that the Y185 plays a key role in the retinal conformational stabilization. It enables us to investigate the conformational changes and activation mechanism of retinal and its binding pocket in the other microbial rhodopsin during photoisomerization process at the atomic level further.