Novel Raman Studies of the Conformation and Dynamics of Biological Molecules

Analytical chemistry, Computational chemistry, Biochemistry

Ford, Steven John

University:University of Glasgow

关键词: Analytical chemistry, Computational chemistry, Biochemistry;   

Others  :  http://theses.gla.ac.uk/74648/1/10992270.pdf

来源: University of Glasgow

【 摘 要 】

Raman optical activity (ROA) is developing into a new, and unique, form of biological spectroscopy and this thesis endeavours to determine the new insights that ROA provides into the conformational and dynamic properties of peptides and proteins. The peptide ROA analysis used small oligopeptides to obtain a clear idea of the fundamental aspects of polypeptide ROA. The ROA signal originating from the vibrations of the amide link were scrutinized particularly closely using the alanyl peptide series Ala2 to Ala5. The amide III vibration (Calpha-H and N-H deformations) proved to be the easiest, and most reliable, band to measure experimentally. The coupling of the Calpha-H and N-H deformations can be seen clearly in both the Raman and the ROA spectra. The amide III Raman and ROA analysis concurs with recent normal mode calculations. As the alanyl series expands delocalization of the amide III band can be seen. It is clear that the delocalization is dependent upon the N-H deformations because no changes are seen in the Calpha-H deformations as the deuterated alanyl peptide (with low frequency N-D deformations) series expands. Other peptides were also studied by ROA, and this gave a greater insight into the peptide ROA observed from all the molecules under examination. Several alanyl ROA assignments were confirmed by the work. The amide III ROA differed between peptides, indicating conformational changes between the molecules. However, the Calpha-H deformations from deuterated peptides were seen to give similar ROA band envelopes in all the peptides studied, indicating again the conformational sensitivity of the amide III ROA signal and the importance of the N-D deformation. Significant amide I and amide II Raman and ROA characteristics were also observed. The protein studies concentrated on hen egg white lysozyme (HEWL) and bovine alpha-lactalbumin (BAL), two well studied and understood proteins. The Raman and ROA spectra of the native, denatured, inhibitor bound, deuterated and partially deuterated states of HEWL have been observed, as have the native, molten globule and metal free states of BAL. It has become apparent that the amide I vibration produces an ROA couplet in most protein states, while the amide III ROA is very sensitive to protein conformation. Several of the ROA bands associated with the amide III vibration have been assigned, and particularly interesting are the bands that are sensitive to rigid, dynamic or disordered structural features. A simplistic theoretical model, that explains some of the ab initio ROA calculations and the general 'couplet' nature of the protein amide III ROA signal, is outlined in the final chapter. Although the ROA signals from proteins cannot be assigned with complete confidence, it is clear that ROA is rapidly developing into a new form of biological spectroscopy, with the exciting possibility of probing the dynamic aspects of protein structure.