Author : Jingshu Guo
Publisher :
ISBN 13 :
Total Pages : 405 pages
Book Rating : 4.:/5 (867 download)
Book Synopsis Structural Analysis of Macromolecular Complexes Using Electrospray Ionization Mass Spectrometry Based Approaches by : Jingshu Guo
Download or read book Structural Analysis of Macromolecular Complexes Using Electrospray Ionization Mass Spectrometry Based Approaches written by Jingshu Guo and published by . This book was released on 2013 with total page 405 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry (MS) has become one of the most robust, reliable and widely used analytical techniques in scientific research due to factors like its speed of analysis, ease of operation, high sensitivity, and applicability to a broad range of analytes. The development of the so-called "soft" ionization methods electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) played a pivotal role in the application of MS to biochemical and biophysical fields enabling comprehensive studies of biomolecules, macromolecules and their complexes. This dissertation presents method development and the application of MS-based strategies for complete sequence and structure determination of hemoglobin (Hb); investigation of the interaction between human Hb and Band 3; characterization of the interaction between Alanyl-tRNA synthetase with tRNA and various ligands; and characterization of various thiol-protected silver nanoclusters. First, the complete primary amino acid sequences of hemoglobin from the endangered species snow leopard (Uncia uncia), Amur tiger (Panthera tigris altaica), Cheetah (Acinonyx jubatus) and Francois' Langur (Trachypithecus francoisi) were determined using a combination of the bottom-up approach to proteomics in combination with single crystal X-ray diffraction. In this approach, MS was used to provide on average 70% coverage of the sequences, which was used in refinement of the diffraction data. The electron density maps from the diffraction data were used to determine the parts of the sequences not covered by MS as well as differentiate between isomeric residues. During the refinement of the X-ray diffraction data, a unique conformational state, the Bform, for naturally occurring ligated hemoglobin was discovered in two feline Hbs. Structural comparisons and possible biological relevance for the B-form will be presented. The binding of human hemoglobin to synthetic peptides corresponding to the Hb-binding sites of human erythrocyte Band 3 is also presented. The effects on binding of ligation and oxidation state of normal human HbA; human sickle HbS; Hb-binding site and peptide length; and Band 3 N-terminal acetylation were investigated. The optimization of MS instrumental parameters and solution conditions for stabilizing and protecting labile thiol-protected silver nanoclusters through ionization, mass analysis and detection is presented. The relatively much more stable and well characterized gold-glutathione cluster Au25(SG)18 was used as a model for instrumental optimization (SG, glutathione). The most crucial instrumental parameters to protecting the clusters were cone gas flow rate, trap/transfer collision energy, and source temperature. Using these optimized parameters and adjustment of solution conditions led to the formula assignment for Ag32(SG)19, the species isolated from band 6 of a polyacrylamide gel electrophoretic separation. Using a similar approach the formula assignment for Ag44L30-4 (L, p-mercaptobenzoic acid) was confirmed. Collision induced dissociation (CID) studies were used to demonstrate that Ag43L28-3, another abundant species in the mass spectra was in fact a fragment of the intact cluster from the facile loss of AgL2-. CID and ion mobility mass spectrometry (IMMS) of Au25(SG)18, Ag32(SG)19, and Ag44L30-4 were used to gain insight into the surface structure and stability of the fragile silver clusters. Finally, the 3'-tRNA binding site on E. coli Alanyl-tRNA synthetase was identified by first crosslinking the 3'-periodate-oxidized tRNAala to the enzyme followed by the bottom-up MS sequencing. Modified lysine residues identified were 74, 526 585, 637, 739 and 648. Lys 74 was in the aminoacylation domain while 526 585, 637, 739 and 648 were in the editing domain. These results highlight an alteration of the 3'-terminal of tRNAala interaction with the enzyme. To study the stability ARS with various ligands, non-hydrolysable adenylate analogs, ASAd and GSAd, were used. Data shows that complete binding of ASAd and GSAd was achieved and that binding occurred in a 1:1 stoichiometry. Binding of ASAd, the analog of the cognate ligand, stabilizes the enzyme against changes in solution pH relative to the apo-enzyme and the enzyme with GSAd bound.
