4 research outputs found
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Intra-molecular cross-linking of acidic residues for protein structure studies.
Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would be useful in developing structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine selective reagents that can cross-link lysine residues to other lysine residues or the amino terminus. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution, and solvent accessibility of the lysines in the protein sequence. To overcome these limitations we have investigated the use of cross-linking reagents that can react with other reactive sidechains in proteins. We used 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E), and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO sidechains can react to form 'zero-length' cross-links with nearby primary amine containing resides, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO sidechains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker ann of variable length. Using these reagents, we have found three new 'zero-length' cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18, and K63-E64). Using the dihydrazide cross-linkers, we have identified 2 new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 angstroms. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry, increasing the probability that the protein target of choice will yield sufficient distance constraints to develop a structural model
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Chemical crosslinking and mass spectrometry studies of the structure and dynamics of membrane proteins and receptors.
Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurement using liquid chromatography-fourier transform mass spectrometry and an automated data analysis pipeline. Assignments were confirmed and, if necessary, resolved, by tandem MS. The relative reactivity of lysine residues participating in cross-links was evaluated by labeling with NHS-esters. A distinct pattern of cross-link formation within the C-terminal domain, and between loop I and the C-terminal domain, emerged. Theoretical distances based on cross-linking were compared to inter-atomic distances determined from the energy-minimized X-ray crystal structure and Monte Carlo conformational search procedures. In general, the observed cross-links can be explained by re-positioning participating side-chains without significantly altering backbone structure. One exception, between C3 16 and K325, requires backbone motion to bring the reactive atoms into sufficient proximity for cross-linking. Evidence from other studies suggests that residues around K325 for a region of high backbone mobility. These findings show that cross-linking studies can provide insight into the structural dynamics of membrane proteins in their native environment
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The interfacial bioscience grand challenge.
This report is broken down into the following 3 sections: (1) Chemical Cross-linking and Mass Spectrometry Applied to Determination of Protein Structure and Dynamics; (2) Computational Modeling of Membrane Protein Structure and Dynamics; and (3) Studies of Toxin-Membrane Interactions using Single Molecule Biophysical Methods
Photoelectron Spectroscopy of Reactive Intermediates
Photoelectron Spectroscopy (PES) has been used to investigate the structure and thermochemistry of a number of alkyl radicals and their corresponding carbonium ions. The radicals have been produced by flash vacuum pyrolysis of alkyl nitrites. The shape of the first band in the photoelectron spectrum of a free radical is related to the structural changes that take place in forming the carbonium ion from the radical. The ionization potentials obtained from the photoelectron spectra of the radicals are combined with gas phase ion thermochemistry data to obtain alkyl radical heats of formation. The thermochemical data thus obtained is used to discuss substituent and structural effects on the stability of radicals and carbonium ions. In many cases the thermolytic decomposition pathways of the alkyl radicals have been elucidated using PES. The application of the PES technique to the analysis of reactive intermediates present in heterogeneous thermolysis mixtures is also discussed.
Chapter 1 presents an introduction to the technique employed in these studies, and a review of the studies performed in this laboratory. Chapter 2 presents results on the thermochemistry and structure of the 1- and 2-adamantyl radicals. The tricyclic 1-adamantyl radical and carbonium ion are important as model bridgehead compounds, and the question of the amount of strain energy caused by the non-planarity of the radical and ion center has been of great interest. The first bands in the photoelectron spectra of the o-, m- and p-methylbenzyl radicals are presented in Chapter 3. The methyl substituent effects on the stabilities of the radicals and ions are discussed.
In Chapter 4 results on investigations of heterogeneous processes in Chemical Vapor Deposition (CVD) systems using the chlorosilanes as feed gases are discussed. SiCl₂ is found to be the major silicon containing reactive intermediate produced by surface reactions at 600 - 1100 °C in CVD systems using dichlorosilane and trichlorosilane as feed gases.
Chapter 5 presents the spectra of the 1- and 2-methylnaphthyl radicals. The relative stabilities of the radicals and carbonium ions are discussed based on proton affinities determined by Fourier transform mass spectrometric equilibrium studies, combined with ionization potentials obtained from photoelectron spectra of the radicals.</p