PubMed · 479049
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D M Gibson, I D Ogden. 1979. A rapid method for purifying bacterial deoxyribonucleic acid.. https://doi.org/10.1111/j.1365-2672.1979.tb00839.x
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Unfolding of marginally stable proteins is a significant factor in commercial application of hydrophobic interaction chromatography (HIC). In this work, hydrogen-deuterium isotope exchange labeling has been used to monitor protein unfolding on HIC media for different stationary phase hydrophobicities and as a function of ammonium sulfate concentration. Circular dichroism and Raman spectroscopy were also used to characterize the structural perturbations experienced by solution phase protein that had been exposed to media and by protein adsorbed on media. As expected, greater instability is seen on chromatographic media with greater apparent hydrophobicity. However, increased salt concentrations also led to more unfolding, despite the well-known stabilizing effect of ammonium sulfate in solution. A thermodynamic framework is proposed to account for the effects of salt on both adsorption and stability during hydrophobic chromatography. Using appropriate estimates of input quantities, analysis with the framework can explain how salt effects on stability in chromatographic systems may contrast with solution stability.
A number of strategies have successfully extended plant proteomics into the bilayer domain. Important benefits can be afforded by including a well-resolved intact protein mass spectrum alongside peptide identification experiments; recent studies of thylakoid membranes have yielded new information on the primary structure and covalent post-translational modification of many of the integral proteins. Intact mass proteomics is advancing through the development of core technologies in separations and mass spectrometry, with the goal of providing comprehensive primary structure coverage that includes transmembrane domains with reliable measurements of protein abundance and turnover. To address the limitations associated with separation technologies such as 2D-gel electrophoresis, alternative systems are being investigated and 2D-liquid chromatography of thylakoid membrane proteins, using both denaturing and non-denaturing first dimensions, has been successful, extending separation space and providing intact protein solutions for electrospray-ionization mass spectrometry and top-down proteomics. High-resolution conventional, and Fourier-transform, mass spectrometry is bringing increasing resolution to tandem mass spectrometry allowing for 'top-down' mass spectrometry of intact proteins. Thus the core chromatographic technologies already developed for intact mass proteomics of integral membrane proteins also allow their 'top-down' analysis. Thylakoid membrane proteins with one and two transmembrane helices have been analysed demonstrating the ability of collision-activated dissociation (CAD) to sequence through transmembrane domains. When analysed by top-down proteomics, the small subunits of the cytochrome b6f complex from Arabidopsis thylakoids reveal novel post-transcriptional/translational modifications including the presence of glutamic acid at position 2 of PetL instead of the proline residue predicted from the gene sequence.
Membrane fractions from Micrococcus luteus catalyze the transfer of mannose from GDP-mannose to mono- and dimannosyldiacylglycerol, mannosylphosphorylundecaprenol (Man-P-Undec), and a membrane-associated lipomannan. This chapter describes the detergent solubilization, partial purification, and properties of Man-P-Undec synthase. The mobility of the mannosyltransferase activity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is a polypeptide with a molecular weight of approx 30.7 kDa. Utilizing the broad specificity of the bacterial mannosyltransferase provides a useful approach for the enzymatic synthesis of a wide variety of Man-P-polyisoprenol products.