Home  About us  Contact
 

Membrane-protein Crystallization (membrane-protein + crystallization)

Distribution by Scientific Domains
Chemistry100%

Selected Abstracts

Effects of impurities on membrane-protein crystallization in different systems

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2009
Christopher A. Kors
When starting a protein-crystallization project, scientists are faced with several unknowns. Amongst them are these questions: (i) is the purity of the starting material sufficient? and (ii) which type of crystallization experiment is the most promising to conduct? The difficulty in purifying active membrane-protein samples for crystallization trials and the high costs associated with producing such samples require an extremely pragmatic approach. Additionally, practical guidelines are needed to increase the efficiency of membrane-protein crystallization. In order to address these conundrums, the effects of commonly encountered impurities on various membrane-protein crystallization regimes have been investigated and it was found that the lipidic cubic phase (LCP) based crystallization methodology is more robust than crystallization in detergent environments using vapor diffusion or microbatch approaches in its ability to tolerate contamination in the forms of protein, lipid or other general membrane components. LCP-based crystallizations produced crystals of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides from samples with substantial levels of residual impurities. Crystals were obtained with protein contamination levels of up to 50% and the addition of lipid material and membrane fragments to pure samples of RC had little effect on the number or on the quality of crystals obtained in LCP-based crystallization screens. If generally applicable, this tolerance for impurities may avoid the need for samples of ultrahigh purity when undertaking initial crystallization screening trials to determine preliminary crystallization conditions that can be optimized for a given target protein. [source]

A rapid method for assessing lipid:protein and detergent:protein ratios in membrane-protein crystallization

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2003
Corrie J. B. DaCosta
A simple procedure for rapidly measuring lipid:protein ratios and detergent concentrations at different stages of the solubilization, purification and crystallization of membrane proteins has been developed. Fourier-transform infrared spectra recorded from 10,µl aliquots of solution using a single-bounce diamond-attenuated total reflectance apparatus exhibit characteristic bands arising from the vibrations of lipid, protein and detergent. Lipid:protein molar ratios as low as 5:1 (for a protein with a molecular weight of 300,kDa) are determined by comparing the ratio of the integrated intensity of the lipid ester carbonyl band near 1740,cm,1 with the protein amide I band near 1650,cm,1. Detergent concentrations at levels well below the critical micellar concentration of most detergents are determined by comparing the integrated intensities of the detergent vibrations, particularly in the 1200,1000,cm,1 region, with a standard curve. Protein amide I band-shape analysis provides insight into the effects of detergents on protein secondary structure. The importance of monitoring detergent concentration changes during simple procedures, such as the concentration of a membrane protein by ultrafiltration, is demonstrated. This analytical tool has been used to rapidly establish protocols for minimizing lipid and detergent levels in solubilized membrane-protein samples. [source]

There is a baby in the bath water: AcrB contamination is a major problem in membrane-protein crystallization

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2008
David Veesler
In the course of a crystallographic study of the Methanosarcina mazei CorA transporter, the membrane protein was obtained with at least 95% purity and was submitted to crystallization trials. Small crystals (<100,µm) were grown that diffracted to 3.42,Å resolution and belonged to space group R32, with unit-cell parameters a = b = 145.74, c = 514.0,Å. After molecular-replacement attempts using available CorA structures as search models failed to yield a solution, it was discovered that the crystals consisted of an Escherichia coli contaminating protein, acriflavine resistance protein B (AcrB), that was present at less than 5% in the protein preparations. AcrB contamination is a major problem when expressing membrane proteins in E. coli since it binds naturally to immobilized metal-ion affinity chromatography (IMAC) resins. Here, the structure is compared with previously deposited AcrB structures and strategies are proposed to avoid this contamination. [source]