ABSTRACT
The second virial coefficient, or B value, is a measurement of how well a protein interacts with itself in solution. These interactions can lead to protein crystallization or precipitation, depending on their strength, with a narrow range of B values (the 'crystallization slot') being known to promote crystallization. A convenient method of determining the B value is by self-interaction chromatography. This paper describes how the light-harvesting complex 1-reaction centre core complex from Allochromatium vinosum yielded single straight-edged crystals after iterative cycles of self-interaction chromatography and crystallization. This process allowed the rapid screening of small molecules and detergents as crystallization additives. Here, a description is given of how self-interaction chromatography has been utilized to improve the crystallization conditions of a membrane protein.
Subject(s)
Bacteria/enzymology , Bacterial Proteins/chemistry , Chromatography, Affinity , Crystallization/methods , Light-Harvesting Protein Complexes/chemistry , Catalytic Domain , Chemical Precipitation , Crystallography, X-Ray , Protein BindingABSTRACT
The C-terminal part of tropomodulin protein 1, isoform A, from Caenorhabditis elegans was expressed in Escherichia coli and purified to homogeneity. Optimized from the initial nanoscreen, crystals grew to dimensions of 0.25 x 0.15 x 0.15 mm at 277 K using 28.0%(v/v) PEG 400 as the precipitant by the hanging-drop vapor-diffusion technique. A data set of 94.9% completeness was collected to a resolution of 1.98 A at 100 K using a synchrotron X-ray source (SER-CAT). The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 31.7, b = 50.6, c = 107.1 A, and contained one molecule per asymmetric unit.
Subject(s)
Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans/metabolism , Recombinant Proteins/chemistry , Amino Acid Sequence , Animals , Crystallization , Crystallography, X-Ray , Escherichia coli/metabolism , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Tropomodulin , X-Ray DiffractionABSTRACT
Cytoskeleton-associated proteins (CAPs) are involved in the organization of microtubules and transportation of vesicles and organelles along the cytoskeletal network. A conserved motif, CAP-Gly, has been identified in a number of CAPs, including CLIP-170 and dynactins. The crystal structure of the CAP-Gly domain of Caenorhabditis elegans F53F4.3 protein, solved by single wavelength sulfur-anomalous phasing, revealed a novel protein fold containing three beta-sheets. The most conserved sequence, GKNDG, is located in two consecutive sharp turns on the surface, forming the entrance to a groove. Residues in the groove are highly conserved as measured from the information content of the aligned sequences. The C-terminal tail of another molecule in the crystal is bound in this groove.
