Abstract

1. Salt Screen Standard Screen # # Hits Hits Precipitant Precipitant Salt Salt Buffer Buffer Like Like 75 73 8 11 30%(w/v) PEG 4000 30%(w/v) PEG 8000 0.2 M Li2SO4 0.2 M (NH4)2SO4 0.1 M Cacodylate pH 6.5 0.1 M Tris pH 8.5 H1 #15 H1 #17 41 75 7 6 30%(w/v) PEG 4000 30%(w/v) PEG 8000 0.2 M Li2SO4 0.2 M Li2SO4 0.1 M Tris pH 8.5 0.1 M NaAcetate pH 4.5 H1 #17 W1 #17 76 85 5 6 2.0 M (NH4)2SO4 20%(w/v) PEG 8000 0.2 M MgAcetate 0.1 M Cacodylate pH 6.5 H1 #32 H1 #18 A B C D 70 89 5 5 1.4 M Na Citrate 30%(w/v) PEG 4000 0.2 M (NH4)2SO4 0.1 M HEPES pH 7.5 0.1 M NaCitrate pH 5.6 H1 #38 H2 #9 43 70 5 4 30%(w/v) PEG 4000 20%(w/v) PEG 8000 0.2 M MgCl2 0.2 M (NH4)2SO4 0.1 M NaCitrate pH 5.6 0.1 M Tris pH 8.5 H2 #9 W2 #2 2 5 20%(w/v) PEG 3000 0.1 M NaCitrate pH 5.5 W1 #06 E F G H I Our model protein-RhoGDI The human RhoGDI is a relatively small cytosolic protein which was identified as a regulator of Rho family GTPases. Basically, they play a crucial role in a variety of cellular function and act as molecular switches between inactive (GDP-bound) and active (GTP-bound) forms. We have chosen RhoGDI as our model protein due to it is well expressed, easy to purify but recalcitrant for crystallization. Ser Thr His Tyr Totals Salt Unique Salt Overlaps Standard Standard Salt Overlaps Standard Standard Overlaps Standard Unique Salt Salt Overlaps Amino acid repertoire is highly encouraging for surface entropy reduction because of Lys/Glu content of nearly 20% what is common among cytosolic regulatory proteins. Preparation of high quality protein crystals by surface entropy reduction: tyrosine as a crystal-contact catalyst.T. Boczek, M. Sikorska, K. Grelewska, M. Pinkowska, M. Zawadzki, D. Cooper and Z.S. DerewendaPSI Center for Structure and Function Innovation and Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, 22908 Abstract Crystallization is a limiting step in macromolecular crystallography. In cases where proteins are particularly recalcitrant to crystallization efforts, mutational modification of surface properties may be essential. We previously suggested that targeted replacement of clusters of residues with high-conformational entropy (lysines, glutamates and/or glutamines) with alanines leads to formation of epitopes that are capable of mediating crystal contacts (1). This is because the entropic cost of immobilizing large side chains at the intermolecular contact regions has been either eliminated, and crystal contacts can be easily formed by the mutated epitopes. This method allowed for successful crystallization and structure determination of a number of novel proteins or to grow crystals diffracting to significantly higher resolution than those of the wild-type form. However, it has not been conclusively demonstrated if alanine constitutes the best choice for replacement of high-entropy residues. Here we present a systematic study of the replacement of nine Lys/Glu-rich patches in RhoGDI with Ser, Thr, His and Tyr residues. All four amino acids are known to occur at interfaces with significantly higher incidence than Lys or Glu/Gln, and therefore can potentially mediate possible weak protein-protein interactions leading to crystal formation. Preliminary data indicate that tyrosines may be particularly helpful, as RhoGDI mutants with Tyr-rich patches give numerous hits in crystallization screens. So far we solved crystal structures of two mutants, K(135,138,141)Y and K(138,141)Y, and we note that Tyr directly mediates crystal contacts through either homotypic (symmetric) or heterotypic (head-to-tail) intermolecular interactions. The quality of these crystals is high, with data extending to 2.4 Å and 2.2 Å, resusing an in-house conventional X-ray source. Results Distribution of HitsStandard Screen Distribution of HitsNaCl Screen The Best Solutions The Best Solutions Tyrosines and histidines directly mediate crystal contact forming Below some representative solved structures are presented. Using SER method we proofed that not only alanine mutation are useful for crystal forming but either histidines and particularly tyrosines. The insight in these mutants structures revealed that new incorporated amino acides playing major role in crystal contact building. However, their nature varys according to mutated patches but generally we observed hydrophobic interactions leading to crystal forming. Amino acids analysis by ProtParam RhoGDI K138K141-Y The Mutant Series 0.1M bicine pH=7.5 30% PEG 6000 32% PEG 8000, 0.22M (NH4)2SO4, 0.1M cacodylate pH=6.5 Resolution 2.1Å Spacegroup P21 (a=32.0,b=55.1,c=38.9,=107.5) Resolution 2.3 A Space group P21( a=39.0.; b=55.8; c=61.2; ß=92.4) Resent studies showed that K A and E A mutations can strongly affect protein crystallization. Here we present a new promising approach to enhancing protein crystalizability by introducing Tyr, His, Ser, Thr residues instead of alanines. We investigated direct relationship between the type of mutation and intermolecular key contacts resulting in crystals. RhoGDI K135K138K141-H Expression, purification and screening process • All RhoGDI mutants were expressed as a GST-fusion protein, cleaved with rTEV, purified and crystallize using vapour diffusion method. • Two different kind of screens were set up: • Standard Screen Drops of Super Screen reagent + protein Reservoir is 100 l of Super Screen reagent • Protein concentration ~15mg/ml RhoGDI fused to GST-taq RhoGDI from Superdex75 1.4 M Na Citrate, 0.1 M HEPES pH 7.0 – #89 Resolution 2.0 Å Spacegroup P3221 (a=b=75.3,c=91.283) • NaCl Screen • Drops of Super Screen reagent + protein • Reservoir is 100 l of 1.5 M NaCl