Lubica Urbanikova

Contribution of hydrogen bonds to protein stability

Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein.

Preparation, Crystallization and Preliminary X-Ray Analysis of the Fab Fragment of Monoclonal Antibody MN423, Revealing the Structural Aspects of Alzheimers Paired Helical Filaments

Monoclonal antibody (mAb) MN423 recognizes Alzheimers disease specific conformation of tau protein assembled into paired helical filaments (PHF). Since the three-dimensional structure of PHF is currently unavailable, the structure of MN423 binding site could provide important information about PHF conformation with the consequences for the Alzheimers disease prevention and cure. Fab fragment of MN423 was prepared and purified. We have identified two different conditions for crystallization of the Fab fragment that yielded two crystal forms. They diffracted to 3.0 and 1.6 A resolution with four and one molecule in the asymmetric unit, respectively. Both crystal forms belonged to the space group P2(1) with unit cell parameters a = 76.4 A, b = 138.4 A, c = 92.4 A, beta = 101.9 degrees , and a = 71.5 A, b = 36.8 A, c = 85.5 A, beta = 113.9 degrees .

Purification, crystallization and preliminary X-ray analysis of two crystal forms of ribonuclease Sa3.

RNase Sa3 produced by Streptomyces aureofaciens strain CCM 3239 belongs to the T1 family of microbial ribonucleases. It is closely related both to RNase Sa, studied in detail earlier, and to RNase Sa2 produced by the same microorganism. The most important property of RNase Sa3 is the relatively high cytotoxic activity, which was not observed for RNase Sa and Sa2. Recombinant RNase Sa3 was overexpressed in Escherichia coli and purified to high homogeneity. The hanging-drop vapour-diffusion method was used for crystallization. The two crystal forms are trigonal P3(1)21 and tetragonal P4(1)2(1)2, with unit-cell parameters a = b = 64.7, c = 69.6 A, gamma = 120 degrees and a = b = 34.0, c = 147.2 A, respectively. They diffract to 2.0 and to 1.7 A resolution, respectively, using synchrotron radiation. The asymmetric units of crystal forms I and II contain one molecule of the enzyme, which corresponds to V(M) = 3.8 A(3) Da(-1) with a solvent content of 68% and V(M) = 1.9 A(3) Da(-1) with a solvent content of 37%, respectively.

Purification, crystallization and preliminary X-ray analysis of the plectin actin-binding domain

Plectin is an abundantly expressed cytoskeletal crosslinking protein of enormous size (>500 kDa) and multiple functions. It represents one of the many members of a large family of actin-binding proteins. The actin-binding domain of mouse plectin was expressed in Escherichia coli and purified to homogeneity. Crystals of the actin-binding domain of plectin were prepared by the hanging-drop method. They belong to space group P2(1), with unit-cell parameters a = 55.92, b = 108.92, c = 63.75 A, beta = 115.25 degrees. Data from a single crystal were collected to 2.0 A resolution at room temperature using synchrotron radiation at EMBL, Hamburg. The asymmetric unit contains two molecules of the protein, which corresponds to V(M) = 3.06 A(3) Da(-1) and a solvent content of 60%. The structure was solved by the molecular-replacement method. In addition, the preparation of selenomethionine-derivative crystals is described.

Crystallization and preliminary X-ray investigation of the complex of RNase Sa with wild-type barstar

RNase Sa, an extracellular ribonuclease produced by Streptomyces aureofaciens, is inhibited by barstar, the natural protein inhibitor of barnase, the ribonuclease of Bacillus amyloliquefaciens. The complex of RNase Sa with wild-type barstar was crystallized by hanging-drop vapour diffusion. It was shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis that RNase Sa and barstar are present in equimolar proportions in the crystals. The crystals are in the hexagonal space group P65 with unit cell dimensions a = b = 56.95, c = 135.8 A. They diffract to 1.7 A resolution at the DESY synchronton source. The asymmetric unit contains one molecule of the complex.