Structural studies of oligometric enzymes

The functions of biological macromolecules are discussed in relation to their structure as linear polymers adopting specific conformations. If such molecules can be crystallised, their three dimensional structure can be determined at atomic resolution by the methods of X-ray crystallography whose applicability to the analysis of protein structures is considered. This is followed by an account of their application in the study of a glycolytic enzyme, triose phosphate isomerase (TIM), leading up to the calculation of an electron density map at 2.5andnbsp;andAring; resolution. The characteristics of crystals of chicken TIM are described and a detailed account is given of the measurement of X-ray diffracted intensities, the determination and refinement of the positions of heavy atom binding in isomorphous derivatives of TIM, and their subsequent use for calculating the phases of the protein structure factors. The search which was conducted for possible heavy atom derivatives produced several which have two sulphydryl sites in common. The problems resulting from these common sites and the systematic errors they can cause in the phase determination are examined in relation to the suitability of different methods of refinement in this situation. The electron density map of TIM at 6andnbsp;andAring; Resolution is described and a preliminary discussion of the 2.5andnbsp;andAring; map is given.</p

Structural studies of oligometric enzymes

The functions of biological macromolecules are discussed in relation to their structure as linear polymers adopting specific conformations. If such molecules can be crystallised, their three dimensional structure can be determined at atomic resolution by the methods of X-ray crystallography whose applicability to the analysis of protein structures is considered. This is followed by an account of their application in the study of a glycolytic enzyme, triose phosphate isomerase (TIM), leading up to the calculation of an electron density map at 2.5andnbsp;andAring; resolution. The characteristics of crystals of chicken TIM are described and a detailed account is given of the measurement of X-ray diffracted intensities, the determination and refinement of the positions of heavy atom binding in isomorphous derivatives of TIM, and their subsequent use for calculating the phases of the protein structure factors. The search which was conducted for possible heavy atom derivatives produced several which have two sulphydryl sites in common. The problems resulting from these common sites and the systematic errors they can cause in the phase determination are examined in relation to the suitability of different methods of refinement in this situation. The electron density map of TIM at 6andnbsp;andAring; Resolution is described and a preliminary discussion of the 2.5andnbsp;andAring; map is given

Investigation of the Role of Copy Number Variation In the Pathogenesis of Type 1 Von Willebrand Disease

Thrombosis and Hemostasi

Copy number variation is a significant contributor to type 1 VWD pathogenesis in the EU MCMDM-1VWD cohort

Thrombosis and Hemostasi

Characterization of large in-frame von Willebrand factor deletions highlights differing pathogenic mechanisms

Copy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion.Thrombosis and Hemostasi