Thermophysical properties of lysozyme (protein) solutions

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/76692/1/AIAA-392-587.pd

A Stable Human-Cell System Overexpressing Cystic Fibrosis Transmembrane Conductance Regulator Recombinant Protein at the Cell Surface

Recent human clinical trials results demonstrated successful treatment for certain genetic forms of cystic fibrosis (CF). To extend treatment opportunities to those afflicted with other genetic forms of CF disease, structural and biophysical characterization of CF transmembrane conductance regulator (CFTR) is urgently needed. In this study, CFTR was modified with various tags, including a His10 purification tag, the SUMOstar (SUMO*) domain, an extracellular FLAG epitope, or an enhanced green fluorescent protein (EGFP), each alone or in various combinations. Expressed in HEK293 cells, recombinant CFTR proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited regulated chloride-channel activity with only modest alterations in channel conductance and gating kinetics. Surface CFTR expression level was enhanced by the presence of SUMO* on the N-terminus. Quantitative mass-spectrometric analysis indicated approximately 10% of the total recombinant CFTR (SUMO*-CFTRFLAG-EGFP) localized to the plasma membrane. Trial purification using dodecylmaltoside for membrane protein extraction reproducibly recovered 178 ± 56 μg SUMO*-CFTRFLAG-EGFP per billion cells at 80% purity. Fluorescence size-exclusion chromatography indicated purified CFTR was monodisperse. These findings demonstrate a stable mammalian cell expression system capable of producing human CFTR of sufficient quality and quantity to augment futrure CF drug discovery efforts, including biophysical and structural studies

Coverage of whole proteome by structural genomics observed through protein homology modeling database

We have been developing FAMSBASE, a protein homology-modeling database of whole ORFs predicted from genome sequences. The latest update of FAMSBASE (http://daisy.nagahama-i-bio.ac.jp/Famsbase/), which is based on the protein three-dimensional (3D) structures released by November 2003, contains modeled 3D structures for 368,724 open reading frames (ORFs) derived from genomes of 276 species, namely 17 archaebacterial, 130 eubacterial, 18 eukaryotic and 111 phage genomes. Those 276 genomes are predicted to have 734,193 ORFs in total and the current FAMSBASE contains protein 3D structure of approximately 50% of the ORF products. However, cases that a modeled 3D structure covers the whole part of an ORF product are rare. When portion of an ORF with 3D structure is compared in three kingdoms of life, in archaebacteria and eubacteria, approximately 60% of the ORFs have modeled 3D structures covering almost the entire amino acid sequences, however, the percentage falls to about 30% in eukaryotes. When annual differences in the number of ORFs with modeled 3D structure are calculated, the fraction of modeled 3D structures of soluble protein for archaebacteria is increased by 5%, and that for eubacteria by 7% in the last 3 years. Assuming that this rate would be maintained and that determination of 3D structures for predicted disordered regions is unattainable, whole soluble protein model structures of prokaryotes without the putative disordered regions will be in hand within 15 years. For eukaryotic proteins, they will be in hand within 25 years. The 3D structures we will have at those times are not the 3D structure of the entire proteins encoded in single ORFs, but the 3D structures of separate structural domains. Measuring or predicting spatial arrangements of structural domains in an ORF will then be a coming issue of structural genomics

Determinants of youth attitudes and skills towards which drinking driving prevention programs should be directed. Volume II: an in-depth review of twelve youth DWI prevention programs. Interim report.

National Highway Traffic Safety Administration, Washington, D.C.Mode of access: Internet.Author corporate affiliation: Pacific Institute for Research and Evaluation, Bethesda, Md.Subject code: EDSubject code: EEASubject code: EODSubject code: QGIHSubject code: SBEGSubject code: YC

Mercury induced modifications in the stereochemistry of the active site through Cys-73 in a serine protease - Crystal structure of the complex of a partially modified proteinase K with mercury at 1.8 Å resolution

298-302Proteinese K(PK) isolated from Tritirachium album Limber was crystallized with HgCl2 in excess. under microgravity conditions. The intensity data were collected at 4°C to 1.8 Ǻ resolution and the final R-factor after refinement for all the reflections was 0. 164. Mercury has been found at two sites with partial occupancies (0.4 and 0.6) which are at distances of 2.48 Ǻ and 2.58 Ǻ respectively from Cys-73 Sγ. The Cys-73 in the enzyme structure is located close to the active site residue. His-69. This region is completely buri ed and is not accessible to the solvent. It is rather tightly packed. Therefore. the binding of mercury distorts the stereochemistry of the neighbouring residues including those be longing to the catalytic tri ad. As a result of this. the Oγ of Ser-224 is displaced by 0.6 Ǻ which causes the inactivation of proteinase K by increasing the H-bond distance to 3.7 Ǻ between Ser-224 Oγ and His-69 Nε2

STS-107 Mission after the Mission: Recovery of Data from the Debris of Columbia

STS-107 was a 16-day, dedicated research mission that included over 80 experiments, spanning many disciplines including biology, physics, chemistry, and earth sciences, including many student experiments. The mission was considered a resounding success until February 1, 2003, when tragedy struck the Columbia and her crew as she re-entered the atmosphere over Texas. During the mission, approximately one third of the overall data was obtained but much more was stored in the flight hardware systems. This paper documents a new set of STS-107 experiment objectives, a "mission after the mission," in which several experiment teams attempted, and, in many cases succeeded, to recover data from their flight hardware, now debris. A description of the data recovery efforts is included for these five experiment facilities: Combustion Module-2, Critical Viscosity of Xenon-2, Commercial Instrumentation Technology Associates Biomedical Experiments-2, Biological Research in Canisters-14, and Commercial Protein Crystal Growth

Structural basis of laminin binding to the LARGE glycans on dystroglycan

Dystroglycan is a highly glycosylated extracellular matrix receptor with essential functions in skeletal muscle and the nervous system. Reduced matrix binding by α-dystroglycan (α-DG) due to perturbed glycosylation is a pathological feature of several forms of muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) synthesizes the matrix-binding heteropolysaccharide [-glucuronic acid-β1,3-xylose- α1,3-]n. Using a dual exoglycosidase digestion, we confirm that this polysaccharide is present on native α-DG from skeletal muscle. The atomic details of matrix binding were revealed by a high-resolution crystal structure of laminin G-like (LG) domains 4-5 of laminin α2 bound to a LARGE-synthesized oligosaccharide. A single glucuronic acid- β1,3-xylose disaccharide repeat straddles a Ca2+ ion in the LG4 domain, with oxygen atoms from both sugars replacing Ca2+-bound water molecules. The chelating binding mode accounts for the high affinity of this protein-carbohydrate interaction. These results reveal a novel mechanism of carbohydrate recognition and provide a structural framework for elucidating the mechanisms underlying muscular dystrophy