Home  About us  Contact
 

Light-scattering Measurements (light-scattering + measurement)

Distribution by Scientific Domains
Chemistry100%

Selected Abstracts

Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis

PROTEIN SCIENCE, Issue 10 2010
Chi Wang
Abstract The lethal genetic disease cystic fibrosis is caused predominantly by in-frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide-binding domain (NBD1) of CFTR, which functions as an ATP-gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light-scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg-ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second-site mutations that increase the solubility of isolated F508del-NBD1 in vitro and suppress the trafficking defect of intact F508del-CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del-CFTR. [source]

Physicochemical consequences of the perdeuteriation of glutathione S -transferase from S. japonicum

PROTEIN SCIENCE, Issue 3 2001
David Brockwell
Abstract Glutathione S -transferase (GST) from Schistosoma japonicum has been prepared in both normal protiated (pGST) and fully deuteriated (dGST) form by recombinant DNA technology. Electrospray mass spectrometry showed that the level of deuteriation in dGST was 96% and was homogeneous across the sample. This result is attributed to the use of a deuterium-tolerant host Escherichia coli strain in the preparation of the protein. 10 heteroatom-bound deuteriums (in addition to the carbon-bound deuteriums) were resistant to exchange when dGST was incubated in protiated buffer. The physicochemical and biological properties of the two proteins were compared. dGST was relatively less stable to heat denaturation and to proteolytic cleavage than was pGST. The midpoint transition temperature for pGST was 54.9°C, whereas that for dGST was 51.0°C. Static light-scattering measurements revealed that the association behavior of dGST is also different from that of pGST. The perdeuteriated enzyme shows a tendency to associate into dimers of the fundamental dimer. This is in contrast with results that have been obtained for other perdeuteriated proteins in which perdeuteriation has been shown to promote dissociation of aggregates. dGST showed a similar Km to pGST; similar results had been obtained previously with bacterial alkaline phosphatase. However, whereas the alkaline phosphatase showed a reduced rate of catalysis on deuteriation, dGST exhibited a slightly higher rate of catalysis than pGST. It is clear that the bulk substitution of deuterium for protium has significant effects on the properties of proteins. Until many more examples have been studied, it will be difficult to predict these effects for any given protein. Nevertheless, deuteriation represents an intriguing method of preparing functional analogs of recombinant proteins. [source]

Online Light Scattering Measurements as a Means to Assess Influence of Extrusion Parameters on Non-reactive Polymer Blend Morphology: Experimental Procedure and Preliminary Results

THE CANADIAN JOURNAL OF CHEMICAL ENGINEERING, Issue 6 2002
Christophe Serra
Abstract The influence of extrusion parameters on the morphology of non-reactive blends has been investigated by means of online light-scattering measurements. A light-scattering device was especially designed to be mounted on a twin screw extruder at different locations along the barrel. The obtained light-scattering patterns were interpreted with respect to the variation of the processing parameters. Preliminary results show that there is little effect of the rotational speed, position along the screw and feed throughput on the morphology but a quite noticeable effect of the blend composition. These results were confirmed by SEM micrographs. On a étudié l'influence des paramètres d'extrusion sur la morphologie de mélanges non réactifs par des mesures de diffusion de la lumière en ligne. Un système de diffusion de la lumière a été spécialement conçu pour être monté sur une extrudeuse bi-vis à différents endroits le long du fourreau. Les modèles de diffusion de la lumière obtenus sont interprétés en tenant compte de la variation des paramètres de procédé. Les résultats préliminaires montrent le peu d'effet de la vitesse rotationnelle, de la position sur la vis et de la capacité d'alimentation sur la morphologie mais un effet assez appréciable de la composition du mélange. Ces résultats sont confirmés par des micrographes en microscopie électronique à balayage. [source]

Crystallization and preliminary X-ray analysis of a novel Trichoderma reesei xylanase IV belonging to glycoside hydrolase family 5

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2004
Tarja Parkkinen
Xylanase IV (XYN IV) is a new recently characterized xylanase from Trichoderma reesei. It is able to degrade several different xylans, mainly producing xylose. XYN IV has been crystallized by the hanging-drop vapour-diffusion method, using PEG 6000 as a precipitant. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 86.3, b = 137.5, c = 196.1,Å, , = , = , = 90°. Assuming a molecular weight of 50.3,kDa, the VM values indicate there to be four XYN IV monomers in an asymmetric unit and the solvent content of the crystals to be 57%. Based on dynamic light-scattering measurements, XYN IV is a dimer in solution. A native data set to 2.8,Å resolution has been collected at a home laboratory and a data set to 2.2,Å resolution has been collected using synchrotron radiation. [source]