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Biophysical Methods (biophysical + methods)
Selected AbstractsConformational properties of bacterial DnaK and yeast mitochondrial Hsp70FEBS JOURNAL, Issue 12 2005-helical subdomain, Role of the divergent C-terminal Among the eukaryotic members of the Hsp70 family, mitochondrial Hsp70 shows the highest degree of sequence identity with bacterial DnaK. Although they share a functional mechanism and homologous co-chaperones, they are highly specific and cannot be exchanged between Escherichia coli and yeast mitochondria. To provide a structural basis for this finding, we characterized both proteins, as well as two DnaK/mtHsp70 chimeras constructed by domain swapping, using biochemical and biophysical methods. Here, we show that DnaK and mtHsp70 display different conformational and biochemical properties. Replacing different regions of the DnaK peptide-binding domain with those of mtHsp70 results in chimeric proteins that: (a) are not able to support growth of an E. coli DnaK deletion strain at stress temperatures (e.g. 42 °C); (b) show increased accessibility and decreased thermal stability of the peptide-binding pocket; and (c) have reduced activation by bacterial, but not mitochondrial co-chaperones, as compared with DnaK. Importantly, swapping the C-terminal ,-helical subdomain promotes a conformational change in the chimeras to an mtHsp70-like conformation. Thus, interaction with bacterial co-chaperones correlates well with the conformation that natural and chimeric Hsp70s adopt in solution. Our results support the hypothesis that a specific protein structure might regulate the interaction of Hsp70s with particular components of the cellular machinery, such as Tim44, so that they perform specific functions. [source] Upward mobility and alternative lifestyles: a report from the 10th biennial meeting on Bacterial Locomotion and Signal TransductionMOLECULAR MICROBIOLOGY, Issue 1 2009Birgit E. Scharf Summary This past January, in Cuernavaca Mexico, a conglomerate of scientists met to discuss the contemporary view of Bacterial Locomotion and Signal Transduction (BLAST). The BLAST meetings represent a field that has its roots in chemotaxis and the flagellum-based motility but now encompass all types of cellular movement and signalling. The topics varied from the interactions between molecules to the interactions between species. We heard about 3D reconstructions of transmembrane chemoreceptors within cells, new biophysical methods for understanding cellular engines, intricate phosphorelays, elaborate gene networks, new messenger molecules and emerging behaviours within complex populations of cells. At BLAST X we gained an appreciation for the lifestyle choices bacteria make, how they get to where they are going and the molecular mechanisms that underlie their decisions. Herein we review the highlights of the meeting. [source] Molecular interactions of isoxazolcurcumin with human serum albumin: Spectroscopic and molecular modeling studiesBIOPOLYMERS, Issue 2 2009Bijaya Ketan Sahoo Abstract Curcumin is a nontoxic natural product with diverse pharmacological potencies. We report the interaction of a potent synthetic derivative of curcumin, isoxazolcurcumin (IOC) with human serum albumin (HSA) using various biophysical methods. The observed fluorescence quenching of HSA by IOC is due to a complex formation by a static quenching process with a quenching constant of the order of 105M,1. The binding affinity and the number of binding sites were obtained from a Scatchard analysis. Thermodynamics reveals that the interaction is entropy driven with predominantly hydrophobic forces. From the observed Förster-type fluorescence resonance energy transfer (FRET), the donor (Trp 214 in HSA) to acceptor (IOC) distance is calculated to be 3.2 nm. The conformational changes of HSA due to the interaction were investigated qualitatively from synchronous fluorescence spectra along with a quantitative estimation of the secondary structure from Fourier Transform Infrared (FTIR) and circular dichroism (CD) spectroscopies. Molecular docking studies were performed to obtain information on the possible residues involved in the interaction process, and changes in accessible surface area of the interacting residues were calculated. The preferred binding site of IOC was analyzed by ligand displacement experiments with 1-anilino-8-naphthalenesulfonate (ANS) and warfarin-bound HSA. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 108,119, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Electrospray-ionization mass spectrometry as a tool for fast screening of protein structural propertiesBIOTECHNOLOGY JOURNAL, Issue 1 2009Rita Grandori Abstract Since the early 1990s, electrospray-ionization mass spectrometry (ESI-MS) has encountered growing interest as a complementary tool to established biochemical and biophysical methods for investigating protein structure and conformation. Nowadays, applications of ESI-MS to protein investigation span from the area of analytical biochemistry to that of structural biology. This review focuses on applications of this technique to the analysis of protein conformational properties and molecular interactions, underscoring their possible relevance for molecular biotechnology, although representing a still very young field. An introductive section presents the major issues related to theoretical and technical aspects of ESI-MS under non-denaturing conditions. Examples from our work and from the literature illustrate which kind of information can be obtained concerning key issues in biotechnology such as stability and aggregation of proteins under both near-native and challenging conditions, and interactions with other proteins, ligands and cofactors. [source] Sequential application of cold and sodium lauryl sulphate decreases irritation and barrier disruption in vivo in humansBRITISH JOURNAL OF DERMATOLOGY, Issue 4 2005J.W. Fluhr Summary Background, Irritant contact dermatitis (ICD) is one of the most frequent types of occupational dermatitis. Different factors are involved in the development of contact dermatitis. In the food-processing industry, the combined exposure to different irritants may be involved in the development of ICD. Few data have been published regarding the irritant potential of sodium lauryl sulphate (SLS) in combination with cold. Objectives, The present study was intended to analyse whether cold exposure and low skin temperature influence the development of ICD. Methods, Twenty (part I) and 12 (part II) healthy volunteers were exposed twice daily for 4 days to SLS alone, different low temperatures alone (4 °C six times for 90 s with an interval of 20 s or 15 °C for 10 min) or a combination of cold and SLS (19·6 µL SLS 1% cm,2, part I; or 52·6 µL SLS 0·5% cm,2, part II) using the tandem repetitive irritation test. Irritant cutaneous reactions were measured by noninvasive biophysical methods with transepidermal water loss as a parameter for permeability barrier function and skin colour reflectance together with visual scoring as parameters for inflammatory reactions. Results, Cold alone caused no significant skin reaction compared with untreated control. Exposure to SLS alone and SLS together with cold (independent of the applied temperature of 4 or 15 °C) twice daily induced a clear irritant reaction and barrier disturbance. Reactions did not differ whether SLS was applied before or after cold. Furthermore, ,tandem application' of cold and SLS diminished the barrier disruption and irritant reaction compared with SLS alone. Conclusions, We conclude that the application of cold may have a protective effect on the development of ICD, at least in our short-term model. [source] | |||||||||||||