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Conformational Exchange (conformational + exchange)
Selected AbstractsConformational exchange in pimonidazole,a hypoxia markerMAGNETIC RESONANCE IN CHEMISTRY, Issue 8 2007Cristina Gabellieri Abstract Pimonidazole is one of a series of nitroimidazole compounds that is widely used as a marker for qualitative and quantitative assessment of tumour hypoxia. We have observed a novel dynamic conformational exchange process in this molecule in aqueous solution. By a combination of 1H, 13C, two-dimensional 1H,1H EXchange SpectroscopY (EXSY) and spectral simulation, we unambiguously attribute the conformational exchange process to flipping of the six-membered heterocyclic ring. Copyright © 2007 John Wiley & Sons, Ltd. [source] Expression, purification, and characterization of Thermotoga maritima membrane proteins for structure determinationPROTEIN SCIENCE, Issue 5 2006Linda Columbus Abstract Structural studies of integral membrane proteins typically rely upon detergent micelles as faithful mimics of the native lipid bilayer. Therefore, membrane protein structure determination would be greatly facilitated by biophysical techniques that are capable of evaluating and assessing the fold and oligomeric state of these proteins solubilized in detergent micelles. In this study, an approach to the characterization of detergent-solubilized integral membrane proteins is presented. Eight Thermotoga maritima membrane proteins were screened for solubility in 11 detergents, and the resulting soluble protein,detergent complexes were characterized with small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and chemical cross-linking to evaluate the homogeneity, oligomeric state, radius of gyration, and overall fold. A new application of SAXS is presented, which does not require density matching, and NMR methods, typically used to evaluate soluble proteins, are successfully applied to detergent-solubilized membrane proteins. Although detergents with longer alkyl chains solubilized the most proteins, further characterization indicates that some of these protein,detergent complexes are not well suited for NMR structure determination due to conformational exchange and protein oligomerization. These results emphasize the need to screen several different detergents and to characterize the protein,detergent complex in order to pursue structural studies. Finally, the physical characterization of the protein,detergent complexes indicates optimal solution conditions for further structural studies for three of the eight overexpressed membrane proteins. [source] Temperature-induced reversible conformational change in the first 100 residues of ,-synucleinPROTEIN SCIENCE, Issue 3 2006Brian C. McNulty Abstract Natively disordered proteins are a growing class of anomalies to the structure,function paradigm. The natively disordered protein ,-synuclein is the primary component of Lewy bodies, the cellular hallmark of Parkinson's disease. We noticed a dramatic difference in dilute solution 1H- 15N Heteronuclear Single Quantum Coherence (HSQC) spectra of wild-type ,-synuclein and two disease-related mutants (A30P and A53T), with spectra collected at 35°C showing fewer cross-peaks than spectra acquired at 10°C. Here, we show the change to be the result of a reversible conformational exchange linked to an increase in hydrodynamic radius and secondary structure as the temperature is raised. Combined with analytical ultracentrifugation data showing ,-synuclein to be monomeric at both temperatures, we conclude that the poor quality of the 1H- 15N HSQC spectra obtained at 35°C is due to conformational fluctuations that occur on the proton chemical shift time scale. Using a truncated variant of ,-synuclein, we show the conformational exchange occurs in the first 100 amino acids of the protein. Our data illustrate a key difference between globular and natively disordered proteins. The properties of globular proteins change little with solution conditions until they denature cooperatively, but the properties of natively disordered proteins can vary dramatically with solution conditions. [source] Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Ulrika W. Hultdin The transcription factor FocB belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic Escherichia coli. Recent findings suggest that FocB-family proteins may form different protein,protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. However, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. The 109-amino-acid FocB transcription factor from the foc gene cluster in E. coli strain J96 has been cloned, expressed and purified. The His6 -tagged fusion protein was captured by Ni2+ -affinity chromatography, cleaved with tobacco etch virus protease and purified by gel filtration. The purified protein is oligomeric, most likely in the form of dimers. NMR analysis guided the crystallization attempts by showing that probable conformational exchange or oligomerization is reduced at temperatures above 293,K and that removal of the highly flexible His6 tag is advantageous. The protein was crystallized using the hanging-drop vapour-diffusion method at 295,K. A native data set to 2.0,Å resolution was collected at 100,K using synchrotron radiation. [source] Microsecond Protein Dynamics Measured by 13C, Rotating-Frame Spin RelaxationCHEMBIOCHEM, Issue 9 2005Patrik Lundström Abstract NMR spin relaxation in the rotating frame (R1,) is a unique method for atomic-resolution characterization of conformational (chemical) exchange processes occurring on the microsecond timescale. We present a rotating-frame13C,relaxation dispersion experiment for measuring conformational dynamics in uniformly13C-labeled proteins. The experiment was validated by using the E140Q mutant of the C-terminal fragment of calmodulin, which exhibits significant conformational exchange between two major conformations, as gauged from previous15N and1H relaxation studies. Consistent with previous work, the present13C, R1,experiment detects conformational-exchange dynamics throughout the protein. The average correlation time of ,,ex,=25±8 ,s is in excellent agreement with those determined previously from1H and15N R1,relaxation data: ,,ex,=19±7 and 21±3 ,s, respectively. The extracted chemical-shift differences between the exchanging states reveal significant fluctuations in dihedral angles within single regions of Ramachandran ,,, space, that were not identified from the1H and15N relaxation data. The present results underscore the advantage of using several types of nuclei to probe exchange dynamics in biomolecules. [source] | ||||||||||