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Protein Regions (protein + regions)
Selected AbstractsAffinity-Based Protein Surface Pattern Formation by Ligand Self-Selection from Mixed Protein SolutionsADVANCED FUNCTIONAL MATERIALS, Issue 19 2009Manish Dubey Abstract Photolithographically prepared surface patterns of two affinity ligands (biotin and chloroalkane) specific for two proteins (streptavidin and HaloTag, respectively) are used to spontaneously form high-fidelity surface patterns of the two proteins from their mixed solution. High affinity protein-surface self-selection onto patterned ligands on surfaces exhibiting low non-specific adsorption rapidly yields the patterned protein surfaces. Fluorescence images after protein immobilization show high specificity of the target proteins to their respective surface patterned ligands. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging further supports the chemical specificity of streptavidin and HaloTag for their surface patterned ligands from mixed protein solutions. However, ToF-SIMS did detect some non-specific adsorption of bovine serum albumin, a masking protein present in excess in the adsorbing solutions, on the patterned surfaces. Protein amino acid composition, surface coverage, density, and orientation are important parameters that determine the relative ToF-SIMS fragmentation pattern yields. ToF-SIMS amino acid-derived ion fragment yields summed to produce surface images can reliably determine which patterned surface regions contain bound proteins, but do not readily discriminate between different co-planar protein regions. Principal component analysis (PCA) of these ToF-SIMS data, however, improves discrimination of ions specific to each protein, facilitating surface protein pattern identification and image contrast. [source] Exploring functional roles of multibinding protein interfacesPROTEIN SCIENCE, Issue 8 2009Manoj Tyagi Abstract Cellular processes are highly interconnected and many proteins are shared in different pathways. Some of these shared proteins or protein families may interact with diverse partners using the same interface regions; such multibinding proteins are the subject of our study. The main goal of our study is to attempt to decipher the mechanisms of specific molecular recognition of multiple diverse partners by promiscuous protein regions. To address this, we attempt to analyze the physicochemical properties of multibinding interfaces and highlight the major mechanisms of functional switches realized through multibinding. We find that only 5% of protein families in the structure database have multibinding interfaces, and multibinding interfaces do not show any higher sequence conservation compared with the background interface sites. We highlight several important functional mechanisms utilized by multibinding families. (a) Overlap between different functional pathways can be prevented by the switches involving nearby residues of the same interfacial region. (b) Interfaces can be reused in pathways where the substrate should be passed from one protein to another sequentially. (c) The same protein family can develop different specificities toward different binding partners reusing the same interface; and finally, (d) inhibitors can attach to substrate binding sites as substrate mimicry and thereby prevent substrate binding. [source] A quantitative strategy to detect changes in accessibility of protein regions to chemical modification on heterodimerizationPROTEIN SCIENCE, Issue 7 2009Mathias Dreger Abstract We describe a method for studying quantitative changes in accessibility of surface lysine residues of the PB1 subunit of the influenza RNA polymerase as a result of association with the PA subunit to form a PB1-PA heterodimer. Our method combines two established methods: (i) the chemical modification of surface lysine residues of native proteins by N -hydroxysuccinimidobiotin (NHS-biotin) and (ii) the stable isotope labeling of amino acids in cell culture (SILAC) followed by tryptic digestion and mass spectrometry. By linking the chemical modification with the SILAC methodology for the first time, we obtain quantitative data on chemical modification allowing subtle changes in accessibility to be described. Five regions in the PB1 monomer showed altered reactivity to NHS-biotin when compared with the [PB1-PA] heterodimer. Mutational analysis of residues in two such regions,at K265 and K481 of PB1, which were about three- and twofold, respectively, less accessible to biotinylation in the PB1-PA heterodimer compared with the PB1 monomer, demonstrated that both K265 and K481 were crucial for polymerase function. This novel assay of quantitative profiling of biotinylation patterns (Q-POP assay) highlights likely conformational changes at important functional sites, as observed here for PB1, and may provide information on protein,protein interaction interfaces. The Q-POP assay should be a generally applicable approach and may detect novel functional sites suitable for targeting by drugs. [source] Diastereomers (RC,SP)- and (RC,RP)- S -methyl P -(3-azidopropyl)- N -[(1R)-1-phenylethyl]phosphonamidothioateACTA CRYSTALLOGRAPHICA SECTION C, Issue 4 2009Lilu Guo Diastereoisomers of the title organophosphorus compound, C12H19N4OPS, denoted RCSP, (I), and RCRP, (II), were structurally characterized and compared. Asymmetric phosphorus compounds are of interest with regard to the use of these systems as possible protein probes via the stereoselective delivery of an azide group tethered to the P atom into key protein regions. The diastereomers were produced in a 1:1 mixture and isolated by chromatography. Although both isomers crystallize in the same space group with superficially similar cell constants, conformational and packing differences are pronounced. Despite the conformational differences, strong intermolecular hydrogen bonding links both isomers into chains parallel to the a axis [N...O = 2.8609,(18) and 2.966,(3),Å in (I) and (II), respectively], with C,H..., interchain interactions of ca 3.5,Å. [source] Molecular impact of MinK on the enantiospecific block of IKs by chromanolsBRITISH JOURNAL OF PHARMACOLOGY, Issue 8 2000C Lerche Slowly activating IKs (KCNQ1/MinK) channels were expressed in Xenopous oocytes and their sensitivity to chromanols was compared to homomeric KCNQ1 channels. To elucidate the contribution of the ,-subunit MinK on chromanol block, a formerly described chromanol HMR 1556 and its enantiomer S5557 were tested for enantio-specificity in blocking IKs and KCNQ1 as shown for the single enantiomers of chromanol 293B. Both enantiomers blocked homomeric KCNQ1 channels to a lesser extent than heteromeric IKs channels. Furthermore, we expressed both WT and mutant MinK subunits to examine the involvement of particular MinK protein regions in channel block by chromanols. Through a broad variety of MinK deletion and point mutants, we could not identify amino acids or regions where sensitivity was abolished or strikingly diminished (>2.5 fold). This could indicate that MinK does not directly take part in chromanol binding but acts allosterically to facilitate drug binding to the principal subunit KCNQ1. British Journal of Pharmacology (2000) 131, 1503,1506; doi:10.1038/sj.bjp.0703734 [source] High-Resolution Solid-State NMR Studies on Uniformly [13C,15N]-Labeled UbiquitinCHEMBIOCHEM, Issue 9 2005Karsten Seidel Abstract Understanding of the effects of intermolecular interactions, molecular dynamics, and sample preparation on high-resolution magic-angle spinning NMR data is currently limited. Using the example of a uniformly [13C,15N]-labeled sample of ubiquitin, we discuss solid-state NMR methods tailored to the construction of 3D molecular structure and study the influence of solid-phase protein preparation on solid-state NMR spectra. A comparative analysis of13C,,13C,, and13C, resonance frequencies suggests that13C chemical-shift variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility. Our results can be refined by additional solid-state NMR techniques and serve as a reference for ongoing efforts to characterize the structure and dynamics of (membrane) proteins, protein complexes, and other biomolecules by high-resolution solid-state NMR. [source] | ||||||||||