Hydrophobic Sites (hydrophobic + site)

Distribution by Scientific Domains


Selected Abstracts


Prediction of the 3D Structure of FMRF-amide Neuropeptides Bound to the Mouse MrgC11 GPCR and Experimental Validation

CHEMBIOCHEM, Issue 13 2007
Jiyoung Heo Dr.
Abstract We report the 3D structure predicted for the mouse MrgC11 (mMrgC11) receptor by using the MembStruk computational protocol, and the predicted binding site for the F-M-R-F-NH2 neuropeptide together with four singly chirally modified ligands. We predicted that the R-F-NH2 part of the tetrapeptide sticks down into the protein between the transmembrane (TM) domains 3, 4, 5, and 6. The Phe (F-NH2) interacted favorably with Tyr110 (TM3), while the Arg makes salt bridges to Asp161 (TM4) and Asp179 (TM5). We predicted that the Met extends from the binding site, but the terminal Phe residue sticks back into an aromatic/hydrophobic site flanked by Tyr237, Leu238, Leu240, and Tyr256 (TM6), and Trp162 (TM4). We carried out subsequent mutagenesis experiments followed by intracellular calcium-release assays that demonstrated the dramatic decrease in activity for the Tyr110Ala, Asp161Ala, and Asp179Ala substitutions, which was predicted by our model. These experiments provide strong evidence that our predicted G protein-coupled receptor (GPCR) structure is sufficiently accurate to identify binding sites for selective ligands. Similar studies were made with the mMrgA1 receptor, which did not bind the R-F-NH2 dipeptide; we explain this to be due to the increased hydrophobic character of the binding pocket in mMrgA1. [source]


MRI tumor characterization using Gd-GlyMe-DOTA-perfluorooctyl-mannose-conjugate (Gadofluorine MÔ), a protein-avid contrast agent

CONTRAST MEDIA & MOLECULAR IMAGING, Issue 3 2006
Hans-Jürgen Raatschen
Abstract The rationale and objectives were to define the MRI tumor-characterizing potential of a new protein-avid contrast agent, Gd-GlyMe-DOTA-perfluorooctyl-mannose-conjugate (Gadofluorine MÔ; Schering AG, Berlin, Germany) in a chemically induced tumor model of varying malignancy. Because of the tendency for this agent to form large micelles in water and to bind strongly to hydrophobic sites on proteins, it was hypothesized that patterns of dynamic tumor enhancement could be used to differentiate benign from malignant lesions, to grade the severity of malignancies and to define areas of tumor necrosis. Gadofluorine M, 0.05,mmol,Gd,kg,1, was administered intravenously to 28 anesthetized rats that had developed over 10 months mammary tumors of varying degrees of malignancy as a consequence of intraperitoneal administration of N -ethyl- N -nitrosourea (ENU), 45,250,mg,kg,1. These tumors ranged histologically from benign fibroadenomas to highly undifferentiated adenocarcinomas. Dynamic enhancement data were analyzed kinetically using a two-compartment tumor model to generate estimates of fractional plasma volume (fPV), apparent fractional extracellular volume (fEV*) and an endothelial transfer coefficient (KPS) for this contrast agent. Tumors were examined microscopically for tumor type, degree of malignancy (Scarff,Bloom,Richardson score) and location of necrosis. Eighteen tumor-bearing rats were successfully imaged. MRI data showed an immediate strong and gradually increasing tumor enhancement. KPS and fEV*, but not fPV obtained from tumors correlated significantly (p,<,0.05) with the SBR tumor grade, r,=,0.65 and 0.56, respectively. Estimates for KPS and fEV* but not fPV were significantly lower in a group consisting of benign and low-grade malignant tumors compared with the group of less-differentiated high-grade tumors (1.61,±,0.64 vs 3.37,±,1.49, p,<,0.01; 0.45,±,0.17 vs 0.78,±,0.24, p,<,0.01; and 0.076,±,0.048 vs 0.121,±,0.088, p,=,0.24, respectively). It is concluded that the protein-avid MRI contrast agent Gadofluorine M enhances tumors of varying malignancy depending on the tumor grade, higher contrast agent accumulation for more malignant lesions. The results show potential utility for differentiating benign and low-grade malignant lesions from high-grade cancers. Copyright © 2006 John Wiley & Sons, Ltd. [source]


Improved conditions for fluorescent staining of proteins with 4,4,-dianilino-1,1,-binaphthyl-5,5,-disulfonic acid in SDS-PAGE

ELECTROPHORESIS, Issue 22 2008
Wei-Tao Cong
Abstract A simple and sensitive fluorescent staining method for the detection of proteins in SDS-PAGE, namely IB (improved 4,4,-dianilino-1,1,-binaphthyl-5,5,-disulfonic acid) stain, is described. Non-covalent hydrophobic probe 4,4,-dianilino-1,1,-binaphthyl-5,5,-disulfonic acid was applied as a fluorescent dye, which can bind to hydrophobic sites in proteins non-specifically. As low as 1,ng of protein band can be detected briefly by 30,min washing followed by 15,min staining without the aiding of stop or destaining step. The sensitivity of the new presented protocol is similar to that of SYPRO Ruby, which has been widely accepted in proteomic research. Comparative analysis of the MS compatibility of IB stain and SYPRO Ruby stain allowed us to address that IB stain is compatible with the downstream of protein identification by PMF. [source]


The role of group bulkiness in the catalytic activity of psychrophile cold-active protein tyrosine phosphatase

FEBS JOURNAL, Issue 17 2008
Hiroki Tsuruta
The cold-active protein tyrosine phosphatase found in psychrophilic Shewanella species exhibits high catalytic efficiency at low temperatures as well as low thermostability, both of which are characteristics shared by many cold-active enzymes. The structure of cold-active protein tyrosine phosphatase is notable for the presence of three hydrophobic sites (termed the CA, Zn-1 and Zn-2 sites) behind the loop structures comprising the catalytic region. To identify the structural components responsible for specific enzyme characteristics, we determined the structure of wild-type cold-active protein tyrosine phosphatase at high resolution (1.1 Å) and measured the catalytic efficiencies of enzymes containing mutations in the three hydrophobic sites. The bulkiness of the amino acid side chains in the core region of the Zn-1 site strongly affects the thermostability and the catalytic efficiency at low temperatures. The mutant enzyme I115M possessed a higher kcat at low temperatures. Elucidation of the crystal structure of I115M at a resolution of 1.5 Å revealed that the loop structures involved in retaining the nucleophilic group and the acid catalyst are more flexible than in the wild-type enzyme. [source]


Chaperone-like activity and hydrophobicity of ,-crystallin

IUBMB LIFE, Issue 11 2006
G. Bhanuprakash Reddy
Abstract ,-Crystallin, a prominent member of small heat shock protein (sHsp) family and a major structural protein of the eye lens is a large polydisperse oligomer of two isoforms, ,A- and ,B-crystallins. Numerous studies have demonstrated that ,-crystallin functions like a molecular chaperone in preventing the aggregation of various proteins under a wide range of stress conditions. The molecular chaperone function of ,-crystallin is thus considered to be vital in the maintenance of lens transparency and in cataract prevention. ,-Crystallin selectively interacts with non-native proteins thereby preventing them from aggregation and helps maintain them in a folding competent state. It has been proposed and generally accepted that ,-crystallin suppresses the aggregation of other proteins through the interaction between hydrophobic patches on its surface and exposed hydrophobic sites of partially unfolded substrate protein. However, a quantifiable relationship between hydrophobicity and chaperone-like activity remains a matter to be concerned about. On an attentive review of studies on ,-crystallin chaperone-like activity, particularly the studies that have direct or indirect implications to hydrophobicity and chaperone-like activity, we found several instances wherein the correlation between hydrophobicity and its chaperone-like activity is paradoxical. We thus attempted to provide an overview on the role of hydrophobicity in chaperone-like activity of ,-crystallin, the kind of evaluation done for the first time. iubmb Life, 58: 632 - 641, 2006 [source]


Effect of K-Casein Deglycosylation on the Acid Coagulability of Milk

JOURNAL OF FOOD SCIENCE, Issue 8 2003
E. Cases
ABSTRACT: The relationship between the content of N-acetylneuraminic acid residues of micellar K-casein and acid coagulability of milk was investigated. At 30 °C, partial deglycosylation of micellar K-casein does not significantly affect the content of micellar proteins, micellar surface charge, and micellar solvation. Casein micelles modified by the release of part of the N-acetylneuraminic acid residues showed a shorter acid gelation time, a higher rate of gel strengthening, and a higher final firmness. This enhancement in the gelation ability of the neuraminidase-treated casein micelles of milk should appear as the result of increase in number of hydrophobic sites on the surface of casein micelle due to enzymatic deglycosylation of micellar K-casein. [source]


3D Pharmacophore Model for Insect Repellent Activity and Discovery of New Repellent Candidates

MOLECULAR INFORMATICS, Issue 5 2005
Apurba
Abstract To better understand the mechanism of insect repellency and to identify new repellent candidates, we performed a three-dimensional quantitative structure-activity (QSAR) study and developed a pharmacophore model for potent repellent activity from a set of eleven known diverse insect repellent compounds by using the CATALYST 3D-QSAR methodology. The protection time for repellent activity of the compounds was taken from an earlier published study. The pharmacophore was found to have three hydrophobic sites (two aliphatic and one aromatic) and a hydrogen-bond acceptor site in specific locations in the three dimensional geometry of the molecules that are critical for potent repellent activity. The pharmacophore showed an excellent correlation (correlation=0.9) between the experimental protection time afforded by the compounds in the training set and their predicted protection time. The validity of the pharmacophore model goes beyond the list in the training set and is found to map well on a variety of other insect repellents. By mapping this model on one of the more potent analogue we have generated a three-dimensional shape based template which allowed a search of our in-house compound database and discover four new potential insect repellent candidates. [source]


Fluorescence Study on the Interaction Between Hypericin and Lens Protein ",-Crystallin"

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2009
Tareq Youssef
Hypericin has been reported as a potent photosensitizing agent exhibiting antiviral, antibacterial, antineoplastic activities. Although its photophysics and mode of action are strongly modulated by the binding protein, detailed information about its mechanism of interaction with possible cellular targets, including proteins, is still lacking. Previous in vitro studies demonstrated that hypericin can be uptaken by intact lens and is able to bind to the major lens protein ",-crystallin." In this study, the mechanism of interaction of this potent drug with ,-crystallin was studied using the chemical denaturant guanidine hydrochloride (GdnHCl) and the hydrophobic surface probe, 8-anilino-1-naphthalenesulfonic acid (ANS). Fluorescence measurements showed that the increased exposure of tryptophan resulting from partial unfolding of ,-crystallin incubated with 1.0 mol L,1 of GdnHCl corresponds to the maximum accessibility of hydrophobic sites to ANS at the same GdnHCl concentration. Interestingly at this additional hydrophobicity of the protein, hypericin exhibited its maximum fluorescence intensity. This in vitro study implied that hydrophobic sites of ,-crystallin play a significant role in its interaction with hypericin. The binding between ,-crystallin and hypericin was found to be enhanced by partial perturbation of the protein. [source]


4-Fluorophenylglycine as a Label for 19F NMR Structure Analysis of Membrane-Associated Peptides

CHEMBIOCHEM, Issue 11 2003
Sergii Afonin
Abstract The non-natural amino acid 4-fluorophenylglycine (4F-Phg) was incorporated into several representative membrane-associated peptides for dual purpose. The 19F-substituted ring is directly attached to the peptide backbone, so it not only provides a well-defined label for highly sensitive 19F NMR studies but, in addition, the D and L enantiomers of the stiff side chain may serve as reporter groups on the transient peptide conformation during the biological function. Besides peptide synthesis, which is accompanied by racemisation of 4F-Phg, we also describe separation of the epimers by HPLC and removal of trifluoroacetic acid. As a first example, 18 different analogues of the fusogenic peptide "B18" were prepared and tested for induction of vesicle fusion; the results confirmed that hydrophobic sites tolerated 4F-Phg labelling. Similar fusion activities within each pair of epimers suggest that the peptide is less structured in the fusogenic transition state than in the helical ground state. In a second example, five doubly labelled analogues of the antimicrobial peptide gramicidin S were compared by using bacterial growth inhibition assays. This cyclic ,-sheet peptide could accommodate both L and D substituents on its hydrophobic face. As a third example, we tested six analogues of the antimicrobial peptide PGLa. The presence of d- 4F-Phg reduced the biological activity of the peptide by interfering with its amphiphilic ,-helical fold. Finally, to illustrate the numerous uses of l- 4F-Phg in 19F NMR spectroscopy, we characterised the interaction of labelled PGLa with uncharged and negatively charged membranes. Observing the signal of the free peptide in an aqueous suspension of unilamellar vesicles, we found a linear saturation behaviour that was dominated by electrostatic attraction of the cationic PGLa. Once the peptide is bound to the membrane, however, solid-state 19F NMR spectroscopy of macroscopically oriented samples revealed that the charge density has virtually no further influence on the structure, alignment or mobility of the peptide. [source]