Protein and Peptide Letters - Volume 15, Issue 1, 2008

A convenient solid phase synthesis of a Thrombin Receptor Glycopeptide Mimetic analogue namely, 1-OMethyl- 2-N-{1’-(argininocarbonyl)-4’-[(4’’-fluoro)-benzylamido]-cyclohexane}-glucosamine using Fmoc/tBu methodology and the 4-Methoxybenzhydryl bromide resin is described. The synthesized analogue was purified by Reverse Phase High Performance Liquid Chromatography (RP-HPLC) and was identified by Electron Spray Ionization-Mass Spectrometry (ESI-MS) and Nuclear Magnetic Resonance (NMR). The synthetic protocol introduced for the first time successfully the acid sensitive 4-Methoxybenzhydryl bromide resin as a scaffold for the synthesis of glycopeptides resulting in high yield reactions. This synthetic procedure could be a general one for the convenient synthesis of such glyco compounds as the method was used for the first time to glycosylate a non peptide mimetic of an important protein sequence, in particular of the thrombin receptor active site S42FLLR46.

Glutathione S-transferase was purified from bovine erythrocytes and some kinetic and characteristic properties of the enzyme were investigated. The purification procedure was composed of preparation of homogenate and Glutathione- Agarose affinity chromatography. Thanks to the procedure, the enzyme was purified 6,800 fold with 97% yield and a specific activity of 136 EU/mg proteins. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), one band with a mass of 27 kDa was found. The native molecular weight of the enzyme was found to be approximately 53 kDa by Sephadex G-100 gel filtration chromatography. Optimum pH, stable pH, optimum temperature, and optimum ionic strength were determined as 7.0, 6.5 in K-phosphate buffer, 20 °C, 0.1 M K-phosphate, respectively. The best activity was obtained with 1-chloro-2,4-dinitrobenzene (CDNB) in a study performed with different substrates. Vmax, Km, and kcat values were calculated as 402.63 ± 4.99 EU/mg proteins, 0.7447 ± 0.0007 mM, and 11436 min-1 for CDNB, and 88.00 ± 2.30 EU/mg proteins, 0.3257 ± 0.0012 mM, and 477 min-1 for GSH, respectively, by using Lineweaver-Burk graphs obtained from 1/V versus 1/[CDNB] and 1/[GSH].

Development of new chemical nucleases is a matter of great interest because of their extensive use in biotechnology and as therapeutic agents. The ATCUN (amino terminal Cu(II) and Ni(II) binding) is a peptide motif that occurs naturally in the serum albumins. The similar peptide motif (GlyAibHis) having unnatural amino acid Aib (α- aminoisobutyric acid) was synthesized and its Cu(II) complex was characterized by ESI-MS and spectrophotometry studies. The reactivity of this complex toward DNA cleavage has been investigated. Cu(II)-GlyAibHis shows the DNA cleavage only in presence of mild oxidizing agents like ascorbate by an oxidative mechanism rather than hydrolytic and follows the pseudo first order kinetics (Kobs = 0.085 min-1). The non-hydrolytic mechanism was further supported by the hydrolysis of pNPP which followed the pseudo first order kinetics (Kobs = 1.98 x 10-2 min-1) having no pH effect.

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body. In brain, they prevent unwanted proteolysis and are involved in several neurodegenerative diseases. In the present study, it has been demonstrated that photoactivated riboflavin and H2O2 induced modifications of high molecular mass goat brain cystatin (HM-GBC) leads to its inactivation and degradation. It was found that the damage with both the oxidants occurred mainly because of the hydroxyl radicals. It has been also proposed that susceptibility of HM-GBC to oxidation by reactive oxygen species generated in vivo arise from oxidative modifications may lead to damage of this significant protein as it is so well pronounced, in vitro.

CYP2C19 is an important member of the cytochrome P-450 enzyme superfamily and plays a significant role in the drug metabolism. In order to gain insights for developing personalized drugs, the structure-activity relationships of two SNPs, W120R and I331V, with the ligands of CEC, Fluvoxamine, Lescol and Ticlopidine were investigated through the structure-activity relationship approach. By means of a series of docking studies, the binding pockets of the two SNPs for the four compounds are explicitly defined that will be very useful for conducting mutagenesis studies, providing insights into personalization of drug treatments and stimulating novel strategies for finding desired personalized drugs.

Coiled coils are important structural modules and domains of protein-protein interactions. Further, they provide the required framework to proteins, like kinesins, myosins and SNAREs, which are either structural or involved in transport of biomolecules. We provide an interactive webserver to measure the strength of interactions between two helices involved in coiled coils. Interactions are measured using non-bonded and electrostatic interactions and the presence of hydrogen bonds and salt bridges. The sum of these interactions is expressed as psuedoenergy, whose ranges have been standardized using all structural entries that are classified to contain coiled coils. The results are displayed conveniently as energy per residue along with options to obtain detailed list of different types of interactions. This webserver can be useful to assess the strength of coiled coil regions, to recognize weak and strong regions, to rationalize the phenotypic behaviour of single residue mutations as well as to design mutation experiments. COILCHECK webserver can be accessed from http://caps.ncbs.res.in/coilcheck/.

Beta-propeller phytases (BPPs) are a special class of enzyme that are mainly isolated from Bacillus and are widely used in animal nutrition, human health and environmental protection. BPPs class exhibits both unique Ca2+- dependent catalytic property and highly strict substrate specificity for the calcium-phytate complex. This review describes the effect of Ca2+ on the catalytic activity, thermal stability, and structural conformation of BPPs.

During analysis of the proteome in the cerebrospinal fluid (CSF) of the Caucasian form of moyamoya disease (MMD), a novel post-translational modification of human transthyretin was observed. Two-dimensional electrophoresis and subsequent peptide sequencing with ESI-MS/MS were performed to discover the γ-carboxylation of the Glu-42 (Gla- 42).

Differences evident in the sequence alignment of human cathepsin-L with shrimp cathepsin-L and silicatein-α suggest the indirect involvement of the heavy to light chain loop (E286 to E289) in the function of these enzymes. Deletion of the loop and adjacent residues S290 to N293, decreased specific protease activity by 81% and 63%, respectively; complete substitution for the corresponding silicatein-α loop decreased activity by 35%. In all cases the Km was largely unchanged. The conformational stability of human procathepsin-L was not altered by deletion of E286 to E289 but increased on deletion of S290 to N293. Therefore, shortening the loop does not change substrate affinity but does influence activity, in part via conformational change.

The hypervariable D3 domain of Salmonella flagellin, composed of the 190-285 segment, is the major determinant of flagellar antigenicity. D3 was cloned and overexpressed in E. coli. Although previous studies concluded that D3 is stabilized by interactions with the D2 domain, our calorimetric experiments have revealed that isolated D3 has a stable tertiary structure which is highly resistant against proteolytic digestion. Repeated heating experiments demonstrated that unfolding of D3 is reversible. Its small size and stable structure makes D3 a promising protein scaffold for the development of artificial binding proteins by directed evolution.

S-adenosylmethionine is a metabolite regulating many biological processes; S-adenosylmethionine effect on ubiquitin-proteasome system (UPS) has not been studied yet. We investigated S-adenosylmethionine effects on UPS activity both in vitro, by inhibitor screening assay, and in rat vascular smooth muscle cells, by Western Blot of proteasomal targets. We found that S-adenosylmethionine inhibited UPS activity.

MiRP1 (encoded by the KCNE2 gene) is one of a family of five single transmembrane domain voltage-gated potassium (Kv) channel ancillary subunits currently under intense scrutiny to establish their position in channel complexes and elucidate α subunit contact points, but its structure is unknown. MiRP1 mutations are associated with inherited and acquired cardiac arrhythmia. Here, synthetic peptides corresponding to human MiRP1 (full-length and separate domains) were structurally analyzed using FTIR and CD spectroscopy. The N-terminal (extracellular) domain was soluble and predominantly non-ordered in aqueous media, but predominantly α-helical in L-α-lysophosphatidylcholine (LPC) micelles. The MiRP1 transmembrane domain was predominantly a mixture of α-helix and non-ordered structure in LPC micelles, with a minor contribution from non-aggregated β-strand. The intracellular C-terminal domain was insoluble in aqueous solution; reconstitution into non-aqueous environments resulted in solubility and adoption of increasing amounts of α-helix, with the solvent order sodium dodecyl sulphate < dimyristoyl L-α-phosphatidylcholine (DMPC) < LPC < trifluoroethanol. Correlation of secondary structure changes with lipid transition temperature during heating suggested that the MiRP1 C-terminus incorporates into DMPC bilayers. Full-length MiRP1 was soluble in SDS micelles and calculated to contain 34% α-helix, 23% β-strand and 43% non-ordered structure in this environment, as determined by CD spectroscopy. Thus, MiRP1 is highly dependent upon hydrophobic interaction via lipid and/or protein contacts for adoption of ordered structure without nonspecific aggregation, consistent with a role as a membrane-spanning subunit within Kv channel complexes. These data will provide a structural framework for ongoing mutagenesis-based in situ structure-function studies of MiRP1 and its relatives.

The polyadenylation factor subunit “Factor Interacting with Poly(A) polymerase” (Fip1) is an important bridging subunit in the eukaryotic polyadenylation complex. To better understand the functioning of Fip1 in Arabidopsis, a random combinatorial screen for peptides that interact with a conserved plant-specific domain in the protein was conducted. A search of the Arabidopsis proteome using these Fip1-binding peptides as queries resulted in the identification of a number of putative Fip1-interacting proteins. One of these was the polyadenylation factor subunit, CstF77. This purported interaction was confirmed by yeast two-hybrid and in vitro assays. Mutation of the motif identified in the phage display screen eliminated the interaction, corroborating the results of the phage display screen. The domain of CstF77 that interacts with Fip1 lies at its extreme C-terminus and is distinct from the part of CstF77 that binds CstF64. Taken together, these results suggest that Fip1 is situated near CstF64 in the polyadenylation complex.

Processing of DNA replication and repair intermediates is a critical aspect of genome stability maintenance. The coordinated action of RecQ-like helicases with structure-specific nucleases such as Flap Endonuclease 1 plays an important role in the processing of certain DNA structures associated with the replication fork, DNA repair, or telomeres. We will summarize our current understanding of how and in what context these interactions take place, with a particular emphasis on the mechanisms of RecQ helicases in processing of key DNA replication and repair intermediates by their protein interactions with FEN-1 and related structure-specific nucleases.

The interaction of calreticulin with amyloid beta (Aβ) was investigated using solid phase and solution binding assays. Calreticulin bound Aβ 1-42 in a time and concentration dependent fashion. The binding was optimal at pH 5 and was stimulated by Ca2+ and inhibited by Zn2+ at pH 7. Interaction took place through the hydrophobic C-terminus of Aβ 1- 42 and the polypeptide binding site of calreticulin. The results are discussed in the light of a reported role of calreticulin as a cell surface scavenger receptor.

Nudix hydrolases catalyze the hydrolysis of nucleoside diphosphates linked to other moieties X, and contain the sequence motif or Nudix box, GX5EX7REUXEEXGU. The mechanisms of Nudix hydrolases are highly diverse in the position on the substrate. In this paper, we examined the sequences and structures of the MutT/Nudix superfamily. And two recent developed methods were employed for data analyses of the superfamily. One is QH method evaluating the similarities among structures for structural phylogeny. The other method is clustering analysis by using the CLANS program that could analysis thousands of sequences of the full dataset rather than the representative sequences of the superfamily. Finally, we proposed a more objective classification of the MutT/Nudix superfamily members based on detailed sequence and structure analyses.

mBanana is a novel monomeric red fluorescent protein mutant. It was cloned and expressed in Escherichia coli with 10 histidine residues at its N-terminal. After cleavage of the His tag by TEV protease, the mBanana was further purified and crystallized by the hanging-drop vapor-diffusion technique. The crystals can diffract to 2.0Å resolution and one set of completed data was collected. It showed that the orthorhombic mBanana crystal was in space group P21 with unit cell parameters (48.629, 42.667, 61.714, 90, 111.676, 90) and contained one molecule in one asymmetric unit.

AxCesD protein required for bacterial cellulose biosynthesis in Acetobacter xylinum was overexpressed in E. coli, purified and crystallized. Single crystals of SeMet-substituted AxCesD were obtained by the sitting-drop vapordiffusion method. The crystal belongs to the primitive trigonal space group P32, with unit-cell parameters a = b = 77.7 Å, and c = 213.9 Å. The asymmetric unit in the crystal was assumed to contain 8 protein molecules giving the Matthews coefficient (VM ) of 2.54 Å3 Da-1. Se-MAD data were collected to 2.3 Å resolution using synchrotron radiations.