Inhibition of Plasmodium falciparum triose-phosphate isomerase by chemical modification of an interface cysteine- electrospray ionization mass spectrometric analysis of differential cysteine reactivities

Plasmodium falciparumtriose-phosphate isomerase, a homodimeric enzyme, contains four cysteine residues at positions 13, 126, 196, and 217 per subunit. Among these, Cys-13 is present at the dimer interface and is replaced by methionine in the corresponding human enzyme. We have investigated the effect of sulfhydryl labeling on the parasite enzyme, with a view toward developing selective covalent inhibitors by targeting the interface cysteine residue. Differential labeling of the cysteine residues by iodoacetic acid and iodoacetamide has been followed by electrospray ionization mass spectrometry and positions of the labels determined by analysis of tryptic fragments. The rates of labeling follows the order Cys-196 is > Cys-13 » Cys-217/Cys-126, which correlates well with surface accessibility calculations based on the enzyme crystal structure. Iodoacetic acid labeling leads to a soluble, largely inactive enzyme, whereas IAM labeling leads to precipitation. Carboxyl methylation of Cys-13 results in formation of monomeric species detectable by gel filtration. Studies with an engineered C13D mutant permitted elucidation of the effects of introducing a negative charge at the interface. The C13D mutant exhibits a reduced stability to denaturants and 7-fold reduction in the enzymatic activity even under the concentrations in which dimeric species are observed

Unusual fluorescence of W168 in Plasmodium falciparum triosephosphate isomerase, probed by single-tryptophan mutants

Plasmodium falciparum triosephosphate isomerase (PfTIM) contains two tryptophan residues, W11 and W168. One is positioned in the interior of the protein, and the other is located on the active-site loop 6. Two single-tryptophan mutants, W11F and W168F, were constructed to evaluate the contributions of each chromophore to the fluorescence of the wild-type (wt) protein and to probe the utility of the residues as spectroscopic reporters. A comparative analysis of the fluorescence spectra of PfTIMwt and the two mutant proteins revealed that W168 possesses an unusual, blue-shifted emission (321 nm) and exhibits significant red-edge excitation shift of fluorescence. In contrast, W11 emits at 332 nm, displays no excitation dependence of fluorescence, and behaves like a normal buried chromophore. W168 has a much shorter mean lifetime (2.7 ns) than W11 (4.6 ns). The anomalous fluorescence properties of W168 are abolished on unfolding of the protein in guanidinium chloride (GdmCl) or at low pH. Analysis of the tryptophan environment using a 1.1-Å crystal structure established that W168 is rigidly held by a complex network of polar interactions including a strong hydrogen bond from Y164 to the indole NH group. The environment is almost completely polar, suggesting that electrostatic effects determine the unusually low emission wavelength of W168. To our knowledge this is a unique observation of a blue-shifted emission from a tryptophan in a polar environment in the protein. The wild-type and mutant proteins show similar levels of enzymatic activity and secondary and tertiary structure. However, the W11F mutation appreciably destabilizes the protein to unfolding by urea and GdmCl. The fluorescence of W168 is shown to be extremely sensitive to binding of the inhibitor, 2-phosphoglycolic acid

Perturbation of the Dimer Interface of Triosephosphate Isomerase and its Effect on Trypanosoma cruzi

Most of the enzymes of parasites have their counterpart in the host. Throughout evolution, the three-dimensional architecture of enzymes and their catalytic sites are highly conserved. Thus, identifying molecules that act exclusively on the active sites of the enzymes from parasites is a difficult task. However, it is documented that the majority of enzymes consist of various subunits, and that conservation in the interface of the subunits is lower than in the catalytic site. Indeed, we found that there are significant differences in the interface between the two subunits of triosephosphate isomerase from Homo sapiens and Trypanosoma cruzi (TcTIM), which causes Chagas disease in the American continent. In the search for agents that specifically inhibit TcTIM, we found that 2,2′-dithioaniline (DTDA) is far more effective in inactivating TcTIM than the human enzyme, and that its detrimental effect is due to perturbation of the dimer interface. Remarkably, DTDA prevented the growth of Escherichia coli cells that had TcTIM instead of their own TIM and killed T. cruzi epimastigotes in culture. Thus, this study highlights a new approach base of targeting molecular interfaces of dimers

Proteomics-A new player in the post-genomic era

291-302In the post-genomic era the concept of personalized medicine and molecular medicine emphasizes the utility of the proteomics approach. Proteomics is the global analysis of cellular proteins and complements the genomics approach. Proteins, in principle do all the work of the cell and ultimately dictate all biological processes and the cellular fate. Proteomics uses a combination of sophisticated techniques including two-dimensional (2D) gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing and bioinformatics to identify and characterize proteins. This review aims at providing the various approaches and pitfalls associated with this technique and gives a brief overview of the utility of this approach in the area of biomedical research

Mass spectrometry and protein structure

205-21

Equilibrium denaturation of buffalo pituitary growth hormone

368-374To understand the structural properties of buffalo growth hormone (buGH), the equilibrium denaturation using guanidinium chloride (GdmCl ) was carried out and was monitored by ultraviolet absorption spectroscopy, intrinsic fluorescence spectroscopy, far UV-circular dichroism and size-exclusion chromatography. The normalized denaturation transition curves for each of the above methods were not coincident, showing that buGH does not follow a simple two state folding mechanism. Further, size-exclusion chromatography also showed the presence of an associated intermediate during the unfolding of buGH. It was observed that in buGH, denaturation resulted in an initial disruption of the tertiary structure, whereas the secondary structure and the degree of compactness were disrupted at a higher concentration of the denaturant. This suggests that buGH follows the hierarchical model of protein folding. </span

Equilibrium denaturation of buffalo pituitary growth hormone

To understand the structural properties of buffalo growth hormone (buGH), the equilibrium denaturation using guanidinium chloride (GdmCl) was carried out and was monitored by ultraviolet absorption spectroscopy, intrinsicfluorescence spectroscopy, far UV-circular dichroism and size-exclusion chromatography. The normalized denaturation transition curves for each of the above methods were not coincident, showing that buGH does not follow a simple two state folding mechanism. Further, size-exclusion chromatography also showed the presence of an associated intermediate during the unfolding of buGH. It was observed that in buGH, denaturation resulted in an initial disruption of the tertiary structure, whereas the secondary structure and the degree of compactness were disrupted at a higher concentration of the denaturant. This suggests that buGH follows the hierarchical model of protein folding

Inhibition of Plasmodium falciparum Triose-phosphate Isomerase by Chemical Modification of an Interface Cysteine: Electrospray Ionization Mass Spectrometric Analysis of Differential Cysteine Reactivities

Plasmodium falciparum triose-phosphate isomerase, a homodimeric enzyme, contains four cysteine residues at positions 13, 126, 196, and 217 per subunit. Among these, Cys-13 is present at the dimer interface and is replaced by methionine in the corresponding human enzyme. We have investigated the effect of sulfhydryl labeling on the parasite enzyme, with a view toward developing selective covalent inhibitors by targeting the interface cysteine residue. Differential labeling of the cysteine residues by iodoacetic acid and iodoacetamide has been followed by electrospray ionization mass spectrometry and positions of the labels determined by analysis of tryptic fragments. The rates of labeling follows the order Cys-196 > Cys-13 >> Cys-217/Cys-126, which correlates well with surface accessibility calculations based on the enzyme crystal structure. Iodoacetic acid labeling leads to a soluble, largely inactive enzyme, whereas IAM labeling leads to precipitation. Carboxyl methylation of Cys-13 results in formation of monomeric species detectable by gel filtration. Studies with an engineered C13D mutant permitted elucidation of the effects of introducing a negative charge at the interface. The C13D mutant exhibits a reduced stability to denaturants and 7-fold reduction in the enzymatic activity even under the concentrations in which dimeric species are observed

<span style="font-size:14.0pt;font-family:Fd35564-Identity-H;mso-bidi-font-family: Fd35564-Identity-H;color:#090909">Physico-chemical characterization of growth hormone from water buffaloes <span style="font-size:14.0pt;font-family: Fd234404-Identity-H;mso-bidi-font-family:Fd234404-Identity-H;color:#090909">(<i style="mso-bidi-font-style:normal">Bubalus bubalis</i>) </span></span>

375-383<span style="font-size:14.0pt;font-family:Fd190096-Identity-H;mso-bidi-font-family: Fd190096-Identity-H;color:#1D1D1D">A purified preparation of growth hormone from pituitaries of water buffaloes <span style="font-size:14.0pt; font-family:Fd122394-Identity-H;mso-bidi-font-family:Fd122394-Identity-H; color:#1D1D1D">(Bubalus bubalis) <span style="font-size:14.0pt;font-family:Fd190096-Identity-H;mso-bidi-font-family: Fd190096-Identity-H;color:#1D1D1D">has been extensively characterized with regard to physico-chemical properties. The molecular size of buffalo GH (buGH) by electrospray ionization mass spectroscopy (ES-MS) was found to be 21394.00 <span style="font-size:14.0pt;font-family:Fd228396-Identity-H;mso-bidi-font-family: Fd228396-Identity-H;color:#1D1D1D">± <span style="font-size:14.0pt; font-family:Fd190096-Identity-H;mso-bidi-font-family:Fd190096-Identity-H; color:#1D1D1D">8.44Da and its stokes radius was determined as 2.3 nm. Size heterogeneity in buffalo GH was checked both by electrophoresis and molecular sieve chromatography using 1251-labelled buffalo GH. Similar size heterogeneity was found in standard preparations of ovine and bovine growth hormones. <span style="font-size:14.0pt;font-family:Fd190096-Identity-H;mso-bidi-font-family: Fd190096-Identity-H;color:#1D1D1D">Isoelectric focussing and chromatofocussing indicated charge heterogeneity in buffalo GH preparation. Major charge isoforms having pI of 7.2, 7.7 and minor forms in the iI range of 5.7 to 7.0 were found. Lectin chromatography on Concanavalin A matrix showed that less than 1 % of buffalo GH was glycosylated. Heterogeneity in NH2-terminal sequence was also observed, with alanine, phenylalanine and methionine as the NH2-terminal residues as checked by dansyl and DABITC methods. Estimation of tryptophan residue indicated that a single tryptophan residue was present. Ellman's method showed presence of two disulfide bridges per mole of buffalo GH. Intrinsic fluorescence spectrum of buffalo GH exhibited <span style="font-size: 14.0pt;font-family:Fd228396-Identity-H;mso-bidi-font-family:Fd228396-Identity-H; color:#1D1D1D">A <span style="font-size:14.0pt;font-family:Fd190096-Identity-H; mso-bidi-font-family:Fd190096-Identity-H;color:#1D1D1D">emission maximum at 337 nm. UV-CD spectrum showed that almost 48% of the secondary structure of buGH was constituted by α-helicity. The T<span style="font-size:14.0pt; font-family:Fd228398-Identity-H;mso-bidi-font-family:Fd228398-Identity-H; color:#1D1D1D">M<span style="font-size:14.0pt;font-family:Fd228398-Identity-H; mso-bidi-font-family:Fd228398-Identity-H;color:#1D1D1D"> <span style="font-size:14.0pt;font-family:Fd190096-Identity-H;mso-bidi-font-family: Fd190096-Identity-H;color:#1D1D1D">of buGH as determined by differential scanning calorimetric (DSC) studies was found to be 63°C.<span style="font-size:14.0pt;font-family:Fd190096-Identity-H;mso-bidi-font-family: Fd190096-Identity-H;color:black"> </span