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Gautam Basu

Publications and source records attributed to Gautam Basu.

12 recordsLinked to original sources
PubMed2006

Cloning, expression, purification, and characterization of Vibrio cholerae transcriptional activator, HlyU.

The HlyU from Vibrio cholerae, involved in the transcriptional regulation of haemolysin genes, plays an important role in the regulation of virulence gene expression. We have cloned, over-expressed and purified HlyU from V. cholerae strain O395 in Escherichia coli, as an N-terminal His6-tagged protein. The purified protein gave a single band at approximately 16 kDa on SDS-PAGE, while the sequence analysis revealed the molecular weight of 15.8 kDa. The molecular mass of HlyU, determined in analytical gel-filtration experiments, was approximately 15.7 kDa, an indication that V. cholerae HlyU is a monomer. HlyU has two cysteine residues (38 and 104); reaction with sulfhydryl reagent resulted in one mol of cysteine residue reacting per mol of HlyU, while the protein denatured in guanidine hydrochloride (GdnHCl) showed the reactivity of both the residues. Circular dichroism (CD) analysis showed HlyU to be predominantly alpha-helical, while fluorescence experiment showed that the only tryptophan residue present in HlyU is solvent exposed. HlyU was found to exhibit a two-state GdnHCl-induced unfolding [DeltaG(NU)(H2O) approximately 6.2 kcal mol-1] when monitored by far-UV CD and intrinsic tryptophan fluorescence.

Bacterial Proteins↗
PubMed2005

Biphasic kinetics of the colchicine-tubulin interaction: role of amino acids surrounding the A ring of bound colchicine molecule.

Isotypes of vertebrate tubulin have variable amino acid sequences, which are clustered at their C-terminal ends. Isotypes bind colchicine at different on-rates and affinity constants. The kinetics of colchicine binding to purified (unfractionated) brain tubulin have been reported to be biphasic under pseudo-first-order conditions. Experiments with individual isotypes established that the presence of beta(III) in the purified tubulin is responsible for the biphasic kinetics. Because the isotypes mainly differ at the C termini, the colchicine-binding kinetics of unfractionated tubulin and the beta(III) isotype, cleaved at the C termini, have been tested under pseudo-first-order conditions. Removal of the C termini made no difference to the nature of the kinetics. Sequence alignment of different beta isotypes of tubulin showed that besides the C-terminal region, there are differences in the main body as well. To establish whether these differences lie at the colchicine-binding site or not, homology modeling of all beta-tubulin isotypes was done. We found that the isotypes differed from each other in the amino acids located near the A ring of colchicine at the colchicine-binding site on beta tubulin. While the beta(III) isotype has two hydrophilic residues (serine(242) and threonine(317)), both beta(II) and beta(IV) have two hydrophobic residues (leucine(242) and alanine(317)). beta(II) has isoleucine at position 318, while beta(III) and beta(IV) have valine at that position. Thus, these alterations in the nature of the amino acids surrounding the colchicine site could be responsible for the different colchicine-binding kinetics of the different isotypes of tubulin.

Alanine↗
PubMed2005

Paramagnetic viral nanoparticles as potential high-relaxivity magnetic resonance contrast agents.

In order to compensate for the inherent high threshold of detectability of MR contrast agents, there has been an active interest in the development of paramagnetic nanoparticles incorporating high payloads of Gd(3+) with high molecular relaxivities. Toward this end, the protein cage of Cowpea chlorotic mottle virus (CCMV), having 180 metal binding sites, is being explored. In vivo CCMV binds Ca(2+) at specific metal binding sites; however, Gd(3+) can also bind at these sites. Using fluorescence resonance energy transfer we have characterized the binding affinity of Gd(3+) to the metal binding sites by competition experiments with Tb(3+). The measured dissociation constant (K(d)) for Gd(3+) bound to the virus is 31 microM. The T(1) and T(2) relaxivities of solvent water protons in the presence of Gd(3+)-bound CCMV were 202 and 376 mM(-1) s(-1), respectively, at 61 MHz Larmor frequency. The unusually high relaxivity values of the Gd(3+)-CCMV are largely a result of the nanoparticle virus size and the large number of Gd(3+) ions bound to the virus. These preliminary results should encourage further investigations into the use of viral protein cages as a new platform for MR contrast agents.

Contrast Media↗
PubMed2004

Electrostatic potential of nucleotide-free protein is sufficient for discrimination between adenine and guanine-specific binding sites.

Despite sharing many common features, adenine-binding and guanine-binding sites in proteins often show a clear preference for the cognate over the non-cognate ligand. We have analyzed electrostatic potential (ESP) patterns at adenine and guanine-binding sites of a large number of non-redundant proteins where each binding site was first annotated as adenine/guanine-specific or non-specific from a survey of primary literature. We show that more than 90% of ESP variance at the binding sites is accounted for by only two principal component ESP vectors, each aligned to molecular dipoles of adenine and guanine. Projected on these principal component vectors, the adenine/guanine-specific and non-specific binding sites, including adenine-containing dinucleotides, show non-overlapping distributions. Adenine or guanine specificities of the binding sites also show high correlation with the corresponding electrostatic replacement (cognate by non-cognate ligand) energies. High correlation coefficients (0.94 for 35 adenine-binding sites and 1.0 for 20 guanine-binding sites) were obtained when adenine/guanine specificities were predicted using the replacement energies. Our results demonstrate that ligand-free protein ESP is an excellent indicator for discrimination between adenine and guanine-specific binding sites and that ESP of ligand-free protein can be used as a tool to annotate known and putative purine-binding sites in proteins as adenine or guanine-specific.

Adenine↗
PubMed2004

Expanded turn conformations: characterization and sequence-structure correspondence in alpha-turns with implications in helix folding.

Like the beta-turns, which are characterized by a limiting distance between residues two positions apart (i, i+3), a distance criterion (involving residues at positions i and i+4) is used here to identify alpha-turns from a database of known protein structures. At least 15 classes of alpha-turns have been enumerated based on the location in the phi,psi space of the three central residues (i+1 to i+3)-one of the major being the class AAA, where the residues occupy the conventional helical backbone torsion angles. However, moving towards the C-terminal end of the turn, there is a shift in the phi,psi angles towards more negative phi, such that the electrostatic repulsion between two consecutive carbonyl oxygen atoms is reduced. Except for the last position (i+4), there is not much similarity in residue composition at different positions of hydrogen and non-hydrogen bonded AAA turns. The presence or absence of Pro at i+1 position of alpha- and beta-turns has a bearing on whether the turn is hydrogen-bonded or without a hydrogen bond. In the tertiary structure, alpha-turns are more likely to be found in beta-hairpin loops. The residue composition at the beginning of the hydrogen bonded AAA alpha-turn has similarity with type I beta-turn and N-terminal positions of helices, but the last position matches with the C-terminal capping position of helices, suggesting that the existence of a "helix cap signal" at i+4 position prevents alpha-turns from growing into helices. Our results also provide new insights into alpha-helix nucleation and folding.

Amino Acid Sequence↗
PubMed2003

Metal binding to cowpea chlorotic mottle virus using terbium(III) fluorescence.

Metals are thought to play a role in the structure of many viruses. The crystal structure of the T=3 icosahedral cowpea chlorotic mottle virus (CCMV) suggests the presence of 180 unique metal-binding sites in the assembled protein cage. Each of these sites is thought to involve the coordination of the metal by five amino acids contributed from two adjacent coat protein subunits. We have used fluorescence resonance energy transfer (FRET), from tryptophan residues proximal to the putative metal-binding sites, to probe Tb(III) binding to the virus. Binding of Tb(III) was investigated on the wild-type virus and a mutant where the RNA binding ability of the virus was removed. Tb(III) binding was observed both in the wild-type virus ( K(d)=19 microM) and the mutant ( K(d)=17 microM), as monitored by the increase in Tb(III) fluorescence (545 nm) and concomitant decrease in tryptophan fluorescence (342 nm). Competitive binding experiments showed Ca(II) to have about 100-fold less affinity for the binding sites ( K(d)=1.97 mM). This is the first direct evidence of metal binding to the putative metal-binding sites, originally suggested from the crystal structure of CCMV.

Binding Sites↗
PubMed2003

Sequence and structure patterns in proteins from an analysis of the shortest helices: implications for helix nucleation.

The shortest helices (three-length 3(10) and four-length alpha), most abundant among helices of different lengths, have been analyzed from a database of protein structures. A characteristic feature of three-length 3(10)-helices is the shifted backbone conformation for the C-terminal residue (phi,psi angles: -95 degrees,0 degrees ), compared to the rest of the helix (-62 degrees,-24 degrees ). The deviation can be attributed to the release of electrostatic repulsion between the carbonyl oxygen atoms at the two C-terminal residues and further stabilization (due to a more linear geometry) of an intrahelical hydrogen bond. A consequence of this non-canonical C-terminal backbone conformation can be a potential origin of helix kinks when a 3(10)-helix is sequence-contiguous at the alpha-helix N-terminal. An analysis of hydrogen bonding, as well as hydrophobic interactions in the shortest helices shows that capping interactions, some of them not observed for longer helices, dominate at the N termini. Further, consideration of the distribution of amino acid residues indicates that the shortest helices resemble the N-terminal end of alpha-helices rather than the C terminus, implying that the folding of helices may be initiated at the N-terminal end, which does not get propagated in the case of the shortest helices. Finally, pairwise comparison of beta-turns and the shortest helices, based on correlation matrices of site-specific amino acid composition, and the relative abundance of these short secondary structural elements, leads to a helix nucleation scheme that considers the formation of an isolated beta-turn (and not an alpha-turn) as the helix nucleation step, with shortest 3(10)-helices as intermediates between the shortest alpha-helix and the beta-turn. Our results ascribe an important role played by shortest 3(10)-helices in proteins with important structural and folding implications.

Amino Acid Motifs↗
PubMed2002

Variants of 3(10)-helices in proteins.

An analysis of the shortest 3(10)-helices, containing three helical residues and two flanking capping residues that participate in two consecutive i + 3 --> i hydrogen bonds, shows that not all helices belong to the classic 3(10)-helix, where the three central residues adopt the right-handed helical conformation (alpha(R)). Three variants identified are: 3L10-helix with all residues in the left-handed helical region (alpha(L)), 3EL10-helix where the first residue is in the extended region followed by two residues in the alpha(L) conformation, and its mirror-image, the 3E'R10-helix. In the context of these helices, as well as the equivalent variants of alpha-helices, the length dependence of the handedness of secondary structures in protein structure is discussed. There are considerable differences in the amino acid preferences at different positions in the various types of 3(10)-helices. Each type of 3(10)-helix can be thought to be made up of an extension of a particular type of beta-turn (made up of residues i to i + 3) such that the (i + 3)th residue assumes the same conformation as the preceding residue. Distinct residue preferences at i and i + 3 positions seem to decide whether a particular stretch of four residues will be a beta-turn or a 3(10)-helix in the folded structure.

Amino Acid Motifs↗
PubMed2002

Direct evidence for alteration of unfolding profile of a helical peptide by far-ultraviolet circular dichroism aromatic side-chain contribution.

Aromatic side-chains are known to contribute to the far-UV circular dichroism (CD) spectra of peptides and proteins. Among other things, this can significantly affect the measured helix propensities of amino acids [Chakrabartty et al., Biochemistry 32 (1993) 5560-5565]. In order to address how interfering side-chain contributions can affect the backbone unfolding transition of a helical peptide, as monitored by [theta;](222) (molar ellipticity at 222 nm), we have studied the unfolding transition of a short designed (alpha-amino isobutyric acid/alanine-based) helical peptide containing an interacting Tyr residue. The guanidinium hydrochloride-induced unfolding of the peptide, as monitored by [theta;](222), showed the presence of a sharp transition superposed over a much broader transition. When the same experiment was performed with a similar peptide that lacked the interacting Tyr residue, the sharp transition disappeared and only the broad transition remained. The sharp transition was assigned to originate from the interacting Tyr side-chain. This demonstrates that conformationally restricted aromatic side-chains that interact with the helical backbone not only can alter the backbone far-UV CD signal, they may also alter the unfolding profiles, monitored by far-UV CD, rendering them unfit for a simple analysis for extracting the appropriate unfolding thermodynamic parameters.

Circular Dichroism↗
PubMed2002

Effect of phosphorylation on the structure and fold of transactivation domain of p53.

Several phosphorylations are known to occur in the N-terminal transactivation domain of human p53. To explore the structural effects of these phosphorylations, we have chemically synthesized the unphosphorylated p53-(1-39) and its three phosphorylated analogs, phosphorylated at Ser-15, Thr-18, and Ser-20. p53-(1-39) and its Ser-15 and Thr-18 phosphorylated analogs were tested for interaction with p300. The order of binding affinities was similar to that derived from biochemical experiments with the whole protein, indicating functional integrity of the domain. Differences in chemical shifts and coupling constants indicate significant structural changes upon phosphorylations. The single tryptophan in the unphosphorylated domain has an emission maximum and a Stern-Volmer constant that are characteristics of tryptophans situated in protein interiors. The diffusion constant is monomer-like, with an axial ratio of 1:7.5, indicating a significant degree of compaction. Upon phosphorylations, the emission maximum and diffusion constant change significantly toward values that indicate more open conformations. Binding of the hydrophobic probe bis-1-anilino-8-naphthalenesulfonate to the unphosphorylated and one of the phosphorylated domains is also significantly different, suggesting different conformations. We propose that phosphorylations switch the largely folded transactivation domain to more open conformations that interact with transcription factors such as p300/cAMP- responsive element-binding protein-binding protein, leading to enhancement of gene expression.

Amino Acid Sequence↗
PubMed2002

Aib-based peptide backbone as scaffolds for helical peptide mimics.

Helical peptides that can intervene and disrupt therapeutically important protein-protein interactions are attractive drug targets. In order to develop a general strategy for developing such helical peptide mimics, we have studied the effect of incorporating alpha-amino isobutyric acid (Aib), an amino acid with strong preference for helical backbone, as the sole helix promoter in designed peptides. Specifically, we focus on the hdm2-p53 interaction, which is central to development of many types of cancer. The peptide corresponding to the hdm2 interacting part of p53, helical in bound state but devoid of structure in solution, served as the starting point for peptide design that involved replacement of noninteracting residues by Aib. Incorporation of Aib, while preserving the interacting residues, led to significant increase in helical structure, particularly at the C-terminal region as judged by nuclear magnetic resonance and circular dichroism. The interaction with hdm2 was also found to be enhanced. Most interestingly, trypsin cleavage was found to be retarded by several orders of magnitude. We conclude that incorporation of Aib is a feasible strategy to create peptide helical mimics with enhanced receptor binding and lower protease cleavage rate.

Amino Acid Sequence↗