Biomedical subjects

S Agarwalla

Publications and source records attributed to S Agarwalla.

7 recordsLinked to original sources

PubMed1997

A novel dCMP methylase by engineering thymidylate synthase.

X-ray crystal structures of binary complexes of dUMP or dCMP with the Lactobacillus caseiTS mutant N229D, a dCMP methylase, revealed that there is a steric clash between the 4-NH2 of dCMP and His 199, a residue which normally H-bonds to the 4-O of dUMP but is not essential for activity. As a result, the cytosine moiety of dCMP is displaced from the active site and the catalytic thiol is moved from the C6 of the substrate about 0.5 A further than in the wild-type TS-dUMP complex. We reasoned that combining the N229D mutation with mutations at residue 199 which did not impinge on the 4-NH2 of dCMP should correct the displacements and further favor methylation of dCMP. We therefore prepared several TS N229D mutants and characterized their steady state kinetic parameters. TS H199A/N229D showed a 10(11) change in specificity for methylation of dCMP versus dUMP. The structures of TS H199A/N229D in complex with dCMP and dUMP confirmed that the position and orientation of bound dCMP closely approaches that of dUMP in wild-type TS, whereas dUMP was displaced from the optimal catalytic binding site.

Binding Sites↗

PubMed1996

Covalent reinforcement of a fragile region in the dimeric enzyme thymidylate synthase stabilizes the protein against chaotrope-induced unfolding.

Urea and guanidinium chloride induced unfolding of thymidylate synthase, a dimeric enzyme, and engineered interface mutants have been monitored by circular dichroism, fluorescence, and size-exclusion chromatography. Equilibrium unfolding studies show biphasic transitions, with a plateau between 3.5 and 5 M urea, when monitored by far-UV CD and fluorescence energy transfer employing an (aminoethylamino) naphthalenesulfonyl (AEDANS) label at the active site residue, Cys198. AEDANS was also specifically incorporated at position Cys155 in the mutant protein T155C. Direct excitation of this extrinsic fluorophore in the wild type protein (labeled at Cys198) and mutant T155C (labeled at Cys155) showed remarkable differences in the unfolding profiles. C155 AEDANS has a transition centered at 3.5 M urea, which is in contrast to Cys 198 AEDANS (5.5 M urea). Unfolding studies monitored by following intrinsic fluorescence of Trp residues which are located in a small structural domain suggest that this region of the protein is intrinsically fragile. The stable equilibrium intermediate is identified to be an ensemble of partially unfolded aggregated species by gel filtration studies. The chaotrope-induced denaturation of TS appears to proceed through a partially unfolded intermediate that is stabilized by aggregation. Dissociation and loss of structure occur concomitantly at high denaturant concentrations. Introduction of two symmetrically positioned disulfide bridges across the dimer interface in the triple mutant T155C/E188C/C244T (TSMox) stabilized the protein against denaturant-induced unfolding. Aggregate formation was completely abolished in the mutant TSMox, which also enhanced the overall structural stability of the protein. Structural reinforcement of the fragile interface in thymidylate synthase results in dramatic stabilization toward chaotrope-induced unfolding.

Binding Sites↗

PubMed1996

Covalent tethering of the dimer interface annuls aggregation in thymidylate synthase.

Thymidylate synthase (TS), a dimeric enzyme, forms large soluble aggregates at concentrations of urea (3.3-5M), well below that required for complete denaturation, as established by fluorescence and size-exclusion chromatography. In contrast to the wild-type enzyme, an engineered mutant of TS (T155C/E188C/C244T), TSMox, in which two subunits are crosslinked by disulfide bridges between residues 155-188' and 188-155' does not show this behavior. Aggregation behavior is restored upon disulfide bond reduction in the mutant protein, indicating the involvement of interface segments in forming soluble associated species. Intermolecular disulfide crosslinking has been used as a probe to investigate the formation of larger non-native aggregates. The studies argue for the formation of large multimeric species via a sticky patch of polypeptide from the dimer interface region that becomes exposed on partial unfolding. Covalent reinforcement of relatively fragile protein-protein interfaces may be a useful strategy in minimizing aggregation of non-native structures in multimeric proteins.

Bacterial Proteins↗

PubMed1994

Thermal stabilization of thymidylate synthase by engineering two disulfide bridges across the dimer interface.

Thermal inactivation of oligomeric enzymes is most often irreversible and is frequently accompanied by precipitation. We have engineered two symmetry related disulfide bridges (155-188' and 188-155') across the subunit interface of Lactobacillus casei thymidylate synthase, at sites chosen on the basis of an algorithm for the introduction of stereochemically unstrained bridges into proteins. In this communication, we demonstrate a remarkable enhancement in the thermal stability of the covalently cross-linked double disulfide containing dimeric enzyme. The mutant enzyme remains soluble and retains secondary structure even at 90 degrees C, in contrast to the wild-type enzyme which precipitates at 52 degrees C. Furthermore, the mutant enzyme has a temperature optimum of 55 degrees C and possesses appreciable enzymatic activity at 65 degrees C. Cooling restores complete activity, in the mutant protein, demonstrating reversible thermal unfolding. The results suggest that inter-subunit crosslinks can impart appreciable thermal stability in multimeric enzymes.

Amino Acid Sequence↗

PubMed1994

Conformation of the flexible bent helix of Leu1-zervamicin in crystal C and a possible gating action for ion passage.

The membrane channel-forming polypeptide, Leu1-zervamicin, Ac-Leu-Ile-Gln-Iva-Ile5-Thr-Aib-Leu-Aib-Hyp10-Gln-Aib-Hyp-Aib-P ro15-Phol (Aib: alpha-aminoisobutyric acid; Iva: isovaline; Hyp: 4-hydroxyproline; Phol: phenylalininol) has been analyzed by x-ray diffraction in a third crystal form. Although the bent helix is quite similar to the conformations found in crystals A and B, the amount of bending is more severe with a bending angle approximately 47 degrees. The water channel formed by the convex polar faces of neighboring helices is larger at the mouth than in crystals A and B, and the water sites have become disordered. The channel is interrupted in the middle by a hydrogen bond between the OH of Hyp (10) and the NH2 of the Gln(11) of a neighboring molecule. The side chain of Gln(11) is wrapped around the helix backbone in an unusual fashion in order that it can augment the polar side of the helix. In the present crystal C there appears to be an additional conformation for the Gln(11) side chain (with approximately 20% occupancy) that opens the channel for possible ion passage. Structure parameters for C85H140N18O22.xH2O.C2H5OH are space group P2(1)2(1)2(1), a = 10.337(2) A, b = 28.387(7) A, c = 39.864(11) A, Z = 4, agreement factor R = 12.99% for 3250 data observed > 3 sigma (F), resolution = 1.2 A.

Amino Acid Sequence↗

PubMed1992

Zervamicins, a structurally characterised peptide model for membrane ion channels.

Voltage dependent membrane channels are formed by the zervamicins, a group of alpha-aminoisobutyric acid containing peptides. The role of polar residues like Thr, Gln and Hyp in promoting helical bundle formation is established by dramatically reduced channel lifetimes for a synthetic apolar analog. Crystal structures of Leu1-zervamicin reveal association of bent helices. Polar contacts between convex faces result in an 'hour glass' like arrangement of an aqueous channel with a central constriction. The structure suggests that gating mechanisms may involve movement of the Gln11 carboxamide group. Gln3 may play a role in modulating the size of the channel mouth.

Amino Acid Sequence↗

PubMed1991

Crystal structure of [Leu1]zervamicin, a membrane ion-channel peptide: implications for gating mechanisms.

Structures in four different crystal forms of [Leu1]zervamicin (zervamicin Z-L, Ac-Leu-Ile-Gln-Iva-Ile5-Thr-Aib-Leu-Aib-Hyp10-Gln-Aib-Hyp-Aib-P ro15-Phol, where Iva is isovaline, Aib is alpha-amino isobutyric acid, Hyp is 4-hydroxyproline, and Phol is phenylalaninol), a membrane channel-forming polypeptide from Emericellopsis salmosynnemata, have been determined by x-ray diffraction. The helical structure is amphiphilic with all the polar moieties on the convex side of the bent helix. Helices are bent at Hyp10 from approximately 30 degrees to approximately 45 degrees in the different crystal forms. In all crystal forms, the peptide helices aggregate in a similar fashion to form water channels that are interrupted by hydrogen bonds between N epsilon H(Gln11) and O delta (Hyp10) of adjacent helices. The Gln11 side chain is folded in an unusual fashion in order to close the channel. Space is available for an extended conformation for Gln11, in which case the channel would be open, suggesting a gating mechanism for cation transport. Structural details are presented for one crystal form derived from methanol/water solution: C85H140N18O22. 10H2O, space group P21, a = 23.068(6) A, b = 9.162(3) A, c = 26.727(9) A, beta = 108.69(2) degrees (standard deviation of last digit is given in parentheses); overall agreement factor R = 10.1% for 5322 observed relfections [magnitude of Fo greater than 3 sigma (F)]; resolution, 0.93 A.

Amino Acid Sequence↗