Biomedical subjects

B K Muralidhara

Publications and source records attributed to B K Muralidhara.

15 recordsLinked to original sources

PubMed2006

Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide.

The previously laboratory-evolved cytochrome P450 2B1 quadruple mutant V183L/F202L/L209A/S334P (QM), which showed enhanced H(2)O(2)-mediated substrate oxidation, has now been shown to exhibit a >3.0-fold decrease in K(m,HOOH) for 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) O-deethylation compared with the parental enzyme L209A. Subsequently, a streamlined random mutagenesis and a high-throughput screening method were developed using QM to screen and select mutants with enhanced tolerance of catalytic activity to temperature and dimethyl sulfoxide (DMSO). Upon screening >3000 colonies, we identified QM/L295H and QM/K236I/D257N with enhanced catalytic tolerance to temperature and DMSO. QM/L295H exhibited higher activity than QM at a broad range of temperatures (35-55 degrees C) and maintained approximately 1.4-fold higher activity than QM at 45 degrees C for 6 h. In addition, QM/L295H showed a significant increase in T(m,app) compared with L209A. QM/L295H and QM/K236I/D257N exhibited higher activity than QM at a broad range of DMSO concentrations (2.5-15%). Furthermore, QM/K236I/D257N/L295H was constructed by combining QM/K236I/D257N with L295H using site-directed mutagenesis and exhibited a >2-fold higher activity than QM at nearly the entire range of DMSO concentrations. In conclusion, in addition to engineering mammalian cytochromes P450 for enhanced activity, directed evolution can also be used to optimize catalytic tolerance to temperature and organic solvent.

Animals↗

PubMed2006

Folding of Desulfovibrio desulfuricans flavodoxin is accelerated by cofactor fly-casting.

Folding of cofactor-binding proteins involves ligand binding in addition to polypeptide folding. We here assess the kinetic folding/binding landscape for Desulfovibrio desulfuricans flavodoxin that coordinates an FMN cofactor. The apo-form folds in a two-step process involving a burst-phase intermediate. Studies on Tyr98Ala and Trp60Ala variants reveal that these aromatics-that stack with the FMN in the holo-form-are not participating in the apo-protein folding pathway. However, these residues are essential for FMN interactions with the unfolded protein during refolding of holo-flavodoxin. Unfolding of wild-type holo-flavodoxin is coupled to FMN dissociation whereas for Tyr98Ala and Trp60Ala holo-variants, FMN dissociates before polypeptide unfolding. Both variants refold as apo-proteins before FMN rebinds. In sharp contrast, refolding of unfolded wild-type holo-flavodoxin is over an order of magnitude faster than that of the apo-form, the pathway does not include a burst-phase intermediate, and the speed is independent of FMN excess ratio. These observations demonstrate that FMN binds rapidly to the unfolded polypeptide and guides folding straight to the native state. As this path to functional D. desulfuricans holo-flavodoxin is faster than if the cofactor binds to pre-folded apo-protein, this is one of few examples where molecular recognition via a "fly-casting" mechanism is kinetically favored.

Alanine↗

PubMed2006

Conformational flexibility of mammalian cytochrome P450 2B4 in binding imidazole inhibitors with different ring chemistry and side chains. Solution thermodynamics and molecular modeling.

Recent x-ray structures of cytochrome P450 2B4 (CYP2B4) reveal an open form that undergoes a large-scale structural transition to a closed form upon binding to 4-(4-chlorophenyl)imidazole (4-CPI). Here, we report for the first time a complete solution thermodynamic study using isothermal titration calorimetry supported by spectroscopic studies to elucidate the conformational flexibility of CYP2B4 in binding imidazole inhibitors with different ring chemistry and side chains: 4-CPI, 1-benzylimidazole (1-BI), 1-CPI, 4-phenylimidazole (4-PI), 1-(2-(benzyloxy)ethyl)imidazole (BEI), and 1-PI. Each of the inhibitors induced type II spectral changes, and IC50 values for enzyme inhibition ranged from 0.1 to 2.4 microM, following the order 1-BI < 4-CPI < 1-CPI < 4-PI < BEI < 1-PI. Calorimetric titrations using monomeric enzyme yielded a 1:1 binding stoichiometry, with the associated KD values ranging from 0.3 to 4.8 microM and following the same rank order as the IC50 values. Changes in enthalpy at 25 degrees C ranged from -6.5 to -8.8 kcal mol(-1). The largest difference in binding entropy (+5.9 versus -4.1 cal mol(-1) K(-1)) was observed between 4-CPI and BEI, respectively, with a 2-fold difference in heat capacity changes (-604 versus -331 cal mol(-1) K(-1)), which is inferred to result from the reduction of apolar surface area of the enzyme ensuing from a conformational change upon 4-CPI binding. Accessibility to acrylamide of the only tryptophan (Trp121), which is located in helix C, was greatly decreased only in protein bound to 4-CPI. Steric restrictions hindered the perfect docking of only BEI to the closed conformation of the enzyme. The thermodynamic signature obtained for structurally similar inhibitors suggests remarkable plasticity of CYP2B4.

Acrylamides↗

PubMed2006

On the precision of experimentally determined protein folding rates and phi-values.

Phi-values, a relatively direct probe of transition-state structure, are an important benchmark in both experimental and theoretical studies of protein folding. Recently, however, significant controversy has emerged regarding the reliability with which phi-values can be determined experimentally: Because phi is a ratio of differences between experimental observables it is extremely sensitive to errors in those observations when the differences are small. Here we address this issue directly by performing blind, replicate measurements in three laboratories. By monitoring within- and between-laboratory variability, we have determined the precision with which folding rates and phi-values are measured using generally accepted laboratory practices and under conditions typical of our laboratories. We find that, unless the change in free energy associated with the probing mutation is quite large, the precision of phi-values is relatively poor when determined using rates extrapolated to the absence of denaturant. In contrast, when we employ rates estimated at nonzero denaturant concentrations or assume that the slopes of the chevron arms (mf and mu) are invariant upon mutation, the precision of our estimates of phi is significantly improved. Nevertheless, the reproducibility we thus obtain still compares poorly with the confidence intervals typically reported in the literature. This discrepancy appears to arise due to differences in how precision is calculated, the dependence of precision on the number of data points employed in defining a chevron, and interlaboratory sources of variability that may have been largely ignored in the prior literature.

Fluorometry↗

PubMed2005

Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: insight into P450 conformational plasticity and membrane interaction.

To better understand ligand-induced structural transitions in cytochrome P450 2B4, protein-ligand interactions were investigated using a bulky inhibitor. Bifonazole, a broad spectrum antifungal agent, inhibits monooxygenase activity and induces a type II binding spectrum in 2B4dH(H226Y), a modified enzyme previously crystallized in the presence of 4-(4-chlorophenyl)imidazole (CPI). Isothermal titration calorimetry and tryptophan fluorescence quenching indicate no significant burial of protein apolar surface nor altered accessibility of Trp-121 upon bifonazole binding, in contrast to recent results with CPI. A 2.3 A crystal structure of 2B4-bifonazole reveals a novel open conformation with ligand bound in the active site, which is significantly different from either the U-shaped cleft of ligand-free 2B4 or the small active site pocket of 2B4-CPI. The O-shaped active site cleft of 2B4-bifonazole is widely open in the middle but narrow at the top. A bifonazole molecule occupies the bottom of the active site cleft, where helix I is bent approximately 15 degrees to accommodate the bulky ligand. The structure also defines unanticipated interactions between helix C residues and bifonazole, suggesting an important role of helix C in azole recognition by mammalian P450s. Comparison of the ligand-free 2B4 structure, the 2B4-CPI structure, and the 2B4-bifonazole structure identifies structurally plastic regions that undergo correlated conformational changes in response to ligand binding. The most plastic regions are putative membrane-binding motifs involved in substrate access or substrate binding. The results allow us to model the membrane-associated state of P450 and provide insight into how lipophilic substrates access the buried active site.

Antifungal Agents↗

PubMed2005

Effect of inorganic phosphate on FMN binding and loop flexibility in Desulfovibrio desulfuricans apo-flavodoxin.

The complex between flavin mononucleotide (FMN) and apo-flavodoxin is dominated by isoalloxazine-stacking interactions and 5'-phosphate hydrogen bonds. We show here that FMN binding to Desulfovibrio desulfuricans apo-flavodoxin is faster and the affinity is higher in the presence of inorganic phosphate as compared to in its absence (I=110 mM, pH 7, 20 degrees C). The transition-state of complex formation was investigated by phi-value analysis using Trp60Ala and Tyr98Ala apo-flavodoxin variants. We find that Tyr98 is highly involved in the FMN/protein transition state independent of inorganic phosphate, whereas the participation of Trp60 is modulated by inorganic phosphate. The phi-value for Trp60 is higher without phosphate, implying that at this condition stronger binding of Trp60 is required in the transition state to assure successful complex formation. Consistent with the experimental data, all-atom molecular dynamic simulations reveal that the presence of an anion in the phosphate subsite restricts the mobility of the Trp60-containing loop in terms of both backbone and side-chain movements, but has no effect on the Tyr98-containing loop. The overall thermodynamic stability of apo-flavodoxin is higher in the presence of inorganic phosphate as compared to in its absence (I=110 mM, pH 7, 20 degrees C). Kinetic experiments reveal that the additional stability originates in slower unfolding. The combined experimental and computational observations demonstrate that phosphate has an ordering effect on the Trp60-containing loop, which positions Trp60 favorably for FMN binding and increases the barrier for protein unfolding.

Apoproteins↗

PubMed2005

How do cofactors modulate protein folding?

Cofactors are essential components of many proteins for biological activity. Characterization of several cofactor-binding proteins has shown that cofactors often have the ability to interact specifically with the unfolded polypeptides. This suggests that cofactor-coordination prior to polypeptide folding may be a relevant path in vivo. By binding before folding, the cofactor may affect both the mechanism and speed of folding. Here, we discuss three different cofactors that modulate protein-folding processes in vitro.

Animals↗

PubMed2004

FMN binding and unfolding of Desulfovibrio desulfuricans flavodoxin: "hidden" intermediates at low denaturant concentrations.

The flavin mononucleotide (FMN) cofactor in Desulfovibrio desulfuricans flavodoxin stays associated with the polypeptide upon guanidine hydrochloride (GuHCl) induced unfolding. Using isothermal titration calorimetry (ITC), we determined the affinity of FMN for the flavodoxin polypeptide as a function of both urea and GuHCl concentrations (pH 7, 25 degrees C). The FMN affinity for folded and GuHCl-unfolded flavodoxin differs 10-fold, which is in agreement with the difference in thermodynamic stability between the apo- and holo-forms. In contrast, the urea-unfolded protein does not interact with FMN and equilibrium unfolding of holo-flavodoxin in urea results in FMN dissociation prior to polypeptide unfolding. ANS-binding, near-UV circular dichroism (CD), acrylamide quenching and FMN-emission experiments reveal the presence of native-like intermediates, not detected by far-UV CD and aromatic fluorescence detection methods, in low concentrations of both denaturants. Time-resolved experiments show that FMN binding is fastest at GuHCl concentrations where the native-like intermediate species is populated.

Circular Dichroism↗

PubMed2004

Thermal unfolding of Apo and Holo Desulfovibrio desulfuricans flavodoxin: cofactor stabilizes folded and intermediate states.

We here compare thermal unfolding of the apo and holo forms of Desulfovibrio desulfuricans flavodoxin, which noncovalently binds a flavin mononucleotide (FMN) cofactor. In the case of the apo form, fluorescence and far-UV circular dichroism (CD) detected transitions are reversible but do not overlap (T(m) of 50 and 60 degrees C, respectively, pH 7). The thermal transitions for the holo form follow the same pattern but occur at higher temperatures (T(m) of 60 and 67 degrees C for fluorescence and CD transitions, respectively, pH 7). The holoprotein transitions are also reversible and exhibit no protein concentration dependence (above 10 microM), indicating that the FMN remains bound to the polypeptide throughout. Global analysis shows that the thermal reactions for both apo and holo forms proceed via an equilibrium intermediate that has approximately 90% nativelike secondary structure and significant enthalpic stabilization relative to the unfolded states. Incubation of unfolded holoflavodoxin at high temperatures results in FMN dissociation. Rebinding of FMN at these conditions is nominal, and therefore, cooling of holoprotein heated to 95 degrees C follows the refolding pathway of the apo form. However, FMN readily rebinds to the apoprotein at lower temperatures. We conclude that (1) a three-state thermal unfolding behavior appears to be conserved among long- and short-chain, as well as apo and holo forms of, flavodoxins and (2) flavodoxin's thermal stability (in both native and intermediate states) is augmented by the presence of the FMN cofactor.

Apoproteins↗

PubMed2003

Can cofactor-binding sites in proteins be flexible? Desulfovibrio desulfuricans flavodoxin binds FMN dimer.

Flavodoxins catalyze redox reactions using the isoalloxazine moiety of the flavin mononucleotide (FMN) cofactor stacked between two aromatic residues located in two peptide loops. At high FMN concentrations that favor stacked FMN dimers in solution, isothermal titration calorimetric studies show that these dimers bind strongly to apo-flavodoxin from Desulfovibrio desulfuricans (30 degrees C, 20 mM Hepes, pH 7, K(D) = 5.8 microM). Upon increasing the temperature so the FMN dimers dissociate (as shown by (1)H NMR), only one-to-one (FMN-to-protein) binding is observed. Calorimetric titrations result in one-to-one binding also in the presence of phosphate or sulfate (30 degrees C, 13 mM anion, pH 7, K(D) = 0.4 microM). FMN remains dimeric in the presence of phosphate and sulfate, suggesting that specific binding of a divalent anion to the phosphate-binding site triggers ordering of the peptide loops so only one isoalloxazine can fit. Although the physiological relevance of FMN and other nucleotides as dimers has not been explored, our study shows that high-affinity binding to proteins of such dimers can occur in vitro. This emphasizes that the cofactor-binding site in flavodoxin is more flexible than previously expected.

Anions↗

PubMed2001

Anion-mediated Fe3+ release mechanism in ovotransferrin C-lobe: a structurally identified SO4(2-) binding site and its implications for the kinetic pathway.

The differential properties of anion-mediated Fe(3+) release between the N- and C-lobes of transferrins have been a focus in transferrin biochemistry. The structural and kinetic characteristics for isolated lobe have, however, been documented with the N-lobe only. Here we demonstrate for the first time the quantitative Fe(3+) release kinetics and the anion-binding structure for the isolated C-lobe of ovotransferrin. In the presence of pyrophosphate, sulfate, and nitrilotriacetate anions, the C-lobe released Fe(3+) with a decelerated rate in a single exponential progress curve, and the observed first order rate constants displayed a hyperbolic profile as a function of the anion concentration. The profile was consistent with a newly derived single-pathway Fe(3+) release model in which the holo form is converted depending on the anion concentration into a "mixed ligand" intermediate that releases Fe(3+). The apo C-lobe was crystallized in ammonium sulfate solution, and the structure determined at 2.3 A resolution demonstrated the existence of a single bound SO(4)(2-) in the interdomain cleft, which interacts directly with Thr(461)-OG1, Tyr(431)-OH, and His(592)-NE2 and indirectly with Tyr(524)-OH. The latter three groups are Fe(3+)-coordinating ligands, strongly suggesting the facilitated Fe(3+) release upon the anion occupation at this site. The SO(4)(2-) binding structure supported the single-pathway kinetic model.

Anions↗

PubMed2000

Anion-mediated iron release from transferrins. The kinetic and mechanistic model for N-lobe of ovotransferrin.

Iron release process of ovotransferrin N-lobe (N-oTf) to anion/chelators has been resolved using kinetic and mechanistic approach. The iron release kinetics of N-oTf were measured at the endosomal pH of 5.6 with three different anions such as nitrilotriacetate, pyrophosphate, and sulfate using stopped flow spectrofluorimetric method, all yielding clear biphasic progress curves. The two observed rate constants and the corresponding amplitudes obtained from the double exponential curve fit to the biphasic curves varied depending on the type and concentration of anions. Several possible models for the iron release kinetic mechanism were examined on the basis of a newly introduced quantitative equation. Results from the curve fitting analyses were consistent with a dual pathway mechanism that includes the competitive iron release from two different protein states, namely, X and Y, with the respective first order rate constants of K(1) and K(2) (X, domain closed holo N-oTf; Y, anion induced different conformer of holo N-oTf). The reversible interconversions of X to Y and Y to X are driven by the second order rate constant k(3) and the first order rate constant K(4), respectively. The obtained rate constants were greatly variable for the three anions depending on the synergistic or nonsynergistic nature. In the light of the anion-binding sites of N-oTf located crystallographically, the compatible mechanistic model that includes competitive anion binding to the iron coordination sites and to a specific anion site is suggested for the dual pathway iron release mechanism.

Anions↗

PubMed2000

Structural and functional consequences of removal of the interdomain disulfide bridge from the isolated C-lobe of ovotransferrin.

The interdomain disulfide bond present in the C-lobe of all the transferrins was postulated to restrict the domain movement resulting in the slow rate of iron uptake and release. In the present study, the conformational stability and iron binding properties of a derivative of the isolated C-lobe of ovotransferrin in which the interdomain disulfide bond, Cys478-Cys671 was selectively reduced and alkylated with iodoacetamide were compared with the disulfide intact form at the endosomal pH of 5.6. Pyrophosphate and chloride mediated iron release kinetics showed no difference between the disulfide-intact and disulfide-reduced/alkylated forms; the two protein forms yielded similar observed rate constants showing an apparent hyperbolic dependency for anion concentrations. The conformational stability evaluated by unfolding and refolding experiments was greater for the disulfide-intact form than for the disulfide-reduced/alkylated form: the deltaG(D)H2O values at 30 degrees C obtained by using urea were 9.0+/-0.8 and 6.0+/-0.4 kJ/mol for the former and latter protein forms, respectively, and the corresponding values obtained by using guanidine hydrochloride were 6.2+/-0.9 and 4.3+/-0.5 kJ/mol. The dissociation constant of iron (kd) was almost the same for the two protein forms, and it varied only subtly with urea concentrations but increased markedly with GdnHCl concentrations. The nonidentical values of deltaG(D)H2O and kd for urea and GdnHCl can be attributed to the ionic nature of the later denaturant, in which chloride anion may influence the structure and iron uptake-release properties of the ovotransferrin C-lobe. Taken together, we conclude that the interdomain disulfide bond has no effect on the iron uptake and release function but significantly decreases the conformational stability in the C-lobe.

Conalbumin↗

PubMed1998

Thermal stabilization of multimeric proteins: a case study with alpha-globulin.

Preferential interaction parameters of multisubunit protein, alpha-globulin and monomeric protein human serum albumin (HSA) were determined in different cosolvents using precision densitymetry. The apparent partial specific volumes were determined under both isomolal and isopotential conditions for alpha-globulin in 0.02 M glycine-NaOH buffer at pH 10 and the values were 0.692+/-0.002 and 0.688+/-0.001, ml/g, respectively, at 20.00+/-0.01 degrees C. From the partial specific volume data with cosolvents the preferential interaction parameter (xi3) and other thermodynamic parameters were calculated at different solvent concentrations. The (xi3) values increased with an increase in the solvent concentration up to 30% and reached a maximum with the values of-0.111+/-0.018 g/g and -0.076+/-0.012 g/g in sucrose and sorbitol, respectively. In glycerol the (xi3) values decreased with an increase in solvent concentration. The above data is further supported by thermal denaturation profiles in which the apparent thermal denaturation temperature (apparent Tm) of alpha-globulin shows an increase from 63 degrees C to higher temperatures in the order of sucrose, sorbitol and glycerol. Alpha-globulin showed coagulation due to protein interaction at temperatures above 50 degree C. The apparent Tm of 63 degrees C for control protein was increased significantly up to 75 degrees C in 40% sorbitol with two fold increase in the delta(S) values showing the increased structural stability of alpha-globulin. At high solvent concentration the protein gets dissociated and the resultant monomers are hydrated which was evident by fluorescence data and the difference spectral results with a 6nm red shift in the emission maximum and 2 nm blue shift in UV-absorption maximum arising out of perturbation of aromatic chromophores. The studies were performed both at native pH of 7.9 where the protein is in its oligomeric form and at pH of 10 where it is dissociated form and the results compared. The data showed that the solvent is excluded more from the protein vicinity in the dissociated state.

Densitometry↗

PubMed1995

Interaction of 3'-O-caffeoyl D-quinic acid with human serum albumin.

The interaction of chlorogenic acid (CGA) with human serum albumin (HSA) was studied from the view-point of thermodynamics and mechanism of binding at pH 6.0. The association constants (Ka) for the HSA-CGA interaction at 10, 25 and 40 degrees C were 6.0 x 10(4), 9.0 x 10(3) and 2 x 10(4) M-1, resulting in delta G of -6.21, -5.80, -6.32 kcal/mol, respectively. These high Ka-values showed that the interaction between CGA and HSA is strong, endothermic and entropically driven. Binding of chlorogenic acid induces conformational change in HSA as indicated by quenching of fluorescence emission intensity along with a red shift in the emission maxima from 338 to 350 nm. This suggested the involvement of the lone tryptophan residue in the region of binding. Far-ultraviolet circular dichroic data showed a decrease in the alpha-helical content of HSA from 56 to 50% upon binding of CGA. These data are also supported by the decrease in the apparent Tm of HSA by 4 degrees C upon binding of CGA causing destabilization of the HSA molecule. The kinetics of the interaction involves a single step in the binding, and the kinetic curve attains equilibrium within 180 +/- 5 s. Data on caffeic acid (CA) and quinic acid (QA), which are the hydrolysis products of the bidentate CGA molecule, indicate that CA interacts more strongly than CGA. CA binds with an association constant of 8 x 10(4) M-1 and with a maximum number of binding sites of four. Microcalorimetric investigation of the interaction of these ligands with HSA suggests that the strength of binding follows the order CA >> CGA >>> QA with a single class of binding sites. The effect of temperature on the binding of CGA to HSA showed that the interaction is dominated by hydrophobic forces and hydrogen bonding.

Binding Sites↗