Improvement of bacterial blight resistance of the high yielding, fine-grain, rice variety, Gangavati sona through marker-assisted backcross breeding.

Gangavati sona (GS) is a high-yielding, fine-grain rice variety widely grown in the Tungabhadra command area in Karnataka, India; however, it is susceptible to bacterial blight (BB). Therefore, the present study was conducted to improve the GS variety for BB resistance. Three BB-resistant genes (xa5, xa13, and Xa21) were introgressed into the genetic background of susceptible cultivar GS through marker-assisted backcrossing (MABB) by using Improved samba Mahsuri (ISM), a popular, high-yielding, bacterial blight resistant rice variety as a donor parent. Foreground selection was carried out using gene-specific markers, viz., xa5FM (xa5), xa13prom (xa13), and pTA248 (Xa21), while background selection was carried out using well-distributed 64 polymorphic microsatellite markers. The true heterozygote F1 was used as the male parent for backcrossing with GS to obtain BC1F1. The process was repeated in BC1F1 generation, and a BC2F1 plant (IGS-5-11-5) possessing all three target genes along with maximum recurrent parent genome (RPG) recovery (86.7%) was selfed to obtain BC2F2s. At BC2F2, a single triple gene homozygote plant (IGS-5-11-5-33) with 92.6% RPG recovery was identified and advanced to BC2F5 by a pedigree method. At BC2F5, the seven best entries were selected, possessing all three resistance genes with high resistance levels against bacterial blight, yield level, and grain quality features equivalent to better than GS. The improved versions of GS will immensely benefit the farmers whose fields are endemic to BB.

Genome-wide survey and characterization of microsatellites in cashew and design of a web-based microsatellite database: CMDB.

The cashew is an edible tree nut crop having a wide range of food and industrial applications. Despite great economic importance, the genome-wide characterization of microsatellites [simple sequence repeats (SSRs)] in cashew is lacking. In this study, we carried out the first comprehensive genome-wide microsatellites/SSRs characterization in cashew and developed polymorphic markers and a web-based microsatellite database. A total of 54526 SSRs were discovered in the cashew genome, with a mean frequency of 153 SSRs/Mb. Among the mined genome-wide SSRs (2-6 bp size motifs), the dinucleotide repeat motifs were dominant (68.98%) followed by the trinucleotides (24.56%). The Class I type of SSRs (≥20 bp) were 45.10%, while Class II repeat motifs (≥12-<20 bp) were 54.89% of the total genomic SSRs discovered here. Further, the AT-rich SSRs occurred more frequently in the cashew genome (84%) compared to the GC-rich SSRs. The validation of the in silico-mined genome-wide SSRs by PCR screening in cashew genotypes resulted in the development of 59 polymorphic SSR markers, and the polymorphism information content (PIC) of the polymorphic SSR markers ranged from 0.19 to 0.84. Further, a web-based database, "Cashew Microsatellite Database (CMDB)," was constructed to provide access to the genome-wide SSRs mined in this study as well as transcriptome-based SSRs from our previous study to the research community through a user-friendly searchable interface. Besides, CMDB provides information on experimentally validated SSRs. CMDB permits the retrieval of SSR markers information with the customized search options. Altogether, the genome-wide SSRs characterization, the polymorphic markers and CMDB database developed in this study would serve as valuable marker resources for DNA fingerprinting, germplasm characterization, genetic studies, and molecular breeding in cashew and related Anacardium species.

De novo assembly and characterization of the draft genome of the cashew (Anacardium occidentale L.).

Cashew is the second most important tree nut crop in the global market. Cashew is a diploid and heterozygous species closely related to the mango and pistachio. Its improvement by conventional breeding is slow due to the long juvenile phase. Despite the economic importance, very little genomics/transcriptomics information is available for cashew. In this study, the Oxford nanopore reads and Illumina reads were used for de novo assembly of the cashew genome. The hybrid assembly yielded a 356.6 Mb genome corresponding to 85% of the estimated genome size (419 Mb). The BUSCO analysis showed 91.8% of genome completeness. Transcriptome mapping showed 92.75% transcripts aligned with the assembled genome. Gene predictions resulted in the identification of 31,263 genes coding for a total of 35,000 gene isoforms. About 46% (165 Mb) of the cashew genome comprised of repetitive sequences. Phylogenetic analyses of the cashew with nine species showed that it was closely related to Mangifera indica. Analysis of cashew genome revealed 3104 putative R-genes. The first draft assembly of the genome, transcriptome and R gene information generated in this study would be the foundation for understanding the molecular basis of economic traits and genomics-assisted breeding in cashew.

Perception, knowledge, and attitude toward mental disorders and psychiatry among medical undergraduates in Karnataka: A cross-sectional study.

CONTEXT: Globally, psychiatry as a subject, psychiatrists as professionals, and patients with psychiatric disorders are subjected to cultural stereotypes and negative attitude by the general population. What is of alarming concern is that these prejudices exist within the medical community as well. AIMS: This study aims at evaluating the perception, knowledge, and attitude toward psychiatric disorders, therapeutic modalities used in psychiatry, psychiatry as a subject and psychiatrists as professionals among undergraduate medical students in Karnataka. SETTINGS AND DESIGN: This is a descriptive, cross-sectional type of study conducted in three medical colleges located in Karnataka. MATERIALS AND METHODS: A sample of 500 students from all three professional phases of MBBS was selected using purposive sampling. A semistructured prevalidated questionnaire was used to assess the perception, knowledge, and attitude of undergraduate medical students toward psychiatric disorders and psychiatry. STATISTICAL ANALYSIS: Data were analyzed using Statistical Package for Social Sciences, version 16.0. RESULTS: The undergraduate medical student population had significant shortcomings in knowledge and attitude pertaining to psychiatric disorders, more glaring in the initial years of education. A comparatively positive opinion was obtained regarding psychiatry as a subject and psychiatrists as professionals, which may reflect the changing trends and concepts, both in society and medical community. CONCLUSION: This study highlights the need for better educational measures at undergraduate level in order to shape a positive attitude of the health care providers towards psychiatry, which is essential for ensuring better care for patients as well as reduction of stigma surrounding psychiatric disorders.

Association between gender, process of care measures, and outcomes in ACS in India: results from the detection and management of coronary heart disease (DEMAT) registry.

BACKGROUND: Studies from high-income countries have shown that women receive less aggressive diagnostics and treatment than men in acute coronary syndromes (ACS), though their short-term mortality does not appear to differ from men. Data on gender differences in ACS presentation, management, and outcomes are sparse in India. METHODS AND RESULTS: The Detection and Management of Coronary Heart Disease (DEMAT) Registry collected data from 1,565 suspected ACS patients (334 women; 1,231 men) from ten tertiary care centers throughout India between 2007-2008. We evaluated gender differences in presentation, in-hospital and discharge management, and 30-day death and major adverse cardiovascular event (MACE; death, re-hospitalization, and cardiac arrest) rates. Women were less likely to present with STEMI than men (38% vs. 55%, p<0.001). Overall inpatient diagnostics and treatment patterns were similar between men and women after adjustment for potential confounders. Optimal discharge management with aspirin, clopidogrel, beta-blockers, and statin therapy was lower for women than men, (58% vs. 65%, p = 0.03), but these differences were attenuated after adjustment (OR = 0.86 (0.62, 1.19)). Neither the outcome of 30-day mortality (OR = 1.40 (0.62, 3.16)) nor MACE (OR = 1.00 (0.67, 1.48)) differed significantly between men and women after adjustment. CONCLUSIONS: ACS in-hospital management, discharge management, and 30-day outcomes did not significantly differ between genders in the DEMAT registry, though consistently higher treatment rates and lower event rates in men compared to women were seen. These findings underscore the importance of further investigation of gender differences in cardiovascular care in India.

Identification and analysis of conserved sequence motifs in cytochrome P450 family 2. Functional and structural role of a motif 187RFDYKD192 in CYP2B enzymes.

Using a multiple alignment of 175 cytochrome P450 (CYP) family 2 sequences, 20 conserved sequence motifs (CSMs) were identified with the program PCPMer. Functional importance of the CSM in CYP2B enzymes was assessed from available data on site-directed mutants and genetic variants. These analyses suggested an important role of the CSM 8, which corresponds to(187)RFDYKD(192) in CYP2B4. Further analysis showed that residues 187, 188, 190, and 192 have a very high rank order of conservation compared with 189 and 191. Therefore, eight mutants (R187A, R187K, F188A, D189A, Y190A, K191A, D192A, and a negative control K186A) were made in an N-terminal truncated and modified form of CYP2B4 with an internal mutation, which is termed 2B4dH/H226Y. Function was examined with the substrates 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC), 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and testosterone and with the inhibitors 4-(4-chlorophenyl)imidazole (4-CPI) and bifonazole (BIF). Compared with the template and K186A, the mutants R187A, R187K, F188A, Y190A, and D192A showed > or =2-fold altered substrate specificity, k(cat), K(m), and/or k(cat)/K(m) for 7-MFC and 7-EFC and 3- to 6-fold decreases in differential inhibition (IC(50,BIF)/IC(50,4-CPI)). Subsequently, these mutants displayed 5-12 degrees C decreases in thermal stability (T(m)) and 2-8 degrees C decreases in catalytic tolerance to temperature (T(50)) compared with the template and K186A. Furthermore, when R187A and D192A were introduced in CYP2B1dH, the P450 expression and thermal stability were decreased. In addition, R187A showed increased activity with 7-EFC and decreased IC(50,BIF)/IC(50,4-CPI) compared with 2B1dH. Analysis of long range residue-residue interactions in the CYP2B4 crystal structures indicated strong hydrogen bonds involving Glu(149)-Asn(177)-Arg(187)-Tyr(190) and Asp(192)-Val(194), which were significantly-reduced/abolished by the Arg(187)-->Ala and Asp(192)-->Alasubstitutions, respectively.

Thermodynamic fidelity of the mammalian cytochrome P450 2B4 active site in binding substrates and inhibitors.

Structural plasticity of mammalian cytochromes P450 (CYP) has recently been explored in our laboratory and elsewhere to understand the ligand-binding promiscuity. CYP2B4 exhibits very different conformations and thermodynamic signatures in binding the small inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) versus the large bifonazole. Using four key active-site mutants (F296A, T302A, I363A, and V367L) that are involved in binding one or both inhibitors, we dissected the thermodynamic basis for the ability of CYP2B4 to bind substrates and inhibitors of different sizes and chemistry. In all cases, 1:1 binding stoichiometry was observed. The inhibitors 4-CPI, 1-(4-chlorophenyl)imidazole, and 1-(2-(benzyloxy)ethyl)imidazole bind to the mutants with a free energy difference (Delta Delta G) of approximately 0.5 to 1 kcal/mol compared with the wild type but with a large entropy-enthalpy compensation of up to 50 kcal/mol. The substrate testosterone binds to all four mutants with a Delta Delta G of approximately 0.5 kcal/mol but with as much as 40 kcal/mol of entropy-enthalpy compensation. In contrast, benzphetamine binding to V367L and F296A is accompanied by a Delta Delta G of approximately 1.5 and 3 kcal/mol, respectively. F296A, I363A, and V367L exhibit very different benzphetamine metabolite profiles, indicating the different substrate-binding orientations in the active site of each mutant. Overall, the findings indicate that malleability of the active site allows mammalian P450s to exhibit a high degree of thermodynamic fidelity in ligand binding.

Thermodynamics of ligand binding to P450 2B4 and P450eryF studied by isothermal titration calorimetry.

Structural plasticity and cooperativity in ligand recognition are two key aspects of the catalytic diversity of cytochrome P450 enzymes. As more mammalian P450 crystal structures have emerged, computational modeling has become a major tool to predict drug metabolism and interactions. There is a need for real solution thermodynamic data to support modeling and crystallographic observations. Using isothermal titration calorimetry (ITC) we successfully evaluated the conformational flexibility of P450 2B4 in binding imidazole inhibitors of different size and chemistry and dissected the stoichiometry and energetics of ligand binding allostery in P450eryF. Thermodynamic signatures obtained by ITC nicely correlated with structural and modeling results. Thus, ITC is a powerful tool to study structure-function relationships in P450s.

Structural and thermodynamic consequences of 1-(4-chlorophenyl)imidazole binding to cytochrome P450 2B4.

The crystal structure of P450 2B4 bound with 1-(4-chlorophenyl)imidazole (1-CPI) has been determined to delineate the structural basis for the observed differences in binding affinity and thermodynamics relative to 4-(4-chlorophenyl)imidazole (4-CPI). Compared with the previously reported 4-CPI complex, there is a shift in the 1-CPI complex of the protein backbone in helices F and I, repositioning the side chains of Phe-206, Phe-297, and Glu-301, and leading to significant reshaping of the active site. Phe-206 and Phe-297 exchange positions, with Phe-206 becoming a ligand-contact residue, while Glu-301, rather than hydrogen bonding to the ligand, flips away from the active site and interacts with His-172. As a result the active site volume expands from 200 A3 in the 4-CPI complex to 280 A3 in the 1-CPI complex. Based on the two structures, it was predicted that a Phe-206-->Ala substitution would alter 1-CPI but not 4-CPI binding. Isothermal titration calorimetry experiments indicated that this substitution had no effect on the thermodynamic signature of 4-CPI binding to 2B4. In contrast, relative to wild-type 1-CPI binding to F206A showed significantly less favorable entropy but more favorable enthalpy. This result is consistent with loss of the aromatic side chain and possible ordering of water molecules, now able to interact with Glu-301 and exposed residues in the I-helix. Hence, thermodynamic measurements support the active site rearrangement observed in the crystal structure of the 1-CPI complex and illustrate the malleability of the active site with the fine-tuning of residue orientations and thermodynamic signatures.

Rational engineering of human cytochrome P450 2B6 for enhanced expression and stability: importance of a Leu264->Phe substitution.

Despite the emerging importance of human P450 2B6 in xenobiotic metabolism, thorough biochemical and biophysical characterization has been impeded as a result of low expression in Escherichia coli. Comparison with similar N-terminal truncated and C-terminal His-tagged constructs (rat P450 2B1dH, rabbit 2B4dH, and dog 2B11dH) revealed that P450 2B6dH showed the lowest thermal stability, catalytic tolerance to temperature, and chemical stability against guanidinium chloride-induced denaturation. Eleven P450 2B6dH mutants were rationally engineered based on sequence comparison with the three other P450 2B enzymes and the solvent accessibility of residues in the ligand-free crystal structure of P450 2B4dH. L198M, L264F, and L390P showed approximately 3-fold higher expression than P450 2B6dH. L264F alone showed enhanced stability against thermal and chemical denaturation compared with P450 2B6dH and was characterized further functionally. L264F showed similar preferential inhibition by pyridine over imidazole derivatives as P450 2B6dH. The Leu(264)-->Phe substitution did not alter the K(s) for inhibitors or the substrate benzphetamine, the K(m) for 7-ethoxy-4-(trifluoromethyl)coumarin, or the benzphetamine metabolite profiles. The enhanced stability and monodisperse nature of L264F made it suitable for isothermal titration calorimetry studies. Interaction of 1-benzylimidazole with L264F yielded a clear binding isotherm with a distinctly different thermodynamic signature from P450 2B4dH. The inhibitor docked differently in the binding pocket of a P450 2B6 homology model than in 2B4, highlighting the different chemistry of the active site of these two enzymes. Thus, L264F is a good candidate to further explore the unique structure-function relationships of P450 2B6 using X-ray crystallography and solution thermodynamics.

Dissecting the thermodynamics and cooperativity of ligand binding in cytochrome P450eryF.

Conformational flexibility and cooperativity in ligand recognition are two key aspects of the catalytic diversity of cytochrome P450 enzymes. In this study, we dissect the ligand binding stoichiometry and energetics of the soluble bacterial P450eryF by isothermal titration calorimetry (ITC) using three allosteric and two non-allosteric ligands of diverse chemistry. Complementary spectral binding studies and sequential, two-ligand docking simulations were performed to help assign the binding sites. Binding of 4-phenylpyridine (4-PP) or 4-(4-chlorophenyl)imidazole (4-CPI) showed 1:1 stoichiometry in ITC, consistent with the lack of cooperativity observed in spectral binding studies. The larger ligands 9-aminophenanthrene (9-AP), 1-pyrenebutanol (1-PB), and alpha-naphthoflavone (ANF) show cooperative spectral binding and yielded 2:1 stoichiometry. The associated thermodynamic parameters for the sites were calculated using a sequential binding mechanism. The binding constant (KD) for the first site was almost two times lower than that of the second site for all three compounds. Ligand binding at site 1 was entropically favored, whereas binding at site 2 was weak and entropically unfavorable. Simulations showed that two molecules of 9-AP, ANF or 1-PB can be adequately docked to two individual sub-sites within a large binding pocket. The absence of hydrophobic tethering and ligand stacking are consistent with the single low affinity binding site observed for 4-CPI and 4-PP. Competitive binding studies with P450eryF preloaded with either 1-PB or ANF showed a decrease in the affinities for 9-AP at both the sites, with large entropy-enthalpy compensation, indicating the ability of the binding pocket to accommodate two ligands of diverse chemistry and enable cooperativity.

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.

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.

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.

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.

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.

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.

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.

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.