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At least 865 records · Page 48Linked to original sources
PubMed2004

Determination of the stable conformation of GABA(A)-benzodiazepine receptor bivalent ligands by low temperature NMR and X-ray analysis.

The stable conformations of GABA(A)-benzodiazepine receptor bivalent ligands 2 and 3 were determined by low temperature NMR spectroscopy and confirmed by single crystal X-ray analysis. The linear conformation was important for these dimers to access the binding site and exhibit potent in vitro affinity as illustrated for alpha5 subtype selective ligand 2 (15 nM). Bivalent ligand 3 with the 5 atom linker folded back upon itself both in solution and in the solid state, moreover, it did not bind to Bz receptors.

Crystallography, X-Ray↗
PubMed2002

Intermediate structural states involved in MRP1-mediated drug transport. Role of glutathione.

Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette transporter family and transports chemotherapeutic drugs as well as diverse organic anions such as leukotriene LTC(4). The transport of chemotherapeutic drugs requires the presence of reduced GSH. By using hydrogen/deuterium exchange kinetics and limited trypsin digestion, the structural changes associated with each step of the drug transport process are analyzed. Purified MRP1 is reconstituted into lipid vesicles with an inside-out orientation, exposing its cytoplasmic region to the external medium. The resulting proteoliposomes have been shown previously to exhibit both ATP-dependent drug transport and drug-stimulated ATPase activity. Our results show that during GSH-dependent drug transport, MRP1 does not undergo secondary structure changes but only modifications in its accessibility toward the external environment. Drug binding induces a restructuring of MRP1 membrane-embedded domains that does not affect the cytosolic domains, including the nucleotide binding domains, responsible for ATP hydrolysis. This demonstrates that drug binding to MRP1 is not sufficient to propagate an allosteric signal between the membrane and the cytosolic domains. On the other hand, GSH binding induces a conformational change that affects the structural organization of the cytosolic domains and enhances ATP binding and/or hydrolysis suggesting that GSH-mediated conformational changes are required for the coupling between drug transport and ATP hydrolysis. Following ATP binding, the protein adopts a conformation characterized by a decreased stability and/or an increased accessibility toward the aqueous medium. No additional change in the accessibility toward the solvent and/or the stability of this specific conformational state and no change of the transmembrane helices orientation are observed upon ATP hydrolysis. Binding of a non-transported drug affects the dynamic changes occurring during ATP binding and hydrolysis and restricts the movement of the drug and its release.

Adenosine Triphosphate↗
PubMed2005

PSSARD: protein sequence-structure analysis relational database.

We have implemented a relational database comprising a representative dataset of amino acid sequences and their associated secondary structure. The representative amino acid sequences were selected according to the PDB_SELECT program by choosing proteins corresponding to protein crystal structure data deposited in the protein data bank that share less than 25% overall pair-wise sequence identity. The secondary structure was extracted from the protein data bank website. The information content in the database includes the protein description, PDB code, crystal structure resolution, total number of amino acid residues in the protein chain, amino acid sequence, secondary structure conformation and its summary. The database is freely accessible from the website mentioned below and is useful to query on any of the above fields. The database is particularly useful to quickly retrieve amino acid sequences that are compatible to any super-secondary structure conformation from several proteins simultaneously.

Amino Acid Sequence↗
PubMed2005

The structure of human apolipoprotein E2, E3 and E4 in solution 1. Tertiary and quaternary structure.

Three recombinant apoE isoforms fused with an amino-terminal extension of 43 amino acids were produced in a heterologous expression system in E. coli. Their state of association in aqueous phase was analyzed by size-exclusion liquid chromatography, sedimentation velocity and sedimentation equilibrium experiments. By liquid chromatography, all three isoforms consisted of three major species with Stokes radii of 4.0, 5.0 and 6.6 nm. Sedimentation velocity confirmed the presence of monomers, dimers and tetramers as major species of each isoform. The association schemes established by sedimentation equilibrium experiments corresponded to monomer-dimer-tetramer-octamer for apoE2, monomer-dimer-tetramer for apoE3 and monomer-dimer-tetramer-octamer for apoE4. Each of the three isoforms exhibits a distinct self-association pattern. The apolipoprotein multi-domain structure was mapped by limited proteolysis with trypsin, chymotrypsin, elastase, subtilisin and Staphylococcus aureus V8 protease. All five enzymes produced stable intermediates during the degradation of the three apoE isoforms, as described for plasma apoE3. The recombinant apoE isoforms, thus, consist of N- and C-terminal domains. The presence of the fusion peptide did not appear to alter the apolipoprotein tertiary organization. However, a 30 kDa amino-terminal fragment appeared during the degradation of the recombinant apoE isoforms resulting from cleavage in the 273-278 region. This region, not accessible in plasma apoE3, results from a different conformation of the C-terminal domain in the recombinant isoforms. A specific pattern for the apoE4 C-terminal domain was observed during the proteolysis. The region 230-260 in apoE4, in contrast to that of apoE3 and apoE2, was not accessible to proteases, probably due to the existence of a longer helix in this region of apoE4 stabilized by an interdomain interaction.

Apolipoprotein E2↗
PubMed1998

Evidence of molten globule like state(s) of interferon gamma in acidic and sodium perchlorate solutions.

Recombinant porcine interferon gamma (IFN gamma) at neutral pH is characterized by a tryptophan (Trp) fluorescence maximum around 343 nm and a rigid conformation, evidenced from tryptophan polarization values. Guanidine HCl shifts the protein emission spectra further to the red and decreases the fluorescence polarization values, indicating denatured IFN gamma in these solutions. In acidic solutions (3 < pH < 4), the emission spectra show a blue shift and lower tryptophan polarization. The midpoint of transition of these fluorescence properties occurs around pH 3.5-3.6. The protein in NaClO4 solution at neutral pH is similarly characterized by a blue shift in the tryptophan fluorescence maxima and low polarization values. The extent of quenching of tryptophan fluorescence by acrylamide is less in acid and in NaClO4 solutions of IFN gamma compared to its native form. This indicates a lower accessibility of the tryptophan in the altered conformation of the protein. The emission spectra of IFN gamma in NaClO4 solution shows a decrease in the tryptophan fluorescence intensity with simultaneous shift of the emission spectra over time. The presence of two conformational forms of IFN gamma in perchlorate solution is evidenced from an isofluorescent point at 315 nm. The change in the conformational state in perchlorate solution is characterized by first order kinetics. The dye anilinonaphthalene sulfonic acid does not bind either to the native IFN gamma or to its denatured form. However, the dye binds to the acid form of IFN gamma, as well as when the protein is present in NaClO4 solution at neutral pH. These observations, together with the results from literature that IFN gamma retains its secondary structure in acid solution to a considerable degree, would suggest that the protein exists as a molten globule-like state in acidic solution. Similarities of the protein fluorescence and 1-anilino-8-naphthalene-sulfonic-acid (ANS) binding properties of the protein in NaClO4 and acid solutions indicate that IFN gamma also exists in a molten globule-like state in perchlorate solution at neutral pH.

Acids↗
PubMed1998

Methionine synthase exists in two distinct conformations that differ in reactivity toward methyltetrahydrofolate, adenosylmethionine, and flavodoxin.

Methionine synthase (MetH) from Escherichia coli catalyzes the synthesis of methionine from homocysteine and methyltetrahydrofolate via two methyl transfer reactions that are mediated by the endogenous cobalamin cofactor. After binding both substrates in a ternary complex, the enzyme transfers a methyl group from the methylcobalamin cofactor to homocysteine, generating cob(I)alamin enzyme and methionine. The enzyme then catalyzes methyl transfer from methyltetrahydrofolate to the cob(I)alamin cofactor, forming methylcobalamin cofactor and tetrahydrofolate prior to the release of both products. The cob(I)alamin form of the enzyme occasionally undergoes oxidation to an inactive cob(II)alamin species; the enzyme also catalyzes its own reactivation. Electron transfer from reduced flavodoxin to the cob(II)alamin cofactor is thought to generate cob(I)alamin enzyme, which is then trapped by methyl transfer from adenosylmethionine to the cobalt, restoring the enzyme to the active methylcobalamin form. Thus the enzyme is potentially able to catalyze two methyl transfers to the cob(I)alamin cofactor: methyl transfer from methyltetrahydrofolate during primary turnover and methyl transfer from adenosylmethionine during activation. It has recently been shown that methionine synthase is constructed from at least four separable regions that are responsible for binding each of the three substrates and the cobalamin cofactor, and it has been proposed that changes in positioning of the substrate binding regions vis-à-vis the cobalamin binding region could allow the enzyme to control which substrate has access to the cofactor. In this paper, we offer evidence that methionine synthase exists in two different conformations that interconvert in the cob(II)alamin oxidation state. In the primary turnover conformation, the enzyme reacts with homocysteine and methyltetrahydrofolate but is unreactive toward adenosylmethionine and flavodoxin. In the reactivation conformation, the enzyme is active toward adenosylmethionine and flavodoxin but unreactive toward methyltetrahydrofolate. The two conformations differ in the susceptibility of the substrate-binding regions to tryptic proteolysis. We propose a model in which conformational changes control access to the cobalamin cofactor and are the primary means of controlling cobalamin reactivity in methionine synthase.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1999

Properties of the mutant Ser-460-Cys implicate this site in a functionally important region of the type IIa Na(+)/P(i) cotransporter protein.

The substituted cysteine accessibility approach, combined with chemical modification using membrane-impermeant alkylating reagents, was used to identify functionally important structural elements of the rat type IIa Na(+)/P(i) cotransporter protein. Single point mutants with different amino acids replaced by cysteines were made and the constructs expressed in Xenopus oocytes were tested for function by electrophysiology. Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein. Transport function of oocytes expressing WT protein was unchanged after exposure to the alkylating reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA, 100 microM), which indicated that native cysteines were inaccessible. However, for one of the mutants (S460C) that showed kinetic properties comparable with the WT, alkylation led to a complete suppression of P(i) transport. Alkylation in 100 mM Na(+) by either cationic ([2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), MTSEA) or anionic [sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES)] reagents suppressed the P(i) response equally well, whereas exposure to methanethiosulfonate (MTS) reagents in 0 mM Na(+) resulted in protection from the MTS effect at depolarized potentials. This indicated that accessibility to site 460 was dependent on the conformational state of the empty carrier. The slippage current remained after alkylation. Moreover, after alkylation, phosphonoformic acid and saturating P(i) suppressed the slippage current equally, which indicated that P(i) binding could occur without cotransport. Pre-steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na(+) dependent, consistent with an intact slippage pathway. Based on an alternating access model for type IIa Na(+)/P(i) cotransport, these results suggest that site 460 is located in a region involved in conformational changes of the empty carrier.

Alkylation↗
PubMed1997

Comparisons of the conformational biases imposed by trans-2,3-methanomethionine and alpha-methylmethionine.

A comparative study of four peptidomimetics of the sequence Phe-Met-Arg-Phe-amide (FMRFa) was performed to compare the conformational bias caused by trans-2,3-methanomethionine and alpha-methylmethionine stereoisomers. The specific compounds studied were {(2S,3S)-cyclo-M} RFa, F{(2R,3R)-cyclo-M} RFa, F{(S)-alpha-Mem} RFa, and F{(R)-alpha-MeM} RFa. Molecular simulations based on CHARMm 22 indicate that gamma-turn, inverse gamma-turn, and alpha-helical conformations about the cyclo-M residue are accessible to the two F{cyclo-M} RFa stereoisomers. Similar calculations for F{(S)-alpha-MeM} RFa, and F{(R)-alpha-MeM}RFa indicate that the alpha-methylamino acids tend to favor alpha-helical conformations. The nmr data is presented for the four peptidomimetics. Most informative were the rotating frame nuclear Overhauser effect cross peaks between the NH protons proximal to the methionine surrogates, and the C beta hydrogens. Overall, these nmr data indicate F{(2S,3S)-cyclo-M} RFa and F{(2R,3R)-cyclo-M} RFa preferentially adopt inverse gamma-turn and gamma-turn conformations, respectively, whereas F{(S)-alpha-MeM} RFa and F{(R)-alpha-MeM} RFa tend to form partial left- and right-handed helical structures (although energy differences between the two turn structures, and between the two helical structures are likely to be small). It is suggested that the wider NH-C alpha-CO angle of cyclopropane amino acids and their more severe steric requirements around the C beta carbons force the peptidomimetic N- and C-termini into the same region of conformational space. This favors C7 turns in the cyclopropane amino acid series relative to the less constrained alpha-methyl derivatives.

FMRFamide↗
PubMed2005

Conformationally sensitive residues in extracellular loop 5 of the Na+/dicarboxylate co-transporter.

The Na+/dicarboxylate co-transporter, NaDC-1, from the kidney and small intestine, transports three sodium ions together with one divalent anion substrate, such as succinate2-. A previous study (Pajor, A. M. (2001) J. Biol. Chem. 276, 29961-29968), identified four amino acids, Ser-478, Ala-480, Ala-481, and Thr-482, near the extracellular end of transmembrane helix (TM) 9 that are likely to form part of the permeation pathway of the transporter. All four cysteine-substituted mutants were sensitive to inhibition by the membrane-impermeant reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate (MTSET) and protected by substrate. In the present study, we continued the cysteine scan through extracellular loop 5 and TM10, from Thr-483 to Val-528. Most cysteine substitutions were well tolerated, although cysteine mutations of some residues, particularly within the TM, produced proteins that were not expressed on the plasma membrane. Six residues in the extracellular loop (Thr-483, Thr-484, Leu-485, Leu-487, Ile-489, and Met-493) were sensitive to chemical labeling by MTSET, depending on the conformational state of the protein. Transport inhibition by MTSET could be prevented by substrate regardless of temperature, suggesting that the likely mechanism of substrate protection is steric hindrance rather than large-scale conformational changes associated with translocation. We conclude that extracellular loop 5 in NaDC-1 appears to have a functional role, and it is likely to be located in or near the substrate translocation pore in the protein. Conformational changes in the protein affect the accessibility of the residues in extracellular loop 5 and provide further evidence of large-scale changes in the structure of NaDC-1 during the transport cycle.

Amino Acid Sequence↗
PubMed2002

Calmodulin activates electron transfer through neuronal nitric-oxide synthase reductase domain by releasing an NADPH-dependent conformational lock.

Neuronal nitric-oxide synthase (nNOS) is activated by the Ca(2+)-dependent binding of calmodulin (CaM) to a characteristic polypeptide linker connecting the oxygenase and reductase domains. Calmodulin binding also activates the reductase domain of the enzyme, increasing the rate of reduction of external electron acceptors such as cytochrome c. Several unusual structural features appear to control this activation mechanism, including an autoinhibitory loop, a C-terminal extension, and kinase-dependent phosphorylation sites. Pre-steady state reduction and oxidation time courses for the nNOS reductase domain indicate that CaM binding triggers NADP(+) release, which may exert control over steady-state turnover. In addition, the second order rate constant for cytochrome c reduction in the absence of CaM was found to be highly dependent on the presence of NADPH. It appears that NADPH induces a conformational change in the nNOS reductase domain, restricting access to the FMN by external electron acceptors. CaM binding reverses this effect, causing a 30-fold increase in the second order rate constant. The results show a startling interplay between the two ligands, which both exert control over the conformation of the domain to influence its electron transfer properties. In the full-length enzyme, NADPH binding will probably close the conformational lock in vivo, preventing electron transfer to the oxygenase domain and the resultant stimulation of nitric oxide synthesis.

Animals↗
PubMed1994

Mapping staphylococcal nuclease conformation using an EDTA-Fe derivative attached to genetically engineered cysteine residues.

Six single cysteine variants of staphylococcal nuclease were reacted with the iron complex of (EDTA-2-aminoethyl) 2-pyridyl disulfide (EPD-Fe) [Ermácora, M. R., Delfino, J. M., Cuenoud, B., Schepartz, A., & Fox, R. O. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387] and used to assess the ability of this cleavage reagent to faithfully report on the structure of nonnative protein states. The act of mutation and modification did not significantly alter the protein's global structure, as measured by CD and enzymatic activity, and only modestly affected its stability. The reaction was conformation dependent and generated specific cleavage products that mapped tertiary interactions present in the folded state. Several parameters relevant to the cleavage reaction and its use as a conformational probe were analyzed. Proximity and solvent accessibility are the most important parameters in determining the cleavage pattern and can be used to predict cleavage sites in the native protein. The cleavage reaction can be performed in the presence of high denaturant concentration, in the presence of SDS, and under a wide range of pH values; thus it can readily be applied to the study of equilibrium folding intermediates. Mass spectrometric analysis combined with N-terminal sequencing identified cleavage products consistent with a single cleavage event per protein molecule and revealed one cleavage mechanism which was not previously considered for protein oxidative degradation, although it was reported for hydroxyl radical induced cleavage of small peptides. Identification of the cleavage sites obtained from each variant allowed a nearest-neighbor mapping of the secondary structural elements of nuclease. Quantitation of specific cleavage products was used to monitor the disruption of the interaction between helices H2 and H3 in equilibrium unfolding experiments. The resulting unfolding curve revealed a local conformational heterogeneity at low denaturant concentration which was not observed when the same transition was monitored by the change in fluorescence of a single nuclease tryptophan. Interestingly, the midpoint of the transition and the second half of the unfolding curve were the same, as monitored by the two probes. This indicates that the lifetime of the reactive oxygen species generated by the cleavage reagent is short compared to the unfolding equilibrium rate constants and that the cleavage technique identifies a native-like folding intermediate not detected by fluorescence. The experiments presented herein demonstrate that EPD-Fe-mediated protein cleavage is an appropriate technique for the study of nonnative protein structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Computer Graphics↗
PubMed1988

Characteristic thermodynamic properties of hydrated water for 20 amino acid residues in globular proteins.

Thermodynamic properties associated with hydrated water of proteins of known three-dimensional structure were computed and average values of hydration free energy, enthalpy, and heat capacity of unfolding for every amino acid residue were obtained. Each amino acid residue had characteristic values; in particular, the quantities for a side chain reflected the character of the amino acid, while those for the main chain were more or less the same except for glycine, alanine, and proline. The major contribution to the quantities was from the end group(s) of a side chain. The following interesting features were found. 1) The hydration quantity of unfolding derived from the native and extended conformations for a protein was approximately equal to the sum of the corresponding average quantities of component amino acid residues in the protein. 2) The profile of a quantity such as hydration free energy of unfolding along the sequence computed from the accessible surface areas of the native and extended conformations showed a strong correlation with the profile obtained by allocating the average value for the amino acid residue at every position on the sequence. The correlation coefficients between two profiles for unfolding quantities of hydration, i.e., free energy, enthalpy, heat capacity, and free energy of side chain are 0.72, 0.62, 0.80, and 0.75, respectively. Thus, every amino acid residue in the native conformation of a globular protein seems to be located in such a position that a thermodynamic quantity for each residue is approximately equal to its average value.

Amino Acids↗
PubMed1994

Zinc destabilizes DNA Watson-Crick pairs at AGCT.

Zinc, a component of a large number of enzymes and an abundant and essential trace metal, alters the structure of AGCT sites in negatively supercoiled DNA. Related sequences show little or no effect. Cobalt and cadmium show reactivities similar to zinc. The conformational change in DNA was fine mapped with haloacetaldehydes using chemical cleavage or primer extension methods. Since a many fold excess of zinc to AGCT sites is required to saturate the reaction, we propose that the zinc-AGT complex is preferentially accessible to the haloacetaldehyde probes due to a conformational deformation. Hence, zinc is the smallest ligand (by approximately 500-fold) that causes a perturbation of a specific DNA sequence.

Base Composition↗
PubMed1997

Differential interactions of the Mg2+ complexes of chromomycin A3 and mithramycin with poly(dG-dC) x poly(dC-dG) and poly(dG) x poly(dC).

The interaction of the two anticancer antibiotics, chromomycin A3 and mithramycin, with the polynucleotides poly(dG-dC) x poly(dC-dG), representative of B-DNA, and poly(dG) x poly(dC), representative of A-DNA, in the presence of Mg2+ is studied by spectroscopic techniques such as absorbance, fluorescence, and dircular dichroism (CD). The studies were done with both drug x Mg2+ complexes, I and II, having 1:1 and 2:1 stoichiometries with respect to drug and Mg2+, respectively [Aich, P., Sen, R., & Dasgupta, D. (1992) Biochemistry 31, 2988-2997]. The objective of the present work is 2-fold. First, an attempt is made to understand the structural basis of the ligand-DNA interaction, particularly the role of DNA backbone conformation with its groove size and the accessibility of the 2-amino group in the minor groove of guanosine. Second, the role of the antibiotic saccharide moieties in the association with DNA was studied. For this purpose, the spectroscopic characterization of the binding was done followed by the evaluation of binding parameters and associated thermodynamics. Analysis of the observed thermodynamics for the ligand-DNA interactions in terms of the different structures of the polynucleotides was done. The salient results are as follows. Complex I does not discriminate significantly among the A- and B-forms of DNA when it binds to them in an entropy-driven process. On the other hand, complex II for both drugs recognizes B- and A-forms of DNA in different ways. This observation implies that the sequence specificity shown by this complex is a sequel to the difference in the parameters such as groove size and accessibility of the guanosine amino group. Another important finding is that binding with the same polynucleotide is not comparable for the complex II of the two drugs. It emphasizes the involvement of the sugar moieties, when the drug x Mg2+ complex binds to DNA. The presence of an acetoxy group in the sugars of chromomycin A3 imparts some distinctive specific features of the association of the chromomycin dimer x Mg2+ complex with DNA. Finally, the results are compared with those available from NMR studies of different drug-oligonucleotide complexes under conditions where complex II is the ligand.

Chromomycin A3↗
PubMed1975

Enzymic and immunochemical properties of lysozyme. IX. Conformation and immunochemistry of derivatives succinylated at certain lysine residues.

Succinylation of lysozyme in the presence of 7 molar excess of [1,4-14C2]-succinic anhydride gave a reaction product which showed at least six components by disc electrophoresis. Chromatography on CM-cellulose enabled the isolation of six homogeneous derivatives. The derivatives were succinylated at the following locations: derivative I, lysines-1 (alpha- and epsilon-NH2), -13, -97 and -116 and the OH group at position 43 (or 36 or 40); derivative II, lysines-1 (alpha- and epsilon-NH2), -13, -96, -116; derivative III, lysines-1 (alpha-and epsilon-NH2), -13, -97, -116; derivative IV, lysines-1 (alpha-NH2), -33, -96 and -116; derivative V, lysines-1 (alpha-NH2), -33 and -96; derivative VI, lysines-33 and -116. Conformational changes were detectable in derivative I by ORD and CD measurements and by accessibility of the disulfide bonds to reduction. On the other hand, the other five succinyl derivatives showed no conformational changes by ORD and CD measurements. However, their disulfide bonds were slightly more accessible to reduction than lysozyme, with the increase being somewhat higher in derivatives I, II and III. Enzymic activity measurements showed that only derivative VI possessed some (10%) enzymic activity. Immunochemical studies with antisera to lysozyme showed that the reactivity of each of the derivatives was lower than the homologous reaction. Correlation of the extent of decrease in immunochemical reaction with the locations of modification and with the results of conformational analysis, led to the conclusion that lysines 33, 96 and 116 are part of antigenic reactive regions in lysozyme. The modification results are also discussed in relation to the three-dimensional structure of lysozyme in solution.

Anhydrides↗
PubMed2001

Probing the open state of cytochrome P450cam with ruthenium-linker substrates.

Cytochromes P450 play key roles in drug metabolism and disease by oxidizing a wide variety of natural and xenobiotic compounds. High-resolution crystal structures of P450cam bound to ruthenium sensitizer-linked substrates reveal an open conformation of the enzyme that allows substrates to access the active center via a 22-A deep channel. Interactions of alkyl and fluorinated biphenyl linkers with the channel demonstrate the importance of exploiting protein dynamics for specific inhibitor design. Large changes in peripheral enzyme structure (F and G helices) couple to conformational changes in active center residues (I helix) implicated in proton pumping and dioxygen activation. Common conformational states among P450cam and homologous enzymes indicate that static and dynamic variability in the F/G helix region allows the 54 human P450s to oxidize thousands of substrates.

Binding Sites↗
PubMed1997

Biophysical investigations on the myb-DNA system.

The oncogene product c-myb is a transcriptional modulator and is known to play important roles in cell growth and differentiation. It binds to DNA in a sequence specific manner and its cognate sequence motifs have been detected in the genes of proteins implying its role in a variety of regulatory functions. The protein has a DNA binding domain consisting of three imperfect repeats with highly conserved tryptophans at regular spacings in each of the repeats. We have carried out a variety of investigations on the structure and interactions of the DNA binding domain of Drosophila c-myb and its cognate DNA target sequences. The domain has been bacterially over-expressed by subcloning a segment of the gene coding for the domain in a pET 11d vector and transforming it into E. coli BL21 (DE3). Circular dichroism of the protein has revealed that the domain is largely helical in nature. Fluorescence investigations indicated that three out of the nine tryptophans are solvent exposed and the others are buried in the interior. The recombinant protein is able to distinguish between specific and non-specific DNA targets in its binding and the interaction is largely electrostatic in nature in both cases. Dynamic fluorescence quenching experiments suggested that the DNA binding sites on the protein for specific and non-specific DNA targets are physically different. Most of the conserved tryptophans are associated with the specific DNA binding site. Simulated annealing and molecular dynamic simulations in a water matrix have been used to predict an energetically favoured conformation for the protein. Calculation of surface accessibilities of the individual residues shows that nearly 60% of the residues are less than 50% accessible to the solvent. Two and three dimensional NMR experiments with isotopically labelled protein have enabled spin system identification for many residue type and the types of residues involved in hydrophobic core formation in the protein. In an attempt to see the DNA surface possibly involved in specific interaction with the protein, a three-dimensional structure of a 12 mer cognate DNA has been determined by NMR in conjunction with restrained energy minimization. The recognition sequence shows interesting structural characteristics that may have important roles in specific interaction.

Amino Acid Sequence↗
PubMed2002

Conformational propensities of protein folding intermediates: distribution of species in the 1S, 2S, and 3S ensembles of the [C40A,C95A] mutant of bovine pancreatic ribonuclease A.

A key problem in experimental protein folding is that of characterizing the conformational ensemble of denatured proteins under folding conditions. We address this problem by studying the conformational propensities of reductively unfolded RNase A under folding conditions, since earlier work has indicated that the equilibrium conformational ensemble of fully reduced RNase A resembles the transient conformational ensemble of a burst-phase folding intermediate of disulfide-intact RNase A. To assess these propensities, the relative disulfide-bond populations of the 1S, 2S, and 3S ensembles of the [C40A,C95A] mutant of RNase A were measured. Thirteen of the fifteen possible disulfide bonds are observed, consistent with earlier results and with the rapid reshuffling and lack of stable tertiary structure in these ensembles. This broad distribution contradicts recent observations by another group, but rigorous cross-checks show unambiguously that our data are self-consistent whereas their data are not. The distributions of disulfide bonds in the wild-type and mutant proteins show a power-law dependence on loop length, with an exponent that is significantly smaller than the exponents of either ideal or excluded-volume polymers. The 65-72 disulfide bond is much more strongly favored than would be predicted by this power law, consistent with earlier peptide studies and the disulfide-bond distributions of the 1S and 2S ensembles in wild-type RNase A. Experimental evidence suggests that this preference results from conformational biases in the backbone, rather than from differing accessibilities or reactivities of the two cysteine residues. In general, the other disulfide species do not deviate significantly from the power-law dependence, indicating that the conformational biases are relatively weak.

Alanine↗