Biomedical subjects

M Bycroft

Publications and source records attributed to M Bycroft.

At least 37 records · Page 2Linked to original sources

PubMed1999

Molecular basis of Celmer's rules: the role of two ketoreductase domains in the control of chirality by the erythromycin modular polyketide synthase.

BACKGROUND: Polyketides are compounds that possess medically significant activities. The modular nature of the polyketide synthase (PKS) multienzymes has generated interest in bioengineering new PKSs. Rational design of novel PKSs, however, requires a greater understanding of the stereocontrol mechanisms that operate in natural PKS modules. RESULTS: The N-acetyl cysteamine (NAC) thioester derivative of the natural beta-keto diketide intermediate was incubated with DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2. The reduction products of the two ketoreductase (KR) domains of DEBS1-TE were a mixture of the (2S, 3R) and (2R,3S) isomers of the corresponding beta-hydroxy diketide NAC thioesters. Repeating the incubation using a DEBS1-TE mutant that only contains KR1 produced only the (2S,3R) isomer. CONCLUSIONS: In contrast with earlier results, KR1 selects only the (2S) isomer and reduces it stereospecifically to the (2S, 3R)-3-hydroxy-2-methyl acyl product. The KR domain of module 1 controls the stereochemical outcome at both methyl-and hydroxyl-bearing chiral centres in the hydroxy diketide intermediate. Earlier work showed that the normal enzyme-bound ketoester generated in module 2 is not epimerised, however. The stereochemistry at C-2 is therefore established by a condensation reaction that exclusively gives the (2R)-ketoester, and the stereo-chemistry at C-3 by reduction of the keto group. Two different mechanisms of stereochemical control, therefore, operate in modules 1 and 2 of the erythromycin PKS. These results should provide a more rational basis for designing hybrid PKSs to generate altered stereochemistry in polyketide products.

Catalysis↗

PubMed1998

Origin of starter units for erythromycin biosynthesis.

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are multifunctional proteins that govern the synthesis of a number of clinically important natural products. The modular arrangement of active sites within these enzymes suggests the possibility of a combinatorial approach to the synthesis of novel bioactive polyketides. The efficacy of combinatorial strategies toward altering the starter unit specificity of polyketide synthases critically depends on controlling the supply of competing endogenous starter acids. Using DEBS 1-TE, a bimodular derivative of DEBS, we aimed to determine whether the beta-ketosynthase (KS) domain responsible for condensation in the first module also has the ability to prime its own biosynthesis by catalyzing the decarboxylation of methylmalonyl-CoA to produce propionyl-CoA. In contrast to earlier reports with a closely similar mini-PKS DEBS 1+TE, we have found that rigorously purified DEBS 1-TE does not catalyze the decarboxylation of methylmalonyl-CoA.

Acyl Coenzyme A↗

PubMed1998

Characterisation of urea-denatured states of an immunoglobulin superfamily domain by heteronuclear NMR.

The structural and dynamic properties of an immunoglobulin superfamily domain (IgSF), Ig 18', have been characterised by NMR at 285 K, in the presence of 4.2 M and 6.0 M urea, respectively. Analysis of chemical shift deviations, 3JHNHalpha coupling constants, sequential NOE pattern, and 15N relaxation data reveals that although the two urea-denatured states are highly disordered, some local turn-like residual structures do exist. Moreover, some distinct differences between the properties of the two denatured states are observed. In 4.2 M urea-denatured Ig 18', regions 80-83 and 86-92 adopt turn-like conformations, furthermore, region 84-93 is involved in slow exchange processes that occur on a micro- to millisecond time-scale. In the 6.0 M urea-denatured state, these turn-like conformations are less occupied, and chemical exchange processes in region 84-93 are largely reduced. In contrast, region 32-36 has persistent turn-like structures in both urea-denatured states. Some correlation between the spectral density function at 0 frequency, Jeff(0), for the urea-denatured states and the secondary structure elements of the folded state have been observed. Except for the terminal regions, residues corresponding to beta-strands have higher Jeff(0) values compared to residues corresponding to loops. The characterisation and comparison of the two urea-denatured states highlight residues that possess properties that may be crucial for the initiation of folding of this domain.

Immunoglobulins↗

PubMed1998

Crystal structure of a calcium-phospholipid binding domain from cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) is a calcium-sensitive 85-kDa enzyme that hydrolyzes arachidonic acid-containing membrane phospholipids to initiate the biosynthesis of eicosanoids and platelet-activating factor, potent inflammatory mediators. The calcium-dependent activation of the enzyme is mediated by an N-terminal C2 domain, which is responsible for calcium-dependent translocation of the enzyme to membranes and that enables the intact enzyme to hydrolyze membrane-resident substrates. The 2.4-A x-ray crystal structure of this C2 domain was solved by multiple isomorphous replacement and reveals a beta-sandwich with the same topology as the C2 domain from phosphoinositide-specific phospholipase C delta 1. Two clusters of exposed hydrophobic residues surround two adjacent calcium binding sites. This region, along with an adjoining strip of basic residues, appear to constitute the membrane binding motif. The structure provides a striking insight into the relative importance of hydrophobic and electrostatic components of membrane binding for cPLA2. Although hydrophobic interactions predominate for cPLA2, for other C2 domains such as in "conventional" protein kinase C and synaptotagmins, electrostatic forces prevail.

Amino Acid Sequence↗

PubMed1998

Evaluating precursor-directed biosynthesis towards novel erythromycins through in vitro studies on a bimodular polyketide synthase.

BACKGROUND: Modular polyketide synthases (PKSs) catalyse the biosynthesis of complex polyketides using a different set of enzymes for each successive cycle of chain extension. Directed biosynthesis starting from synthetic diketides is a potentially valuable route to novel polyketides. We have used a purified bimodular derivative of the erythromycin-producing polyketide synthase (DEBS 1-TE) to study chain extension starting from a variety of diketide analogues and, in some cases, from the alternative acyl-CoA thioester substrates. RESULTS: Chain initiation in vitro by DEBS 1-TE module 2 using a synthetic diketide analogue as a substrate was tolerant of significant structural variation in the starter unit of the synthetic diketide, but other changes completely abolished activity. Interestingly, a racemic beta-keto diketide was found to be reduced in situ on the PKS and utilised in place of its more complex hydroxy analogue as a substrate for chain extension. The presence of a diketide analogue strongly inhibited chain initiation via the loading module. Significantly higher concentrations of diketide N-acetylcysteamine analogues than their corresponding acyl-CoA thioesters are required to achieve comparable yields of triketide lactones. CONCLUSIONS: Although a broad range of variation in the starter residue is acceptable, the substrate specificity of module 2 of a typical modular PKS in vitro is relatively intolerant of changes at C-2 and C-3. This will restrict the usefulness of approaches to synthesise novel erythromycins using synthetic diketides in vivo. The use of synthetic beta-keto diketides in vivo deserves to be explored.

Catalysis↗

PubMed1997

The molecular basis of Celmer's rules: the stereochemistry of the condensation step in chain extension on the erythromycin polyketide synthase.

Modular polyketide synthases (PKSs), for example, the 6-deoxyerythronolide B synthase (DEBS) responsible for synthesis of the aglycone core of the macrolide antibiotic erythromycin, generate an impressive diversity of asymmetric centers in their polyketide products. However, as noted by Celmer, macrolides have the same absolute configuration at all comparable stereocenters. Understanding how the stereochemistry of chain extension in controlled is therefore crucial to determining the common mechanism of action of these enzymes. We aimed to elucidate the molecular basis of Celmer's rules through in vitro studies with DEBS 1-TE, a bimodular derivative of DEBS from Saccharopolyspora erythraea, which uses (2S)-methylmalonyl-coenzyme. A to produce both D- and L-methyl centers in its triketide lactone product. We show here that condensation of (2S)-methylmalonyl-CoA in module 2 proceeds with decarboxylative inversion without cleavage of the C-H bond adjacent to the methyl group; in contrast, in module 1 the chain extension process involves loss of the hydrogen attached to C-2 of the methylmalonyl-CoA precursor. The production of the D-methyl center in module 2 without loss of hydrogen from the asymmetric center of the (2S)-methylmalonyl-CoA establishes that condensation takes place with inversion of configuration as in fatty acid biosynthesis. The loss of the key hydrogen from the (2S)-methylmalonyl-CoA to produce the L-methyl center generated in module 1 implies that an additional obligatory epimerization step takes place in that module. The nature and timing of the epimerization remain to be established.

Acyl Coenzyme A↗

PubMed1997

The solution structure of the S1 RNA binding domain: a member of an ancient nucleic acid-binding fold.

The S1 domain, originally identified in ribosomal protein S1, is found in a large number of RNA-associated proteins. The structure of the S1 RNA-binding domain from the E. coli polynucleotide phosphorylase has been determined using NMR methods and consists of a five-stranded antiparallel beta barrel. Conserved residues on one face of the barrel and adjacent loops form the putative RNA-binding site. The structure of the S1 domain is very similar to that of cold shock protein, suggesting that they are both derived from an ancient nucleic acid-binding protein. Enhanced sequence searches reveal hitherto unidentified S1 domains in RNase E, RNase II, NusA, EMB-5, and other proteins.

Amino Acid Sequence↗

PubMed1996

Structure and stability of an immunoglobulin superfamily domain from twitchin, a muscle protein of the nematode Caenorhabditis elegans.

The NMR solution structure of an immunoglobulin superfamily module of twitchin (Ig 18') has been determined and the kinetic and equilibrium folding behaviour characterised. Thirty molecular coordinates were calculated using a hybrid distance geometry-simulated annealing protocol based on 1207 distance and 48 dihedral restraints. The atomic rms distributions about the mean coordinate for the ensemble of structures is 0.55( +/- 0.09) A for backbone atoms and 1.10( +/- 0.08) A for all heavy atoms. The protein has a topology very similar to that of telokin and the titin Ig domains and thus it falls into the I set of the immunoglobulin superfamily. The close agreement between the predicted and observed structures of Ig 18' demonstrates clearly that the I set profile can be applied in the structure prediction of immunoglobulin-like domains of diverse modular proteins. Folding studies reveal that the protein has relatively low thermodynamic stability, deltaG(H2O)U-F = 4.0 kcal mol(-1) at physiological pH. Unfolding studies suggest that the protein has considerable kinetic stability, the half life of the unfolding is greater than 40 minutes in the absence of denaturant.

Amino Acid Sequence↗

PubMed1996

Equilibrium dissociation and unfolding of the dimeric human papillomavirus strain-16 E2 DNA-binding domain.

The equilibrium unfolding reaction of the C-terminal 80-amino-acid dimeric DNA-binding domain of human papillomavirus (HPV) strain 16 E2 protein has been investigated using fluorescence, far-UV CD, and equilibrium sedimentation. The stability of the HPV-16 E2 DNA-binding domain is concentration-dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The conformational stability of the protein, delta GH2O, was found to be 9.8 kcal/mol at pH 5.6, with the corresponding equilibrium unfolding/dissociation constant, Ku, being 6.5 x 10(-8) M. Equilibrium sedimentation experiments give a Kd of 3.0 x 10(-8) M, showing an excellent agreement between the two different techniques. Denaturation by temperature followed by the change in ellipticity also shows a concomitant disappearance of secondary and tertiary structures. The Ku changes dramatically at physiologically relevant pH's: with a change in pH from 6.1 to 7.0, it goes from 5.5 x 10(-8) M to 4.4 x 10(10) M. Our results suggest that, at the very low concentration of protein where DNA binding is normally measured (e.g., 10(-11) M), the protein is predominantly monomeric and unfolded. They also stress the importance of the coupling between folding and DNA binding.

Base Sequence↗

PubMed1996

The dimeric DNA binding domain of the human papillomavirus E2 protein folds through a monomeric intermediate which cannot be native-like.

The dimeric DNA binding domain of the human papillomavirus E2 protein displays a two-state concerted unfolding and dissociation, with no detectable monomeric intermediate species accumulated at equilibrium. We investigated the kinetic folding mechanism of the dimeric domain using stopped-flow spectroscopic techniques and observed a fast forming monomeric intermediate, followed by a slower bimolecular reaction. Both phases involve secondary structure rearrangements of similar magnitude. Our results support a folding pathway in which the formation of an early monomeric intermediate, with characteristics of hydrophobic collapse, is followed by a bimolecular step encompassing association and folding. The interwoven folding topology of this particular type of dimeric beta-barrel found in the E2 DNA binding domain strongly suggests that any monomeric species formed could not be native-like.

DNA-Binding Proteins↗

PubMed1995

A comparison of the pH, urea, and temperature-denatured states of barnase by heteronuclear NMR: implications for the initiation of protein folding.

The denatured states of barnase that are induced by urea, acid, and high temperature and acid have been assigned and characterised by high resolution heteronuclear NMR. The assignment was completed using a combination of triple-resonance and magnetisation-transfer methods. The latter was facilitated by selecting a suitable mutant of barnase (Ile-->Val51) which has an appropriate rate of interconversion between native and denatured states in urea. 3J NH-C alpha H coupling constants were determined for pH and urea-denatured barnase and intrinsic "random coil" coupling constants are shown to be different for different residue types. All the denatured states are highly unfolded. But, a consistent series of weak correlations in chemical shift, NOESY and coupling constant data provides evidence that the acid-denatured state has some residual structure in regions that form the first and second helices and the central strands of beta-sheet in the native protein. The acid/temperature-denatured states has less structure in these regions, and the urea-denatured state, less still. These observations may be combined with detailed analyses of the folding pathway of barnase from kinetic studies to illuminate the relevance of residual structure in the denatured states of proteins to the mechanism of protein folding. First, the folding of barnase is known to proceed in its later stages through structures in which the first helix and centre of the beta-sheet are extensively formed. Thus, embryonic initiation sites for these do exist in the denatured states and so could well develop into true nuclei. Second, it has been clearly established that the second helix is unfolded in these later states, and so residual structure in this region of the protein is non-productive. These data fit a model of protein folding in which local nucleation sites are latent in the denatured state and develop only when they make interactions elsewhere in the protein that stabilise them during the folding process. Thus, studies of the structure of denatured states pinpoint where nucleation sites may be, and the kinetic and protein engineering studies show which ones are productive.

Amides↗

PubMed1995

NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5.

The double-stranded RNA binding domain (dsRBD) is an approximately 65 amino acid motif that is found in a variety of proteins that interact with double-stranded (ds) RNA, such as Escherichia coli RNase III and the dsRNA-dependent kinase, PKR. Drosophila staufen protein contains five copies of this motif, and the third of these binds dsRNA in vitro. Using multinuclear/multidimensional NMR methods, we have determined that staufen dsRBD3 forms a compact protein domain with an alpha-beta-beta-beta-alpha structure in which the two alpha-helices lie on one face of a three-stranded anti-parallel beta-sheet. This structure is very similar to that of the N-terminal domain of a prokaryotic ribosomal protein S5. Furthermore, the consensus derived from all known S5p family sequences shares several conserved residues with the dsRBD consensus sequence, indicating that the two domains share a common evolutionary origin. Using in vitro mutagenesis, we have identified several surface residues which are important for the RNA binding of the dsRBD, and these all lie on the same side of the domain. Two residues that are essential for RNA binding, F32 and K50, are also conserved in the S5 protein family, suggesting that the two domains interact with RNA in a similar way.

Amino Acid Sequence↗

PubMed1995

Assignment of the backbone 1H,15N,13C NMR resonances and secondary structure of a double-stranded RNA binding domain from the Drosophila protein staufen.

NMR spectroscopy has been used to determine the secondary structure of one of the double-stranded RNA binding domains from the Drosophila protein staufen. The domain has an alpha beta beta beta alpha arrangement of secondary structure, with the beta strands forming an antiparallel beta sheet. The secondary structure differs from that found in the RNP RNA binding domain.

Amino Acid Sequence↗

PubMed1994

Toward solving the folding pathway of barnase: the complete backbone 13C, 15N, and 1H NMR assignments of its pH-denatured state.

The structures of the major folding intermediate, the transition state for folding, and the folded state of barnase have been previously characterized. We now add a further step toward a complete picture of the folding of barnase by reporting the backbone 15N, 13C, and 1H NMR assignments for barnase unfolded at pH 1.8 and 30 degrees C. These assignments, which were obtained from a combination of heteronuclear magnetization transfer and backbone triple-resonance NMR experiments, constitute the first stage in the structural characterization of this denatured state by NMR. Interresidue nuclear Overhauser effect contacts and deviations from 1H random-coil chemical shifts provide evidence for stable residual structure. The structured regions span residues in the native protein that contain its major alpha-helix and central strands of the beta-sheet. Earlier experiments have shown that these regions are predominantly intact in the major folding intermediate and that their docking is partly rate determining in folding.

Amino Acid Sequence↗

PubMed1994

Three-dimensional solution structure and 13C assignments of barstar using nuclear magnetic resonance spectroscopy.

We present the high-resolution solution structure and 13C assignments of wild-type barstar, an 89 amino acid residue polypeptide inhibitor of barnase, derived from heteronuclear NMR techniques. These were obtained from measurements on unlabeled, uniformly 15N- and 13C/15N-labeled, and 10% 13C-labeled barstar samples that have both cysteines (at positions 40 and 82) fully reduced. In total, 30 structures were calculated by hybrid distance geometry-dynamical simulated annealing calculations. The atomic rms distribution about the mean coordinate positions is 0.42 A for all backbone atoms and 0.90 A for all atoms. The structure is composed of three parallel alpha-helices packed against a three-stranded parallel beta-sheet. A more poorly defined helix links the second beta-strand and the third major alpha-helix. The loop involved in binding barnase is extremely well defined and held rigidly by interactions from the main body of the protein to both ends and the middle of the loop. This structure will be used to aid protein engineering studies currently taking place on the free and bound states of barstar and barnase.

Bacterial Proteins↗

PubMed1993

Local breathing and global unfolding in hydrogen exchange of barnase and its relationship to protein folding pathways.

We have measured the rate constants for exchange of amide protons in 15N-labeled wild-type barnase and a disulfide mutant that is more stable by 2 kcal.mol-1. The relative rate constants for exchange for wild type and mutant should reflect the changes in the equilibrium constants for local or global unfolding. The values for regions whose structure has been shown to be unaffected by the mutation fall into three subsets: those that are essentially unaffected by the mutation and so presumably exchange by local breathing; those where the energies change by close to 2 kcal.mol-1 and so presumably require global unfolding for exchange; and intermediate values that probably reflect a mixture of local and global unfolding in wild-type barnase. Amide protons that require the full change in unfolding energy are predominantly in the beta-sheet, which forms early in folding, but also include two that are involved in tertiary interactions that are known not to be formed until late in the folding pathway. Exchange in the major helix, which is known to form early, is largely unaffected by mutation and so exchanges by local breathing. There is thus no direct relationship between hydrogen-exchange behavior and the protein folding pathway. However, experiments on mutants of varying stability may provide further evidence on the sequence of events in folding.

Amino Acid Sequence↗

PubMed1993

Structure and dynamics of barnase complexed with 3'-GMP studied by NMR spectroscopy.

The binding of 3'-GMP to the ribonuclease, barnase, has been studied using heteronuclear 2D and 3D NMR spectroscopy. The 1H and 15N NMR spectra of barnase complexed with 3'-GMP have been assigned. 2D and 3D NOESY spectra have been used to identify inter- and intramolecular NOEs, and a solution structure for the barnase-3'-GMP complex has been calculated. The position of the guanine ring of the ligand is reasonably well defined in the structures. The guanine ring forms hydrogen bonds with the NH protons of Ser57 and Arg59. These residues are located in a loop that is conserved among the microbial guanine-specific ribonucleases. The 2'-hydroxyl of 3'-GMP is close to His102 and Glu73, which have been shown to be involved in catalysis. The phosphate group of 3'-GMP is close to a number of positively charged residues that have also been shown to be important for activity. The position of the sugar moiety of 3'-GMP is less well defined in the structures. Structures calculated for the complex could not simultaneously satisfy all the observed intermolecular NOEs for the sugar protons, suggesting that the sugar samples several conformations when bound to barnase. The binding of 3'-GMP to barnase in solution is similar to that observed in the crystal structures of nucleotides bound to related ribonucleases. 3'-GMP binding causes no major conformational change in barnase. In contrast to the small structural changes that occur, there is a significant decrease in the rates of hydrogen/deuterium exchange and aromatic ring rotation in the active site of barnase upon ligand binding.

Bacterial Proteins↗

PubMed1993

Assignment of the backbone 1H and 15N NMR resonances and secondary structure characterization of barstar.

Barstar, a polypeptide inhibitor of ribonucleases, has been studied by 2D and 3D NMR techniques using uniformly 15N-labeled protein. Backbone (15NH-C alpha H-C beta H) resonances were assigned for all but 5 of the 89 residues. Dihedral angle and deuterium exchange studies were used in conjunction with medium range inter-proton NOEs to characterize the secondary structure of barstar. The protein is composed of four alpha-helices and three short stretches of extended strand. By further analysis of the NOE data three of the helices were found to be parallel to each other with the single disulphide bond linking the second and fourth helices at their C-terminal ends.

Amino Acid Sequence↗