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1.
Nat Commun ; 13(1): 2124, 2022 04 19.
Article in English | MEDLINE | ID: mdl-35440652

ABSTRACT

Synthetic molecular machines hold tremendous potential to revolutionize chemical and materials sciences. Their autonomous motion controlled by external stimuli allows to develop smart materials whose properties can be adapted on command. For the realisation of more complex molecular machines, it is crucial to design building blocks whose properties can be controlled by multiple orthogonal stimuli. A major challenge is to reversibly switch from forward to backward and again forward light-driven rotary motion using external stimuli. Here we report a push-pull substituted photo-responsive overcrowded alkene whose function can be toggled between that of a unidirectional 2nd generation rotary motor and a molecular switch depending on its protonation and the polarity of its environment. With its simplicity in design, easy preparation, outstanding stability and orthogonal control of distinct forward and backward motions, we believe that the present concept paves the way for creating more advanced molecular machines.


Subject(s)
Motion
2.
J Magn Reson ; 164(1): 19-27, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12932451

ABSTRACT

In this article we present methodology for simulating protein dynamics while imposing restraints derived from NMR measurements on partially ordered molecules. Such measurements may include residual dipolar couplings and chemical-shift anisotropies. We define a restraint potential for use in molecular dynamics and energy minimization. The presented potential is consistent with the simultaneously optimized molecular order tensor. Restraining can be performed with time and ensemble averaging. We performed a large number of molecular dynamics simulations of the histidine containing phosphocarrier protein with restraints on backbone N-H vector orientations derived from residual dipolar couplings. From these simulations it is evident that the use of time- or ensemble-averaged restraints is essential to leave the fluctuations of the restrained vectors unaffected. Without averaging the fluctuations of the restrained vectors are reduced significantly. This also has the effect of decreasing the apparent molecular order-parameter tensor.


Subject(s)
Algorithms , Bacterial Proteins , Computer Simulation , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Motion , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Anisotropy , Models, Statistical , Reproducibility of Results , Sensitivity and Specificity , Stress, Mechanical
3.
J Mol Biol ; 308(5): 993-1009, 2001 May 18.
Article in English | MEDLINE | ID: mdl-11352587

ABSTRACT

The determination by NMR of the solution structure of the phosphorylated enzyme IIB (P-IIB(Chb)) of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli is presented. Most of the backbone and side-chain resonances were assigned using a variety of mostly heteronuclear NMR experiments. The remaining resonances were assigned with the help of the structure calculations.NOE-derived distance restraints were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In addition, combinations of ambiguous restraints were used to resolve ambiguities in the NOE assignments. By combining sets of ambiguous and unambiguous restraints into new ambiguous restraints, an error function was constructed that was less sensitive to information loss caused by assignment uncertainties. The final set of structures had a pairwise rmsd of 0.59 A and 1.16 A for the heavy atoms of the backbone and side-chains, respectively. Comparing the P-IIB(Chb) solution structure with the previously determined NMR and X-ray structures of the wild-type and the Cys10Ser mutant shows that significant differences between the structures are limited to the active-site region. The phosphoryl group at the active-site cysteine residue is surrounded by a loop formed by residues 10 through 16. NOE and chemical shift data suggest that the phosphoryl group makes hydrogen bonds with the backbone amide protons of residues 12 and 15. The binding mode of the phosphoryl group is very similar to that of the protein tyrosine phosphatases. The differences observed are in accordance with the presumption that IIB(Chb) has to be more resistant to hydrolysis than the protein tyrosine phosphatases. We propose a proton relay network by which a transfer occurs between the cysteine SH proton and the solvent via the hydroxyl group of Thr16.


Subject(s)
Cysteine/metabolism , Disaccharides/metabolism , Escherichia coli/enzymology , Nuclear Magnetic Resonance, Biomolecular , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Binding Sites , Crystallography, X-Ray , Hydrogen Bonding , Models, Molecular , Mutation , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Phosphorylation , Protein Structure, Secondary , Protons , Solvents , Substrate Specificity , Thermodynamics
5.
J Biomol NMR ; 13(4): 357-68, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10383197

ABSTRACT

Protein disulfide isomerase (PDI) is a multifunctional protein of the endoplasmic reticulum, which catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. It consists of four domains designated a, b, b and a. Both a and a domains contains an active site with the sequence motif -Cys-Gly-His-Cys-involved directly in thiol-disulfide exchange reactions. As expected these domains have structures very similar to the ubiquitous redox protein thioredoxin. A low-resolution NMR structure of the b domain revealed that this domain adopts a fold similar to the PDI a domain and thioredoxin [Kemmink, J., Darby, N.J., Dijkstra, K., Nilges, M. and Creighton, T.E. (1997) Curr. Biol. 7, 239-245]. A refined ensemble of solution structures based on the input of 1865 structural restraints shows that the structure of PDI b is well defined throughout the complete protein except for about 10 residues at the C-terminus of the sequence. 15N relaxation data show that these residues are disordered and not part of this structural domain. Therefore the domain boundaries of PDI can now be fixed with reasonable precision. Structural comparison of the PDI b domain with thioredoxin and PDI a reveals several features important for thiol-disulfide exchange activity.


Subject(s)
Protein Disulfide-Isomerases/chemistry , Protein Structure, Secondary , Amino Acid Sequence , Binding Sites , Carbon Isotopes , Computer Simulation , Humans , Models, Molecular , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular , Recombinant Proteins/chemistry , Solutions
6.
Protein Sci ; 8(1): 5-12, 1999 Jan.
Article in English | MEDLINE | ID: mdl-10210178

ABSTRACT

The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the interleukin-6-type cytokines. It is a member of the cytokine-receptor superfamily predicted to consist of six domains in its extracellular part. The second and third domain constitute the cytokine-binding module defined by a set of four conserved cysteines and a WSXWS motif, respectively. The three-dimensional structure of the carboxy-terminal domain of this region was determined by multidimensional NMR. The domain consists of seven beta-strands constituting a fibronectin type III-like topology. The structure reveals that the WSDWS motif of gp130 is part of an extended tryptophan/arginine zipper which modulates the conformation of the CD loop.


Subject(s)
Antigens, CD/chemistry , Membrane Glycoproteins/chemistry , Receptors, Cytokine/chemistry , Amino Acid Sequence , Cytokine Receptor gp130 , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Sequence Homology, Amino Acid
7.
J Biol Chem ; 273(33): 20785-94, 1998 Aug 14.
Article in English | MEDLINE | ID: mdl-9694823

ABSTRACT

The thermal stability and domain interactions in the mannitol transporter from Escherichia coli, enzyme IImtl, have been studied by differential scanning calorimetry. To this end, the wild type enzyme, IICBAmtl, as well as IICBmtl and IICmtl, were reconstituted into a dimyristoylphosphatidylcholine lipid bilayer. The changes in the gel to liquid crystalline transition of the lipid indicated that the protein was inserted into the membrane, disturbing a total of approximately 40 lipid molecules/protein molecule. The thermal unfolding profile of EIImtl exhibited three separate transitions, two of which were overlapping, that could be assigned to structural domains in the protein. Treatment with trypsin, resulting in the degradation of the water-soluble part of the enzyme while leaving the binding and translocation capability of the enzyme intact, resulted in a decrease of the Tm and enthalpy of unfolding of the membrane-embedded C domain. This effect was much more apparent in the presence of the substrate but only partly so in the presence of the substrate analog perseitol. These results are consistent with a recently proposed model (Meijberg, W., Schuurman-Wolters, G. K., and Robillard, G. T. (1998) J. Biol. Chem. 273, 7949-7946), in which the B domain takes part in the conformational changes during the substrate binding process.


Subject(s)
Escherichia coli/enzymology , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Base Sequence , Calorimetry, Differential Scanning , DNA Primers , Enzyme Stability , Escherichia coli Proteins , Mannitol/chemistry , Models, Chemical , Monosaccharide Transport Proteins , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Phosphorylation , Protein Conformation , Protein Folding , Substrate Specificity
8.
Biochemistry ; 37(2): 622-37, 1998 Jan 13.
Article in English | MEDLINE | ID: mdl-9425085

ABSTRACT

The folding of an 85-residue protein, the histidine-containing phosphocarrier protein HPr, has been studied using a variety of techniques including DSC, CD, ANS fluorescence, and NMR spectroscopy. In both kinetic and equilibrium experiments the unfolding of HPr can be adequately described as a two-state process which does not involve the accumulation of intermediates. Thermodynamic characterization of the native and the transition states has been achieved from both equilibrium and kinetic experiments. The heat capacity change from the denatured state to the transition state (3. 2 kJ mol-1 K-1) is half of the heat capacity difference between the native and denatured states (6.3 kJ mol-1 K-1), while the solvent accessibility of the transition state (0.36) indicates that its compactness is closer to that of the native than that of the denatured state. The high value for the change in heat capacity upon unfolding results in the observation of cold denaturation at moderate denaturant concentrations. Refolding from high denaturant concentrations is, however, slow. The rate constant of folding in water, (14.9 s-1), is small compared to that reported for other proteins of similar size under similar conditions. This indicates that very fast refolding is not a universal character of small globular proteins which fold in the absence of detectable intermediates.


Subject(s)
Bacterial Proteins/chemistry , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Protein Folding , Calorimetry, Differential Scanning , Circular Dichroism , Guanidine , Kinetics , Models, Chemical , Protein Denaturation , Species Specificity , Temperature , Thermodynamics
9.
Proteins ; 29(2): 240-51, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9329088

ABSTRACT

A method is presented that generates random protein structures that fulfil a set of upper and lower interatomic distance limits. These limits depend on distances measured in experimental structures and the strength of the interatomic interaction. Structural differences between generated structures are similar to those obtained from experiment and from MD simulation. Although detailed aspects of dynamical mechanisms are not covered and the extent of variations are only estimated in a relative sense, applications to an IgG-binding domain, an SH3 binding domain, HPr, calmodulin, and lysozyme are presented which illustrate the use of the method as a fast and simple way to predict structural variability in proteins. The method may be used to support the design of mutants, when structural fluctuations for a large number of mutants are to be screened. The results suggest that motional freedom in proteins is ruled largely by a set of simple geometric constraints.


Subject(s)
Protein Conformation , Proteins/chemistry , Magnetic Resonance Spectroscopy , Proteins/metabolism
10.
Protein Sci ; 6(2): 304-14, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9041631

ABSTRACT

The assignment of the side-chain NMR resonances and the determination of the three-dimensional solution structure of the C10S mutant of enzyme IIBcellobiose (IIBcel) of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli are presented. The side-chain resonances were assigned nearly completely using a variety of mostly heteronuclear NMR experiments, including HCCH-TOCSY, HCCH-COSY, and COCCH-TOCSY experiments as well as CBCACOHA, CBCA(CO)NH, and HBHA(CBCA)(CO)NH experiments. In order to obtain the three-dimensional structure, NOE data were collected from 15N-NOESY-HSQC, 13C-HSQC-NOESY, and 2D NOE experiments. The distance restraints derived from these NOE data were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In an iterative procedure, additional NOE assignments were derived from the calculated structures and new structures were calculated. The final set of structures, calculated with approximately 2000 unambiguous and ambiguous distance restraints, has an rms deviation of 1.1 A on C alpha atoms. IIBcel consists of a four stranded parallel beta-sheet, in the order 2134. The sheet is flanked with two and three alpha-helices on either side. Residue 10, a cysteine in the wild-type enzyme, which is phosphorylated during the catalytic cycle, is located at the end of the first beta-strand. A loop that is proposed to be involved in the binding of the phosphoryl-group follows the cysteine. The loop appears to be disordered in the unphosphorylated state.


Subject(s)
Escherichia coli/enzymology , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Amino Acid Sequence , Carbon Isotopes , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Nitrogen Isotopes , Protons , Solutions
11.
Proteins ; 26(3): 314-22, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8953652

ABSTRACT

Recently, we developed a method (Amadei et al., J. Biomol. Str. Dyn. 13: 615-626; de Groot et al., J. Biomol. Str. Dyn. 13: 741-751, 1996) to obtain an extended sampling of the configurational space of proteins, using an adapted form of molecular dynamics (MD) simulations, based on the essential dynamics (ED) (Amadei et al., Proteins 17:412-425, 1993) method. In the present study, this ED sampling technique is applied to the histidine-containing phosphocarrier protein HPr from Escherichia coli. We find a cluster of conformations that is an order of magnitude larger than that found for a usual MD simulation of comparable length. The structures in this cluster are geometrically and energetically comparable to NMR structures. Moreover, on average, this large cluster satisfies nearly all NMR-derived distance restraints.


Subject(s)
Bacterial Proteins/chemistry , Computer Simulation , Models, Molecular , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Escherichia coli , Magnetic Resonance Spectroscopy , Protein Conformation , Protein Structure, Secondary , Reproducibility of Results
12.
Protein Sci ; 5(3): 442-6, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8868480

ABSTRACT

The structure of the phosphorylated form of the histidine-containing phosphocarrier protein HPr from Escherichia coli has been solved by NMR and compared with that of unphosphorylated HPr. The structural changes that occur upon phosphorylation of His 15, monitored by changes in NOE patterns, 3JNHH alpha-coupling constants, and chemical shifts, are limited to the region around the phosphorylation site. The His15 backbone torsion angles become strained upon phosphorylation. The release of this strain during the phosphoryl-transfer to Enzyme II facilitates the transport of carbohydrates across the membrane. From an X-ray study of Streptococcus faecalis HPr (Jia Z, Vandonselaar M, Quail JW, Delbaere LTJ, 1993, Nature 361:94-97), it was proposed that the observed torsion-angle strain at residue 16 in unphosphorylated S. faecalis HPr has a role to play in the protein's phosphocarrier function. The model predicts that this strain is released upon phosphorylation. Our observations on E. coli HPr in solution, which shows strain only after phosphorylation, and the fact that all other HPrs studied thus far in their unphosphorylated forms show no strain either, led us to investigate the possibility that the crystal environment causes the strain in S. faecalis HPr. A 1-ns molecular dynamics simulation of S. faecalis HPr, under conditions that mimic the crystal environment, confirms the observations from the X-ray study, including the torsion-angle strain at residue 16. The strain disappeared, however, when S. faecalis HPr was simulated in a water environment, resulting in an active site configuration virtually the same as that observed in all other unphosphorylated HPrs. This indicates that the torsion-angle strain at Ala 16 in S. faecalis HPr is a result of crystal contacts or conditions and does not play a role in the phosphorylation-dephosphorylation cycle.


Subject(s)
Bacterial Proteins/chemistry , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Bacterial Proteins/metabolism , Computer Simulation , Crystallography, X-Ray , Enterococcus faecalis/chemistry , Escherichia coli/chemistry , Magnetic Resonance Spectroscopy , Models, Molecular , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Phosphorylation , Water/metabolism
13.
Protein Sci ; 4(12): 2587-93, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8580850

ABSTRACT

A genetically engineered protein consisting of the 120 residues at the N-terminus of human protein disulfide isomerase (PDI) has been characterized by 1H, 13C, and 15N NMR methods. The sequence of this protein is 35% identical to Escherichia coli thioredoxin, and it has been found also to have similar patterns of secondary structure and beta-sheet topology. The results confirm that PDI is a modular, multidomain protein. The last 20 residues of the N-terminal domain of PDI are some of those that are similar to part of the estrogen receptor, yet they appear to be an intrinsic part of the thioredoxin fold. This observation makes it unlikely that any of the segments of PDI with similarities to the estrogen receptor comprise individual domains.


Subject(s)
Isomerases/chemistry , Magnetic Resonance Spectroscopy , Thioredoxins/chemistry , Amino Acid Sequence , Escherichia coli/chemistry , Humans , Molecular Sequence Data , Protein Disulfide-Isomerases , Protein Structure, Secondary , Receptors, Estrogen/chemistry , Sequence Homology
14.
J Biomol NMR ; 6(1): 33-40, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7545042

ABSTRACT

Nuclear Overhauser effect (NOE) measurements on molecules in solution provide information about only the ensemble-averaged properties of these molecules. An algorithm is presented that uses a list of NOEs to produce an ensemble of molecules that on average agrees with these NOEs, taking into account the effect of surrounding spins on the buildup of each NOE ('spin diffusion'). A simplified molecular dynamics simulation on several copies of the molecule in parallel is restrained by forces that are derived directly from differences between calculated and measured NOEs. The algorithm is tested on experimental NOE data of a helical peptide derived from bovine pancreatic trypsin inhibitor.


Subject(s)
Peptides/chemistry , Algorithms , Amino Acid Sequence , Animals , Aprotinin/chemistry , Aprotinin/genetics , Cattle , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptides/genetics , Protein Conformation , Protein Structure, Secondary , Solutions , Thermodynamics
15.
J Mol Biol ; 246(1): 180-93, 1995 Feb 10.
Article in English | MEDLINE | ID: mdl-7853396

ABSTRACT

The solution structure of the phosphorylated form of the histidine-containing phosphocarrier protein, HPr, from Escherichia coli has been determined by NMR in combination with restrained molecular dynamics simulations. The structure of phospho-HPr (P-HPr) results from a molecular dynamics simulation in water, using time-dependent distance restraints to attain agreement with the measured NOEs. Experimental restraints were identified from both three-dimensional 1H-1H-15N HSQC-NOESY and two-dimensional 1H-1HNOESY spectra, and compared with those of the unphosphorylated form. Structural changes upon phosphorylation of HPr are limited to the active site, as evidenced by changes in chemical shifts, in 3JNHH alpha-coupling constants and NOE patterns. Chemical shift changes were obtained mainly for protons that were positioned close to the phosphoryl group attached to the His15 imidazole ring. Differences could be detected in the intensity of the NOEs involving the side-chain protons of His15 and Pro18, resulting from a change in the relative position of the two rings. In addition, a small change could be detected in the three-bond J-coupling between the amide proton and the H alpha proton of Thr16 and Arg17 upon phosphorylation, in agreement with the changes of the phi torsion angle of these two residues obtained from time-averaged restrained molecular dynamics simulations in water. The proposed role of the torsion-angle strain at residue 16 in the mechanism of Streptococcus faecalis HPr is not supported by these results. In contrast, phosphorylation seems to introduce torsion angle strain at residue His15. This strain could facilitate the transfer of the phosphoryl group to the A-domain at enzyme II. The phospho-histidine is not stabilised by hydrogen bonds to the side-chain group of Arg17; instead stable hydrogen bonds are formed between the phosphate group and the backbone amide protons of Thr16 and Arg17, which show the largest changes in chemical shift upon phosphorylation, and a hydrogen bond involving the side-chain O gamma proton of Thr16. HPr accepts the phosphoryl group from enzyme I and donates it subsequently to the A domain of various enzyme II species. The binding site for EI on HPr resembles that of the A domain of the mannitol-specific enzyme II, as can be concluded from the changes on the amide proton and nitrogen chemical shifts observed via heteromolecular single-quantum coherence spectroscopy.


Subject(s)
Bacterial Proteins/chemistry , Escherichia coli/chemistry , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Bacterial Proteins/metabolism , Binding Sites , Computer Graphics , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Structure , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Phosphorylation
16.
J Biomol NMR ; 6(1): 106-11, 1995 Jul.
Article in English | MEDLINE | ID: mdl-22911579

ABSTRACT

We address the question how well proteins can be modelled on the basis of NMR data, when these data are incorporated into the protein model using distance restraints in a molecular dynamics simulation. We found, using HPr as a model protein, that distance restraining freezes the essential motion of proteins, as defined by Amadei et al. [Amadei, A., Linssen, A.B.M. and Berendsen, H.J.C. (1993) Protein Struct. Funct. Genet., 17, 412-425]. We discuss how modelling protocols can be improved in order to solve this problem.

17.
J Mol Biol ; 237(5): 544-59, 1994 Apr 15.
Article in English | MEDLINE | ID: mdl-8158637

ABSTRACT

The solution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli has been determined by NMR in combination with distance geometry and restrained molecular dynamics. The structure is based on 1520 experimental restraints identified from both three-dimensional 1H-1H-13C and 1H-1H-15N nuclear Overhauser effect multiple-quantum coherence spectroscopy and two-dimensional 1H-1H nuclear Overhauser effect spectra. Thirty-two four-dimensional coordinate frames were produced by metric matrix distance geometry, subjected to distance bounds driven dynamics, projected into three-dimensional space and again subjected to distance-bounds driven dynamics. These 32 distance geometry structures were refined further by restrained molecular dynamics (40 ps) in the GROMOS in vacuo force field. All 32 structures reached acceptable energy minima while satisfying the imposed restraints. Two of these structures were subjected to a further 200 ps of molecular dynamics simulation in water, using time-dependent distance restraining, followed by a 200 ps free simulation without any distance restraining. The resulting structure is very similar to the X-ray structure of Bacillus subtilis HPr, but differs mainly in the position of the two loops containing the active site histidine residue 15 and residues 53 to 57 relative to the rest of the structure. The unfavorable phi torsion angle that was found for residue 16 in the active center of unphosphorylated Streptococcus faecalis HPr was proposed to play a role in the activity of the protein. Although present at the early stages of the structure calculations, this torsion-angle strain disappeared in the final model obtained from molecular dynamics simulations in water using time-averaged distance restraining and upon releasing the distance restraints. This suggests that the strain may be an artifact of crystallization conditions instead of an essential element in the phosphorylation/dephosphorylation process.


Subject(s)
Bacterial Proteins/chemistry , Escherichia coli/chemistry , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Protein Structure, Secondary , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Models, Chemical , Models, Molecular , Water/chemistry
18.
Protein Sci ; 3(2): 282-90, 1994 Feb.
Article in English | MEDLINE | ID: mdl-8003964

ABSTRACT

The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBcellobiose of the Escherichia coli cellobiose-specific phosphoenol-pyruvate-dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple resonance experiments, the HNCA and HN(CO)CA experiments, correlating backbone 1H, 15N, and 13C alpha resonances, and the HN(CA)CO and HNCO experiments, correlating backbone 1H,15N and 13CO resonances. Heteronuclear 1H-NOE 1H-15N single quantum coherence (15N-NOESY-HSQC) spectroscopy and heteronuclear 1H total correlation 1H-15N single quantum coherence (15N-TOCSY-HSQC) spectroscopy were used to resolve ambiguities arising from overlapping 13C alpha and 13CO frequencies and to check the assignments from the triple resonance experiments. This procedure, together with a 3-dimensional 1H alpha-13C alpha-13CO experiment (COCAH), yielded the assignment for all observed backbone resonances. The secondary structure was determined using information both from the deviation of observed 1H alpha and 13C alpha chemical shifts from their random coil values and 1H-NOE information from the 15N-NOESY-HSQC. These data show that enzyme IIBcellobiose consists of a 4-stranded parallel beta-sheet and 5 alpha-helices. In the wild-type enzyme IIBcellobiose, the catalytic residue appears to be located at the end of a beta-strand.


Subject(s)
Escherichia coli/enzymology , Magnetic Resonance Spectroscopy , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Amino Acid Sequence , Base Sequence , Molecular Sequence Data , Mutagenesis , Protein Structure, Secondary
19.
Protein Sci ; 2(8): 1331-41, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8401218

ABSTRACT

This report presents the backbone assignments and the secondary structure determination of the A domain of the Escherichia coli mannitol transport protein, enzyme-IImtl. The backbone resonances were partially assigned using three-dimensional heteronuclear 1H NOE 1H-15N single-quantum coherence (15N NOESY-HSQC) spectroscopy and three-dimensional heteronuclear 1H total correlation 1H-15N single-quantum coherence (15N TOCSY-HSQC) spectroscopy on uniformly 15N enriched protein. Triple-resonance experiments on uniformly 15N/13C enriched protein were necessary to complete the backbone assignments, due to overlapping 1H and 15N frequencies. Data obtained from three-dimensional 1H-15N-13C alpha correlation experiments (HNCA and HN(CO)CA), a three-dimensional 1H-15N-13CO correlation experiment (HNCO), and a three-dimensional 1H alpha-13C alpha-13CO correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of virtually all observed backbone resonances. Determination of the secondary structure of IIAmtl is based upon NOE information from the 15N NOESY-HSQC and the 1H alpha and 13C alpha secondary chemical shifts. The resulting secondary structure is considerably different from that reported for IIAglc of E. coli and Bacillus subtilis determined by NMR and X-ray.


Subject(s)
Escherichia coli/enzymology , Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry , Protein Conformation , Protein Structure, Secondary , Amino Acid Sequence , Bacillus subtilis/enzymology , Enterobacter/enzymology , Escherichia coli Proteins , Magnetic Resonance Spectroscopy/methods , Molecular Sequence Data , Monosaccharide Transport Proteins , Salmonella typhimurium/enzymology , Sequence Homology, Amino Acid , X-Ray Diffraction
20.
J Mol Biol ; 230(1): 312-22, 1993 Mar 05.
Article in English | MEDLINE | ID: mdl-7680725

ABSTRACT

A synthetic peptide corresponding to the 15 N-terminal residues of bovine pancreatic trypsin inhibitor, with serine replacing the two cysteine residues, has been characterized by 1H-nuclear magnetic resonance spectroscopy. This peptide has a very disordered conformation that is essentially the same when it is part of the analogue of the (30-51) one-disulphide intermediate in folding. This confirms the conclusion of a previous paper, that the (30-51) intermediate is partially folded, with the N-terminal segment disordered. Local elements of non-random conformation were observed in the peptide. Especially prominent was an apparently electrostatic interaction between the face of the aromatic ring of Tyr10 and the amide group of Gly12, which caused the latter to have a very anomalous chemical shift. A similar interaction was observed in shorter peptides, especially in tetrapeptides with the sequences Tyr/Phe-X-Gly-Y. The local nature of this interaction indicates that it should be a general feature in peptides and in unfolded proteins with such a sequence.


Subject(s)
Trypsin Inhibitors/chemistry , Amides/chemistry , Amino Acid Sequence , Animals , Aprotinin , Cattle , In Vitro Techniques , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Pancreas/enzymology , Peptides/chemistry
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