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C M Dobson

Publications and source records attributed to C M Dobson.

At least 199 records · Page 11Linked to original sources
PubMed1992

A study of D52S hen lysozyme-GlcNAc oligosaccharide complexes by NMR spectroscopy and electrospray mass spectrometry.

The production of a mutant hen lysozyme is described in which Asp-52, one of the catalytically important residues, is replaced by Ser. The mutant enzyme has very low catalytic activity but NMR studies show that its structure is closely similar to that of the wild-type protein. NMR experiments also show that well defined complexes are formed with GlcNAc4 and GlcNAc6 bound in the active site of the mutant enzyme. These complexes have been examined using electrospray mass spectrometry (ESMS). The most intense peaks arise from the uncomplexed protein indicating that dissociation takes place in the mass spectrometer under the conditions used here. Peaks from minor species corresponding to complexes between the protein and the oligosaccharides are, however, also observed. The possibility that the latter arise from novel covalent enzyme-saccharide complexes is discussed.

Acetylglucosamine↗
PubMed1992

Hydrogen exchange in native and denatured states of hen egg-white lysozyme.

The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme have been measured at pH 7.5 and 30 degrees C by 2D NMR methods. These constitute the most protected subset of amides, with exchange half lives some 10(5)-10(7) times longer than anticipated from studies of small model peptides. The observed distribution of rates under these conditions can be rationalized to a large extent in terms of the hydrogen bonding of individual amides and their burial from bulk solvent. Exchange rates have also been measured in a reversibly denatured state of lysozyme; this was made possible under very mild conditions, pH 2.0 35 degrees C, by lowering the stability of the native state through selective cleavage of the Cys-6-Cys-127 disulfide cross-link (CM6-127 lysozyme). In this state the exchange rates for the majority of amides approach, within a factor of 5, the values anticipated from small model peptides. For a few amides, however, there is evidence for significant retardation (up to nearly 20-fold) relative to the predicted rates. The pattern of protection observed under these conditions does not reflect the behavior of the protein under strongly native conditions, suggesting that regions of native-like structure do not persist significantly in the denatured state of CM6-127 lysozyme. The pattern of exchange rates from the native protein at high temperature, pH 3.8 69 degrees C, resembles that of the acid-denatured state, suggesting that under these conditions the exchange kinetics are dominated by transient global unfolding. The rates of folding and unfolding under these conditions were determined independently by magnetization transfer NMR methods, enabling the intrinsic exchange rates from the denatured state to be deduced on the basis of this model, under conditions where the predominant equilibrium species is the native state. Again, in the case of most amides these rates showed only limited deviation from those predicted by a simple random coil model. This reinforces the view that these denatured states of lysozyme have little persistent residual order and contrasts with the behavior found for compact partially folded states of proteins, including an intermediate detected transiently during the refolding of hen lysozyme.

Amides↗
PubMed1991

Secondary structure and topology of human interleukin 4 in solution.

Human interleukin 4 (IL-4) has been studied by 2D and 3D NMR techniques using uniformly 15N-labeled recombinant protein. Assignment of resonances for all but 3 of the 130 residues of the recombinant protein has been achieved, enabling the secondary structure of the protein to be defined. This consists of four major alpha-helical regions and one short section of double-stranded antiparallel beta-sheet. Analysis of distance and angle restraints derived from NMR experiments has enabled the overall molecular topology to be determined. This is related to that found for other four-helix proteins but has several distinctive features including cross-linking of helices by means of three disulfide bonds and a short section of beta-sheet. The structural analysis gives support to the hypothesis that many helical cytokines have a common fold and provides a basis for understanding the biological function of IL-4.

Amino Acid Sequence↗
PubMed1991

Antigen mobility in the combining site of an anti-peptide antibody.

The interaction between a high-affinity antibody, raised against a peptide incorporating the loop region of hen egg lysozyme (residues 57-84), and a peptide antigen corresponding to this sequence, has been probed by proton NMR. The two-dimensional correlated spectroscopy spectrum of the antibody-antigen complex shows sharp, well-resolved resonances from at least half of the bound peptide residues, indicating that the peptide retains considerable mobility when bound to the antibody. The strongly immobilized residues (which include Arg-61, Trp-62, Trp-63, and Ile-78) do not correspond to a contiguous region in the sequence of the peptide. Examination of the crystal structure of the protein shows that these residues, although remote in sequence, are grouped together in the protein structure, forming a hydrophobic projection on the surface of the molecule. The antibody binds hen egg lysozyme with only a 10-fold lower affinity than the peptide antigen. We propose that the peptide could bind to the antibody in a conformation that brings these groups together in a manner related to that found in the native protein, accounting for the high crossreactivity.

Amino Acid Sequence↗
PubMed1991

Solution structure of neuronal bungarotoxin determined by two-dimensional NMR spectroscopy: sequence-specific assignments, secondary structure, and dimer formation.

The solution structure of neuronal bungarotoxin (nBgt) has been studied by using two-dimensional 1H NMR spectroscopy. Sequence-specific assignments for over 95% of the backbone resonances and 85% of the side-chain resonances have been made by using a series of two-dimensional spectra at four temperatures. From these assignments over 75% of the NOESY spectrum has been assigned, which has in turn provided 582 distance constraints. Twenty-seven coupling constants (NH-alpha CH) were determined from the COSY spectra, which have provided dihedral angle constraints. In addition, hydrogen exchange experiments have suggested the probable position of hydrogen bonds. The NOE constraints, dihedral angle constraints, and the rates of amide proton exchange suggest that a triple-stranded antiparallel beta sheet is the major component of secondary structure, which includes 25% of the amino acid residues. A number of NOE peaks were observed that were inconsistent with the antiparallel beta-sheet structure. Because we have confirmed by sedimentation equilibrium that nBgt exists as a dimer, we have reinterpreted these NOE constraints as intermolecular interactions. These constraints suggest that the dimer consists of a six-stranded antiparallel beta sheet (three from each monomer), with residues 55-59 forming the dimer interface.

Amino Acid Sequence↗
PubMed1991

A nuclear magnetic resonance study of the hydrogen-exchange behaviour of lysozyme in crystals and solution.

Amide hydrogen/deuterium exchange behaviour has been studied for all of the peptide amides of hen lysozyme by means of two-dimensional n.m.r. spectroscopy. The amides have been grouped into four categories on the basis of their rates of exchange in solution at pH 4.2 and 7.5. The distribution of the amides into the different categories has been examined in the light of the crystallographic structural information, considering the type of secondary structure, the nature of hydrogen bonding and the distance from the protein surface. None of these features was found to determine uniquely the pattern of hydrogen exchange rates within the protein. The exchange behaviour of the individual amides could, however, in general be rationalized by a combination of these features. Hydrogen exchange was also monitored in both tetragonal and triclinic crystals of lysozyme, by allowing exchange to take place in the crystals prior to dissolution and recording of n.m.r. spectra under conditions where further exchange was minimized. This enabled direct comparison to be made of the exchange behaviour in the crystals and solution. A reduction in exchange rate was observed in the crystalline state relative to solution for a substantial number of amides and distinct differences between exchange in the different crystals could be observed. These differences between the solution and the different crystal states do not, however, correlate in a simple manner with proximity to intermolecular contacts in the crystals. However, the existence of these contacts, which are on the surface of the protein molecule, have a profound effect on the exchange of amides in the interior of the protein. The results indicate that the spectrum of fluctuations giving rise to hydrogen exchange may be significantly altered by the intermolecular interactions present within the crystalline state.

Amides↗
PubMed1991

Demonstration by NMR of folding domains in lysozyme.

Although there has been much speculation on the pathways of protein folding, only recently have experimental data on the topic been available. The study of proteins under conditions where species intermediate between the fully folded and unfolded states are stable has provided important information, for example about the disulphide intermediates in BPTI, cis/trans proline isomers of RNase A3 and the molten globule state of alpha-lactalbumin. An alternative approach to investigating folding pathways has involved detection and characterization of transient conformers in refolding studies using stopped-flow methods coupled with NMR measurements of hydrogen exchange. The formation of intermediate structures has been detected in the early stages of folding of cytochrome c, RNaseA and barnase. For alpha-lactalbumin, hydrogen exchange kinetics monitored by NMR proved to be crucial for identifying native-like structural features in the stable molten globule state. An analogous partially folded protein stable under equilibrium conditions has not been observed for the structurally homologous protein hen egg-white lysozyme, although there is evidence that a similar but transient state is formed during refolding. Here we describe NMR experiments based on competition between hydrogen exchange and the refolding process which not only support the existence of such a transient species for lysozyme, but enable its structural characteristics to be defined. The results indicate that the two structural domains of lysozyme are distinct folding domains, in that they differ significantly in the extent to which compact, probably native-like, structure is present in the early stages of folding.

Animals↗
PubMed1991

Analysis of phi and chi 1 torsion angles for hen lysozyme in solution from 1H NMR spin-spin coupling constants.

Three-bond 3JHN alpha coupling constants have been determined for 106 residues and 3J alpha beta coupling constants have been measured for 57 residues of the 129-residue protein hen egg white lysozyme. These NMR data have been compared with torsion angles defined in the tetragonal and the triclinic crystal forms of the protein. For most residues the measured 3JHN alpha values were consistent with the phi torsion angles found in both crystal forms; the RMS difference between the coupling constants calculated by using the tetragonal crystal structure phi angles and the experimental 3JHN alpha values is 0.88 Hz. Thus there appears to be no significant averaging of the phi torsion angle either in the interior or at the surface of the protein. For 41 of the residues where 3J alpha beta coupling constants have been determined, the values are consistent with a single staggered conformation about the chi 1 torsion angle and there is complete agreement between the NMR data in solution and the torsion angles defined in the crystalline state. In contrast, for the other 16 residues where 3J alpha beta coupling constant values have been measured, the data indicate extensive motional averaging about the chi 1 torsion angle. These residues occur largely on the surface of the protein and examination of the crystal structures shows that many of these residues adopt a different conformation in the triclinic and tetragonal crystal forms and have high crystallographic temperature factors. It appears, however, that in solution conformational flexibility of the side chains of surface residues is significantly more pronounced than in individual crystal structures.

Amino Acids↗
PubMed1991

A photochemically induced dynamic nuclear polarization study of denatured states of lysozyme.

Photochemically induced dynamic nuclear polarization (photo-CIDNP) techniques have been used to examine denatured states of lysozyme produced under a variety of conditions. 1H CIDNP difference spectra of lysozyme denatured thermally, by the addition of 10 M urea, or by the complete reduction of its four disulfide bonds were found to differ substantially not only from the spectrum of the native protein but also from that expected for a completely unstructured polypeptide chain. Specifically, denatured lysozyme showed a much reduced enhancement of tryptophan relative to tyrosine than did a mixture of blocked amino acids with the same composition as the intact protein. By contrast, the CIDNP spectrum of lysozyme denatured in dimethyl sulfoxide solution was found to be similar to that expected for a random coil. It is proposed that nonrandom hydrophobic interactions are present within the denatured states of lysozyme in aqueous solution and that these reduce the reactivity of tryptophan residues relative to tyrosine residues. Characterization of such interactions is likely to be of considerable significance for an understanding of the process of protein folding.

Amino Acids↗
PubMed1991

Domain interactions in human plasminogen studied by proton NMR.

The NMR spectrum of miniplasminogen (V443-plasminogen) under conditions of acidic pH reveals a subset of particularly well-resolved resonances whose chemical shift values are closely similar to those of isolated kringle 5. The temperature dependence of the spectrum indicates that this set of resonances disappears in a single cooperative unfolding transition appropriate for kringle 5, whilst other broader resonances from the protease domain persist to higher temperature. These results provide evidence for significant structural and motional independence of the kringle and protease domains in spite of the short linker between these domains. The NMR spectrum of Glu1-plasminogen is closely similar to that of miniplasminogen under the same conditions. This suggests that the domain independence observed in miniplasminogen is maintained in the intact molecule.

Humans↗
PubMed1991

A three-disulphide derivative of hen lysozyme. Structure, dynamics and stability.

A three-disulphide derivative of hen egg-white lysozyme was made by selective reduction and carboxymethylation of one of the four original disulphide bridges. N-Terminal sequencing and two-dimensional 1H-n.m.r. spectroscopy revealed that the disulphide bridge linking cysteine residues 6 and 127 had been modified and that the three remaining disulphide bonds were native-like in nature. Analysis of COSY and NOESY spectra indicated that the three-disulphide lysozyme (CM6.127-lysozyme retains the same secondary and tertiary structure as its four-disulphide counterpart; its stability to pH and temperature is, however, dramatically decreased. N.m.r. spectroscopy was used to characterize the thermal folding and unfolding transition of CM6.127-lysozyme. Not only is the transition still a highly co-operative event, but the enthalpy change associated with folding and unfolding resembles that of intact lysozyme when their differences in thermal stability are taken into consideration. The significance of these results in terms of the folding process of lysozyme is discussed. By contrast with authentic lysozyme, CM6.127-lysozyme was found to exist in an unfolded state at pH 2 at room temperature. N.m.r. spectroscopy and c.d. were used to characterize this state. Unlike their homologous relative, alpha-lactalbumin, which exists in a partially folded molten globule state under these conditions, only residual non-native-like structure persists in the acid-unfolded state of CM6.127-lysozyme. These results indicate that the difference in folding behaviour of lysozyme and alpha-lactalbumin cannot be accounted for simply by their differences in thermal stability.

Animals↗
PubMed1991

NMR spectroscopy and protein folding: studies of lysozyme and alpha-lactalbumin.

The description of the folding process for any protein has as a principal objective the characterization of the structural changes that occur during the transition from a disordered state to a highly ordered state. It is now generally accepted that folding occurs via some pathway or pathways which can be described in terms of intermediate, partially folded states. Three complementary strategies have emerged for obtaining structural information about intermediate states. The first involves characterization of species generated transiently during refolding of denatured proteins, either in real time or by means of trapping experiments. The second involves the study of those partially folded states, such as the increasingly recognized molten globule state, which are stable under equilibrium conditions. The third strategy involves the design and study of peptide models of folding intermediates. NMR spectroscopy, because of its ability to provide information at the molecular level about protein structure and dynamics in solution, plays a crucial role in each of these strategies. We describe results from our own studies of lysozyme and alpha-lactalbumin to illustrate the scope and potential of NMR spectroscopy in studies of protein folding.

Amides↗
PubMed1991

Hydrophobic clustering in nonnative states of a protein: interpretation of chemical shifts in NMR spectra of denatured states of lysozyme.

Chemical shifts of resonances of specific protons in the 1H NMR spectrum of thermally denatured hen lysozyme have been determined by exchange correlation with assigned native state resonances in 2D NOESY spectra obtained under conditions where the two states are interconverting. There are subtle but widespread deviations of the measured shifts from the values which would be anticipated for a random coil; in the case of side chain protons these are virtually all net upfield shifts and it is shown that this may be the averaged effect of interactions with aromatic rings in a partially collapsed denatured state. In a very few cases, notably that of two sequential tryptophan residues, it is possible to interpret these effects in terms of specific, local interresidue interactions. Generally, however, there is no correlation with either native state shift perturbations or with sequence proximity to aromatic groups. Diminution of most of the residual shift perturbations on reduction of the disulfide cross-links confirms that they are not simply effects of residues adjacent in the sequence. Similar effects of chemical denaturants, with the disulfides intact, demonstrate that the shift perturbations reflect an enhanced tendency to side chain clustering in the thermally denatured state. The temperature dependences of the shift perturbations suggest that this clustering is noncooperative and is driven by small, favorable enthalpy changes. While the extent of conformational averaging is clearly much greater than that observed for a homologous protein, alpha-lactalbumin, in its partially folded "molten globule" state, the results clearly show that thermally denatured lysozyme differs substantially from a random coil, principally in that it is partially hydrophobically collapsed.

Cluster Analysis↗
PubMed1991

Relaxation data in NMR structure determination: model calculations for the lysozyme-Gd3+ complex.

The effect of including paramagnetic relaxation data as additional restraints in the determination of protein tertiary structures from NMR data has been explored by a systematic series of model calculations. The system used for testing the method was the 2.0 A resolution tetragonal crystal structure of hen egg white lysozyme (129 amino acid residues) and structures were generated using a version of the hybrid "distance geometry-dynamic simulated annealing" procedure. A limited set of 769 NOEs was used as restraints in all the calculations; the strengths of these were categorized into three classes on the basis of distances observed in the crystal structure. The values of 50 phi angles were also restrained on the basis of amide-alpha coupling constants calculated from the X-ray structure. Five sets of 12 structures were determined using differing sets of paramagnetic relaxation data as restraints additional to those involving the NOE and coupling constant data. The paramagnetic relaxation data were modeled on the basis of the distances of defined protons from the crystallographic binding site of Gd3+ in lysozyme. Analysis of the results showed that the relaxation data significantly improved the correspondence between the set of generated structures and the crystal structure, and that the more well defined the relaxation data, the more significant the improvement in the quality of the structures. The results suggest that the inclusion of paramagnetic relaxation restraints could be of significant value for the experimental determination of protein structures from NMR data.

Gadolinium↗