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Biomedical subjects

C M Dobson

Publications and source records attributed to C M Dobson.

At least 127 records · Page 7Linked to original sources
PubMed1997

Identification of the epitopes of calcitonin gene-related peptide (CGRP) for two anti-CGRP monoclonal antibodies by 2D NMR.

The interactions between calcitonin gene-related peptide and FAB fragments prepared from two different high-affinity anti-CGRP monoclonal antibodies (CB3 and CD1) have been studied at physiological pH using the ability of 1H NMR to detect selectively regions of dynamic flexibility. The 37-residue peptide retains considerable flexibility in regions of its sequence when bound to both antibodies; in each case, more than half of the residues can be seen to have linewidths little perturbed from those of the free peptide. However the regions where substantial broadening of resonances occur, attributed to substantially reduced motional freedom of the peptide resulting from interactions within the antibody combining site, differ greatly in the two cases. In the complex with CB3 the results indicate that the restricted residues lie exclusively within the C-terminal half of the peptide, and include residues 25 to 32 and the terminal two residues (36 and 37). By contrast, in the complex with CD1, the conformationally restricted residues appear to lie predominantly within the N-terminal half of the CGRP molecule, particularly residues 4-16, although several residues in the middle section of the sequence (22-31) have reduced conformational freedom. These findings, consistent with the results from immunological assays, add considerably to our knowledge of the epitopes.

Antibodies, Monoclonal↗
PubMed1997

A residue-specific NMR view of the non-cooperative unfolding of a molten globule.

Molten globules are partially folded forms of proteins that are thought to be general intermediates in protein folding. Nonetheless, there is limited structural information about such species because they possess conformational heterogeneity and complex dynamical properties that lead to extreme line broadening in NMR spectra. Here we use a 2-D NMR approach that overcomes this difficulty by detecting the unfolding of individual residues in a molten globule in increasing concentrations of denaturant. The results show that the structure in the low pH form of alpha-lactalbumin (alpha-LA) is not formed cooperatively. Moreover, a core region remains collapsed under extremely denaturing conditions, even when the majority of the polypeptide chain is completely unfolded. Our results support a model for protein folding in which the core provides a template for correct assembly of the remainder of the structure.

Circular Dichroism↗
PubMed1996

Protein folding monitored at individual residues during a two-dimensional NMR experiment.

An approach is described to monitor directly at the level of individual residues the formation of structure during protein folding. A two-dimensional heteronuclear nuclear magnetic resonance (NMR) spectrum was recorded after the rapid initiation of the refolding of a protein labeled with nitrogen-15. The intensities and line shapes of the cross peaks in the spectrum reflected the kinetic time course of the folding events that occurred during the spectral accumulation. The method was used to demonstrate the cooperative nature of the acquisition of the native main chain fold of apo bovine alpha-lactalbumin. The general approach, however, should be applicable to the investigation of a wide range of chemical reactions.

Circular Dichroism↗
PubMed1996

Rapid refolding of a proline-rich all-beta-sheet fibronectin type III module.

Fibronectin type III modules contain approximately 90 residues and are an extremely common building block of animal proteins. Despite containing a complex all-beta-sheet topology and eight prolines, the refolding of the 10th type III module of human fibronectin has been found to be very rapid, with native core packing, amide hydrogen bonding, and backbone conformation all recovered within 1 s at 5 degrees C. These observations indicate that this domain can overcome many structural characteristics often thought to slow the folding process.

Circular Dichroism↗
PubMed1996

Molecular characterisation of a thermoactive beta-1,3-glucanase from Oerskovia xanthineolytica.

Molecular characterisation of a lytic thermoactive beta-1,3-glucanase from Oerskovia xanthineolytica LL-G109 has been performed. A molecular mass of 27 195.6 +/- 1.3 Da and an isoelectric point of 4.85 were determined by electrospray mass spectrometry and from its titration curve, respectively. Its thermoactivity profile shows it to be a heat-stable enzyme with a temperature optimum of 65 degrees C. The secondary structure content of the protein was estimated by circular dichroism to be approx. 25% alpha-helix, 7% random coil, and 68% beta-sheet and beta-turn structure. Nuclear magnetic resonance spectra confirm the high content of beta-structure. Furthermore, the presence of a compact hydrophobic core is indicated by the presence of slowly exchanging amide hydrogens and the enzyme's relatively high resistance to proteolysis. The N-terminal sequences of the intact protein and of a tryptic peptide each exhibit significant similarity to family 16 of glycosyl hydrolases whose overall fold is known to contain almost exclusively beta-sheets and surface loops. Moreover, the sequenced tryptic peptide appears to encompass residues of the Oerskovia xanthineolytica glucanase active site, since it contains a portion of the family 16 active-site motif E-[L/I/V]-D-[L/I/V]-E.

Amino Acid Sequence↗
PubMed1996

Insight into a random coil conformation and an isolated helix: structural and dynamical characterisation of the C-helix peptide from hen lysozyme.

A 17 residue peptide corresponding to the C-helix of hen lysozyme (residues 86 to 102) has been investigated in detail to assess the factors that determine its conformation in both aqueous and trifluoroethanol (TFE) solutions. A thorough characterisation of the peptide by CD and NMR techniques under both conditions has been performed including the determination of complete NMR proton sequential assignments, and measurement of NOE effects, 3JHN alpha coupling constants, temperature coefficients and residue-specific hydrogen-exchange rates. In water, the peptide adopts a largely unstructured conformation and NMR data, particularly coupling constants and chemical shift deviations, have been shown to agree closely with predictions from a model for a random coil based on the phi, psi distributions in a protein database. This indicates that under these conditions the intrinsic conformational preferences of the individual amino acid residues are the dominating factors that determine the population of conformers adopted. With increasing concentrations of TFE a cooperative transition to an extensively helical conformation occurs and the resultant changes in C alpha H chemical shifts have been shown to correlate with the changes in phi, psi populations. Using NOE and coupling constant data for this state, an ensemble of structures has been calculated and provides a model for a helix in the absence of tertiary interactions. In this model fluctuations, which increase in amplitude towards the termini, occur about the average helical phi, psi angles and are responsible for increasing the values of 3JHN alpha coupling constants above those anticipated for a static helix. The residue-specific rates of hydrogen exchange for the peptide in 50% TFE-d, are consistent with such a model, the maximum protection from exchange being observed for residues in the centre of the helix.

Amino Acid Sequence↗
PubMed1996

Main-chain dynamics of a partially folded protein: 15N NMR relaxation measurements of hen egg white lysozyme denatured in trifluoroethanol.

15N NMR relaxation measurements have been used to study the dynamic behaviour of the main-chain of hen lysozyme in a partially folded state, formed in a 70% (v/v) trifluoroethanol (TFE)/30% water mixture at 37 degrees C and pH 2. This state is characterised by helical secondary structure in the absence of extensive tertiary interactions. The NMR relaxation data were interpreted by mapping of spectral density functions and by derivation of segmental as well as global order parameters. The results imply that the dynamics of lysozyme in TFE can, at least for the great majority of residues, be adequately described by internal motions which are superimposed on all overall isotropic tumbling of the molecule. Although the dynamic behaviour shows substantial variations along the polypeptide chain, it correlates well with the conformational preferences identified in the TFE state by other NMR parameters. Segments of the polypeptide chain which are part of persistent helical structures are highly restricted in their motion (S2 > 0.8 , with effective internal correlation times tau(e) < 200 ps) but are also found to experience conformational exchange on a millisecond timescale. Regions which are stabilised in less persistent helical structure possess greater flexibility (0.6 < S2 < 0.8, 200 ps < tau(e) < 1 ns) and those which lack defined conformational preferences are highly flexible (S2 < 0.6, tau(e) approximately 1 ns). The dynamic behaviour of the main-chain was found to be correlated with other local features of the polypeptide chain, including hydrophobicity and the position of the disulphide bridges. Despite the absence of extensive tertiary interactions, preferential stabilisation of native-like secondary structure by TFE results in a pattern of main-chain dynamics which is similar to that of the native state.

Animals↗
PubMed1996

Analysis of main chain torsion angles in proteins: prediction of NMR coupling constants for native and random coil conformations.

Using a data base of 85 high resolution protein crystal structures the distributions of main chain torsion angles, both in secondary structure and in coil regions where no secondary structure is present, have been analysed. These torsion angle distributions have been used to predict NMR homonuclear and heteronuclear coupling constants for residues in secondary structure using known Karplus relationships. For alpha helices, 3(10) helices and beta strands mean predicted 3JHN alpha coupling constants are 4.8, 5.6 and 8.5 Hz, respectively. These values differ significantly from those expected for the ideal phi angles (3.9, 3.0 and 8.9 Hz; phi = -57 degrees, -49 degrees, -139 degrees for alpha and 3(10) helices and beta strands (antiparallel), respectively) in regular secondary structure, but agree well with available experimental NMR data for nine proteins. The crystallographic data set has also been used to provide a basis for interpreting coupling constants measured for peptides and denatured proteins. Using a model for a random coil, in which all residues adopt distributions of phi, psi angles equivalent to those seen for residues in the coil regions of native folded proteins, predicted 3JHN alpha values for different residue types have been found to range from 5.9 Hz and 6.1 Hz for glycine and alanine, respectively, to 7.7 Hz for valine. A good correlation has been found between the predicted 3JHN alpha coupling constants for this model and experimental values for a set of peptides that other evidence suggest are highly unstructured. For other peptides, however, deviations from the predictions of the model are clear and provide evidence for additional interactions within otherwise disordered states. The values of homonuclear and heteronuclear coupling constants derived from the protein data base listed here therefore provide a basis not only for analysing the secondary structure of native proteins in solution but for assessing and interpreting the extent of structure present in peptides and non-native states of proteins.

Databases, Factual↗
PubMed1996

The domain organization of streptokinase: nuclear magnetic resonance, circular dichroism, and functional characterization of proteolytic fragments.

Streptococcus equisimilis streptokinase (SK) is a bacterial protein of unknown tertiary structure and domain organization that is used extensively to treat acute myocardial infarction following coronary thrombosis. Six fragments of SK were generated by limited proteolysis with chymotrypsin and purified. NMR and CD experiments have shown that the secondary and tertiary structure present in the native molecule is preserved within all fragments, except the N-terminal fragment SK7. NMR spectra demonstrate the presence in SK of three structurally autonomous domains and a less structured C-terminal "tail." Cleavage within the N-terminal domain generates an N-terminal fragment, SK7, which remains noncovalently associated with the remainder of the molecule; in isolation, SK7 adopts an unfolded conformation. The abilities of these fragments to induce active site formation within human plasminogen upon formation of their heterodimeric complex were assayed. The lowest mass SK fragment exhibiting Plg-dependent activator activity was shown to be SK27 (mass 27,000, residues 147-380), which contains both central and C-terminal domains, although this activity was reduced approximately 6,000-fold relative to that of full-length SK. The activity of a 36,000 mass fragment, SK36 (residues 64-380), which differs from SK27 in possessing a portion of the N-terminal domain, was reduced to 0.1-1.0% of that of SK. Other fragments (masses 7,000, 11,000, 16,000, 17,000, 25,000, and 26,000), representing either single domains or single domains extended by portions of other domains, were inactive. However, SK7 (residues 1-63), at a 100-fold molar excess concentration, greatly potentiated the activities of SK27 and SK36, by up to 50- and > 130-fold, respectively. These findings demonstrate that all of SK's three domains are essential for native-like SK activity. The central and C-terminal domains mediate plasminogen-binding and active site-generating functions, whereas the N-terminal domain mediates an activity-potentiating function.

Binding Sites↗
PubMed1996

Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance.

Streptococcus equisimilis streptokinase (SK) is a single-chain protein of 414 residues that is used extensively in the clinical treatment of acute myocardial infarction due to its ability to activate human plasminogen (Plg). The mechanism by which this occurs is poorly understood due to the lack of structural details concerning both molecules and their complex. We reported recently (Parrado J et al., 1996, Protein Sci 5:693-704) that SK is composed of three structural domains (A, B, and C) with a C-terminal tail that is relatively unstructured. Here, we report thermal unfolding experiments, monitored by CD and NMR, using samples of intact SK, five isolated SK fragments, and two two-chain noncovalent complexes between complementary fragments of the protein. These experiments have allowed the unfolding processes of specific domains of the protein to be monitored and their relative stabilities and interdomain interactions to be characterized. Results demonstrate that SK can exist in a number of partially unfolded states, in which individual domains of the protein behave as single cooperative units. Domain B unfolds cooperatively in the first thermal transition at approximately 46 degrees C and its stability is largely independent of the presence of the other domains. The high-temperature transition in intact SK (at approximately 63 degrees C) corresponds to the unfolding of both domains A and C. Thermal stability of domain C is significantly increased by its isolation from the rest of the chain. By contrast, cleavage of the Phe 63-Ala 64 peptide bond within domain A causes thermal destabilization of this domain. The two resulting domain portions (A1 and A2) adopt unstructured conformations when separated. A1 binds with high affinity to all fragments that contain the A2 portion, with a concomitant restoration of the native-like fold of domain A. This result demonstrates that the mechanism whereby A1 stimulates the plasminogen activator activities of complementary SK fragments is the reconstitution of the native-like structure of domain A.

Circular Dichroism↗
PubMed1996

Time-resolved biophysical methods in the study of protein folding.

Many of the biophysical techniques developed to characterize native proteins at equilibrium have now been adapted to the structural and thermodynamic characterization of transient intermediate populations during protein folding. Recent advances in these techniques, the use of novel methods of initiating refolding, and a convergence of theoretical and experimental approaches are leading to a detailed understanding of many aspects of the folding process.

Animals↗
PubMed1996

Collapse and cooperativity in protein folding.

The folding of a polypeptide chain is associated both with compactness and cooperativity within local and global regions of the protein structure, and with the formation of the native-like molecular architecture. Recent experiments shed light on these issues and their relationships to the pathways of protein folding.

Animals↗
PubMed1996

The concept of a random coil. Residual structure in peptides and denatured proteins.

Non-native states of proteins are of increasing interest because of their relevance to issues such as protein folding, translocation and stability. A framework for interpreting the wealth of experimental data for non-native states emerging from rapid advances in experimental techniques involves comparison with a "random coll' state, which possesses no structure except that inherent in the local interactions. We review here the concept of a random coil, from its global to its local properties. In particular, we focus on the description of a random coil in terms of statistical distributions in psi, phi space. We show that such a model, in combination with experimental data, provides insight into the structural properties of polypeptide chains and has significance for understanding protein folding and for molecular design.

Animals↗
PubMed1996

Native-like secondary structure in a peptide from the alpha-domain of hen lysozyme.

BACKGROUND: To gain insight into the local and nonlocal interactions that contribute to the stability of hen lysozyme, we have synthesized two peptides that together comprise the entire alpha-domain of the protein. One peptide (peptide 1-40) corresponds to the sequence that forms two alpha-helices, a loop region, and a small beta-sheet in the N-terminal region of the native protein. The other (peptide 84-129) makes up the C-terminal part of the alpha-domain and encompasses two alpha-helices and a 3(10) helix in the native protein. RESULTS: As judged by CD and a range of NMR parameters, peptide 1-40 has little secondary structure in aqueous solution and only a small number of local hydrophobic interactions, largely in the loop region. Peptide 84-129, by contrast, contains significant helical structure and is partially hydrophobically collapsed. More specifically, the region corresponding to helix C in native lysozyme is disordered, whereas regions corresponding to the D and 3(10) helices in the native protein are helical in this peptide. The structure in peptide 84-129 is at least partly stabilized by interactions between residues in the two helical regions, as suggested by further NMR analysis of three short peptides corresponding to the individual helices in this region of the native protein. CONCLUSIONS: Stabilization of structure in the sequence 1-40 appears to be facilitated predominantly by long-range interactions between this region and the sequence 84-129. In native lysozyme, the existence of two disulphide bonds between the N- and C-terminal halves of the alpha-domain is likely to be a major factor in their stabilization. The data show, however, that native-like secondary structure can be generated in the C-terminal portion of the alpha-domain by nonspecific and nonnative interactions within a partially collapsed state.

Amino Acid Sequence↗
PubMed1996

Investigation of protein folding by mass spectrometry.

Mass spectrometry is emerging as one of the most exciting new techniques being applied to studies of protein folding. Recent developments in soft ionization techniques enable intact proteins to be generated in the gas phase from aqueous solution, and fragmentation methods are providing a means of obtaining sequence-specific information. These techniques, particularly in combination with established methods such as NMR spectroscopy, allow the investigation of both covalent and noncovalent events that occur during refolding processes. One important type of application involves mass spectrometry in an analytical role, for example, in characterizing the products of oxidative refolding experiments. Mass spectrometry can also be used to reveal fundamentally new information about the conformational properties of folding intermediates, both in isolation and in complexes with molecular chaperones, through the exploitation of hydrogen-deuterium exchange phenomena. Of particular interest is the insight this approach can provide into the cooperativity of structure formation and the distribution of intermediates at different steps along folding pathways. In this paper we describe recent insights into protein folding resulting from the application of mass spectrometry and discuss the future potential of the method for studies in this area.

Chaperonin 60↗
PubMed1995

Different subdomains are most protected from hydrogen exchange in the molten globule and native states of human alpha-lactalbumin.

alpha-Lactalbumin (alpha-LA) is a two-domain, calcium-binding protein that forms one of the best studied molten globules. We present here amide hydrogen exchange studies of the molten globule formed by human alpha-LA at pH 2 and compare these results with a similar study of the native state at pH 6.3. The most persistent structure in the molten globule is localized in the helical domain, consistent with previous results. However, the helices most protected from hydrogen exchange in the molten globule are, in the native state, less protected from exchange than other regions of the protein. The molten globule appears to contain major elements of the native fold, but formation of the fully native state requires stabilization of structure around the calcium-binding site and domain interface.

Amino Acid Sequence↗
PubMed1995

Characterization of conformational preferences in a partly folded protein by heteronuclear NMR spectroscopy: assignment and secondary structure analysis of hen egg-white lysozyme in trifluoroethanol.

2D and 3D heteronuclear NMR methods have been used to characterize the structure of hen egg-white lysozyme in a partially folded state using uniformly 15N-labeled protein. This state is formed by the denaturation of the protein in 70% trifluoroethanol (TFE)/30% water (v/v) and is characterized by substantial helical secondary structure in the absence of extensive tertiary interactions. 15N-filtered 3D NOESY and TOCSY experiments have allowed the sequential assignment of resonances for all but 2 of the 126 main chain amide nitrogen atoms and of the majority of main and side chain proton resonances. The conformation of the polypeptide chain was characterized by analysis of the pattern of NOEs, H alpha chemical shift perturbations, 3J(HN, H alpha)-coupling constants, and hydrogen exchange protection. These NMR parameters are highly complementary and are consistent with a model for the TFE state in which six regions of the polypeptide chain are substantially ordered in helical conformations. The structure in different regions however, shows different levels of persistency. Five of the helices exhibit significant protection of amide hydrogens against exchange with solvent and are located in regions of the polypeptide which are helical in the native state. By contrast, helical structures of greater flexibility are observed both as extensions to the native-like helices and as a nonnative structure in the region of the molecule which forms the C-terminal part of the beta-sheet in the native state. No specific structural preferences are detected in regions corresponding to the long loop and to the N-terminal part of the beta-sheet of native lysozyme. A combination of local features of the polypeptide chain, including the predicted propensities of residues for helix formation and for their participation in N- and C-terminal helix capping interactions, allows the conformational behavior of the polypeptide chain of hen lysozyme to be rationalized for this partially folded state. The analysis implies that the nonnative structures are a result of interactions which are local to the polypeptide chain. These, and the highly persistent native-like structures, give insight into species which form early during folding.

Amides↗