The folding of an enzyme. V. H/2H exchange-nuclear magnetic resonance studies on the folding pathway of barnase: complementarity to and agreement with protein engineering studies.

Two major methods are currently being used to characterize transient intermediates during protein folding at the level of individual residues. Nuclear magnetic resonance (n.m.r.) measurements on the protection of peptide NH hydrogens against exchange with solvent during refolding can provide information about secondary structure formation. Protein engineering and kinetics can provide direct information about intramolecular interactions of protein side-chains and indirect evidence on secondary structure. These procedures have provided the most complete pictures so far about protein folding intermediates. Both methods have been applied to the characterization of an intermediate in the refolding of barnase. Although the two methods give complementary information, there are some regions of the protein where the methods overlap well. We show that, with one possible exception that is obscure, n.m.r. and protein engineering give identical results for those interactions that can be analysed by both methods. This suggests that these are valid approaches for the study of protein folding intermediates in the case of barnase and that the combination of the methods is a powerful analytical procedure. Information provided by n.m.r. data that is complementary to the protein engineering experiments is: (1) early formation of the C terminus of helix2; (2) early formation of helix3; (3) early formation of several beta-turns (46-49, 101-104 in loop5); and (5) partial formation of loop5. Confirmatory evidence of protein engineering data on the intermediate is: (1) helix1 is complete from residues 10 to 18; (2) the interactions between all beta-strands are present; (3) part of loop2 is not formed; (4) part of loop3 is formed; and (5) some specific tertiary interactions are not made. For some interactions the protein engineering and H/2H exchange methods overlap directly. The information obtained for direct overlap is self consistent.     

Probing protein structure by solvent perturbation of nuclear magnetic resonance spectra. Nuclear magnetic resonance spectral editing and topological mapping in proteins by paramagnetic relaxation filtering.

Soluble spin labels, which "bleach" the surface proton resonances of a protein to n.m.r. measurements, can provide useful information about protein conformation and dynamics. The use of the soluble nitroxide, TEMPOL, has been explored to show the correlation of the paramagnetic perturbations of protein two-dimensional n.m.r. data with proton exposure to the free radical in hen egg-white lysozyme. The results demonstrate that the nitroxide approaches the protein randomly, and that the extent of the observed paramagnetic effects reflects the native folding pattern of the protein. A correlation of spectral simplification with the known tertiary structure establishes the feasibility of new strategies for topological mapping of surface and buried protons of the protein. Application to the elucidation of protein structure and to the study of dynamical processes is discussed.     

Substituent distribution on O,N-carboxymethylchitosans by 1H and 13C n.m.r.

The use of the n.m.r. method in the investigation of chitosan carboxymethylation was evaluated. It seems to be the most effective technique to determine concurrently the degree and the position of substitution of the carboxymethylated chitosan derivatives. The 13C-n.m.r., by the DEPT method, 1H-1H and 1H-13C-n.m.r. correlations give much valuable information from the chemical shifts of the complex carboxymethylchitosan spectra. The relative reactivity of the functional groups of chitosan towards carboxymethylation was also determined assuming a higher reactivity of the C-6 position.     

Formation of n.m.r.-invisible ADP during renal ischaemia in rats.

Measurement of the adenine nucleotide and inorganic phosphate content of normoxic and ischaemic kidney in vivo has been made, comparing enzymic assay (after freeze-clamping and acid extraction) with quantification by 31P-n.m.r. Both methods give similar results for ATP, and n.m.r. quantification of Pi gives a value 25-50% of that obtained by enzymic assay. ADP, which is largely invisible to n.m.r. in the normoxic kidney, remains invisible during ischaemia despite a 2-3 fold rise in enzymically assayed ADP. N.m.r. and enzymic assay of the acid extracts give similar values for all metabolites measured. The question of ADP binding in the kidney is discussed, as are the implications for the metabolic regulation of ADP-dependent reactions.     

Protein structure by solid state nuclear magnetic resonance. Residues 40 to 45 of bacteriophage fd coat protein

The three-dimensional structure of part of the coat protein in the filamentous bacteriophage fd is described by nuclear magnetic resonance (n.m.r.). Residues 40 to 45 are in a somewhat distorted alpha-helix. This n.m.r. approach for determining protein structure relies on the spectral manifestations of chemical shift and heteronuclear dipolar couplings in a symmetrical assembly of protein subunits oriented parallel to the applied magnetic field. The angles between individual peptide linkages and the filament axis of the virion constitute the basic source of structural information. These angles are directly related to x, y, z co-ordinates for describing the protein structure.     

An evaluation of the combined use of nuclear magnetic resonance and distance geometry for the determination of protein conformations in solution

An evaluation of the potential of nuclear magnetic resonance (n.m.r.) as a means of determining polypeptide conformation in solution is performed with the aid of a new distance geometry program which is capable of computing complete spatial structures for small proteins from n.m.r. data. Ten sets of geometric constraints which simulate the results available from n.m.r. experiments of varying precision and completeness were extracted from the crystal structure of the basic pancreatic trypsin inhibitor, and conformers consistent with these constraints were computed. Comparison of these computed structures with each other and with the original crystal structure shows that it is possible to determine the global conformation of a polypeptide chain from the distance constraints which are available from n.m.r. experiments. The results obtained with the different data sets also provide a standard by which the quality of protein structures computed from n.m.r. data can be evaluated when no crystal structure is available, and indicate directions in which n.m.r. experiments for protein structure determination could be further improved.     

Fluorescent probes in membrane studies.

A number of spectroscopic techniques are suitable for studying biological membranes. Of these, fluorescence has the sensitivity and time resolution for following membrane events associated with nerve excitation. In this paper, the nature of the information derived from measurements of the fluorescence properties of externally introduced chromophores in membranes is examined. In particular, the locations of various probes are described on the basis of nuclear magnetic resonance (n.m.r.) experiments in model situations. Then the motional characteristics of the probe molecules (rotation and diffusion) are discussed. Finally experiments designed to relate the detailed observations that can be made in lipid bilayers using n.m.r. and fuorescence measurements to those (more limited in nature) that can be made in membranes are described.     

The structure and function of protein modules.

Analysis of protein sequences shows that many proteins in multicellular organisms have evolved by a process of exon shuffling, deletion and duplication. These exons often correspond to autonomously folding protein modules. Many extracellular enzymes have this modular structure; for example, serine proteases involved in blood-clotting, fibrinolysis and complement. The main role of these modules is to confer specificity by protein-protein interactions. Lack of structural information about such proteins has required a new strategy for studying the structure and function of protein modules. The strategy involves the production of individual modules by protein expression techniques, determination of their structure by high resolution nuclear magnetic resonance and definition of functional patches on the modules by site-directed mutagenesis and biological assays. The structures of the growth factor module, the fibronectin type 1 module and the complement module are briefly described. The possible functional roles of modules in various proteins, including the enzymes factor IX and tissue plasminogen activator, are discussed.     

Modelling the polypeptide backbone with 'spare parts' from known protein structures

An automatic procedure for building a protein polyalanine backbone from C alpha positions and 'spare parts' retrieved from a data base of 66 high-resolution protein structures is described. Protein backbones are constructed from overlapping fragments of variable length, which allows the backbone of regular secondary structure elements to be built in one block. The procedure is shown to yield backbones which compare very favourably with those from highly refined X-ray structures (r.m.s. deviation between generated and crystal structures less than 1A). The method is furthermore quite insensitive to experimental errors in C alpha positions as well as to the size of the data base, and is seen to yield valuable insight into the relationships between sequence and 3-D structure: one example on triose phosphate isomerase, a beta-barrel protein, shows that beta alpha loops can be considered as structurally more uncommon than alpha beta loops. The 'spare parts' approach is also found to be useful for general-purpose modelling of local structural changes produced by insertion or deletion of residues. It should, however, be used with caution. Crude selection criteria based solely on fragment length and geometric fit to the loop base regions yield realistic backbones in about two-thirds of the test cases (r.m.s. deviations from refined crystal structure approximately 1A). In the remaining cases, sequence information, in particular the presence of glycine residues which tend to adopt more unusual backbone conformations, must be considered to obtain comparable results.     

N.m.r. imaging of intact biological systems

Internal images of structured objects may be obtained with n.m.r. by labelling component parts with different magnetic field strengths and therefore recognizably different n.m.r. frequencies. A linear field gradient generates a one-dimensional projection of nuclear density and a variety of techniques are employed to manipulate this one-dimensional probe to yield internal images in two and three dimensions. In the past few years, n.m.r. imaging, sometimes also called zeugmatography or spin mapping, has been applied progressively to provide proton images of small phantoms, fruit, vegetables and small animals, and finally to in vivo imaging of the human body; it promises to provide a valuable means of interior investigation of intact biological systems generally. For medical imaging the method is non-invasive, does not use ionizing radiations, appears to be without hazard and penetrates bony cavities without attenuation. Furthermore, other n.m.r. parameters, for example, relaxation times and fluid flow, may also be mapped; there is evidence that the relaxation times from tumours are significantly longer than those from corresponding normal tissue. Effort to date has mostly been concentrated on proton n.m.r., but some work has been done with other nuclei. Three examples are shown of n.m.r. images of intact biological systems: a fruit, an animal and a human system. The discussion includes the quantitative nature of the images, tissue discrimination, the relation between the resolution in the image and image acquisition time, attenuation and phase shift of the r.f. field in the biological tissue, and magnets suitable for n.m.r. imaging. In principle, all conventional n.m.r. techniques can be combined with n.m.r. methods in order to investigate heterogeneous systems. Overhauser imaging is briefly discussed.     

N.m.r. studies of red cells

Recent n.m.r. studies of intact red cells are described. With 1H n.m.r. the normal high resolution spectra of red cells, even at high fields, are relatively uninformative because the very large number of resonances from the cells merge into a broad envelope. If a simple 90-tau-180 degree spin echo pulse sequence is used, however, many resonances can all be resolved. These include signals from haemoglobin histidines, glutathione, lactate and pyruvate. 13C and 31P signals have also been seen with a spectrometer converted to observe these nuclei essentially simultaneously. N.m.r. is well suited to monitor the time course of events after a perturbation of the cell system. Lactate increase, glutathione recovery after oxidation and alkylation of glutathione by iodoacetate can all be observed directly in red cell suspensions by means of 1H spin echo n.m.r. This method has also been used to measure isotope exchange (1H-2H) of lactate and of pyruvate at both the C-3 and the C-2 positions, and some of these exchange rates can be interpreted in terms of the activity of specific enzymes in the cells. 1H spin echo n.m.r. has also been used to obtain information about the transport rates of small molecules into cells. By means of the 13C/31P spectrometer and [13C-1] glucose, the 13C enrichment of 2,3-diphosphoglycerate (2,3-DPG) can be monitored at the same time as the levels of 2,3-DPG, ATP and inorganic phosphate are observed by 31P n.m.r.     

Sequence-specific 1H-n.m.r. assignments and peptide backbone conformation in rat epidermal growth factor.

The solution conformation of rat epidermal growth factor (EGF) has been investigated by proton n.m.r. techniques. Two-dimensional proton n.m.r. experiments have allowed sequential resonance assignments to be made for most protons. On the basis of these assignments, two regions of anti-parallel beta-sheet structure have been derived from the n.m.r. data. A beta-sheet segment running from about V19 to V23 (capital letters refer to amino acids in the single-letter notation) is folded onto a beta-sheet segment running from R28 to N32 and joined by a chain reversal from E24 to D27. A second region involves a beta-turn from V34 to Y37, which starts a short beta-sheet up to G39, followed by a chain reversal up to Q43, which leads to folding of the C-terminal beta-sheet segment, i.e. H44-R45, running antiparallel to the short Y37 beta-sheet segment. The N-terminal segment up to G18 exists in a multiple bend conformation and is folded on to the V29-V23/R28-N32 beta-sheet such that Y10, Y13, Y22 and Y29 are proximal to each other. Structural comparison of rat, murine and human EGFs indicates a number of highly conserved structural features common to at least these species of EGF.     

Ionization of tyrosine and lysine residues in native and modified horse cytochrome c.

1H-n.m.r. and 13C-n.m.r. spectroscopy of horse cytochrome c and 1H-n.m.r. spectroscopy of the lysine-modified proteins N epsilon-acetimidyl-, N epsilon-amidino-, N epsilon-trifluoroacetyl- and N epsilon-maleyl-cytochrome c have shown that, although the lysine modifications do not greatly perturb the protein structure at pH7 and 27 degrees C, at higher temperature or at alkaline pH some parts of the structure are markedly perturbed. At pH7 and 27 degrees C the region of the protein about Ile-57 is affected in all the modified proteins, though not all to the same degree. N epsilon-Maleylation most seriously affects the protein structure, and the fully maleylated protein is readily unfolded. At 27 degrees C all four of the tyrosine residues of native horse cytochrome c have pKa values above 11, but in N epsilon-acetimidyl-cytochrome c the pKa of one tyrosine residue is 10.2.     

Secondary structure prediction and determination of proteins--a review

The rapid increase in sequence data in combination with a greater understanding of the forces regulating protein structure has been the impetus for an upsurge in the development of theoretical prediction methods. These methods have afforded protein chemists the ability to identify and quantify the various secondary structures along the protein chain. Concurrently, various physico-chemical techniques have been developed such as nuclear Overhauser enhancement n.m.r. and laser Raman spectroscopy. In addition, traditional methods such as infrared and circular dichroism spectroscopy have been refined. Although both predictive and physico-chemical techniques are limited in the types of secondary structure they are capable of determining, they have provided valuable information with regards to protein folding and topology in the absence of X-ray data, and have formed the basis for the development of improved methods for secondary structure determination. This paper reviews some of the predictive and physico-chemical methods presently used to determine protein secondary structure.     

Dihydrofolate reductase. 1H resonance assignments and coenzyme-induced conformational changes

Lactobacillus casei dihydrofolate reductase has been studied in solution by one and two-dimensional 1H nuclear magnetic resonance (n.m.r.) spectroscopy at 500 MHz. By using a combination of n.m.r. methods in conjunction with the crystal structure of the enzyme-methotrexate-NADPH complex, resonances have been assigned for 32 of the 162 residues of the enzyme. These are widely distributed throughout the structure of the protein, and include all the histidine and tyrosine residues, as well as several valine, leucine, isoleucine and phenylalanine residues. The assignments have been made for the enzyme-methotrexate and enzyme-methotrexate-NADP+ complexes as well as the enzyme-methotrexate-NADPH complex. Comparison of assigned resonances in the spectra of the three complexes has permitted a preliminary assessment of structural differences between them. The beta-sheet "core" of the protein is unaffected by coenzyme binding, but two regions of the structure that undergo coenzyme-induced conformation changes have been identified. These are the loop comprising residues 13 to 23, and alpha-helix C (residues 42 to 49).     

Determination of the nuclear magnetic resonance solution structure of the DNA-binding domain (residues 1 to 69) of the 434 repressor and comparison with the X-ray crystal structure.

The DNA-binding domain of the phage 434 repressor consisting of N-terminal residues 1 to 69 (434 repressor(1-69)), was expressed in Escherichia coli with natural isotope abundance, uniform 15N-labeling and biosynthetically directed fractional 13C-labeling in extent of about 10%. With these protein preparations the three-dimensional structure was determined in solution. The techniques used were nuclear magnetic resonance (n.m.r.) spectroscopy for the collection of conformational constraints, calculation of the protein structure from the n.m.r. data with the program DIANA and structure refinements by restrained energy minimization with a modified version of the program AMBER. A group of 20 conformers characterizes a well-defined structure for residues 1 to 63, with an average of 0-6 A for the root-mean-square deviations (RMSD) calculated for the backbone atoms of the individual conformers relative to the mean co-ordinates. The spatial structure of C-terminal residues 64 to 69 is not defined by the n.m.r. data. The molecular architecture of the 434 repressor(1-69) in solution includes five alpha-helices extending from residues 2 to 13, 17 to 24, 28 to 35, 45 to 52 and 56 to 60, which enclose a well-defined hydrophobic core. The n.m.r. structure is closely similar to the reported crystal structure of the 434 repressor(1-69), with an RMSD value of 1.1 A for the backbone atoms of residues 1 to 63. Small differences between the two structures in regions of the first helix and the loop between helices 3 and 4 were analyzed relative to possible correlations with protein-protein contacts in the crystal lattice and the different milieus of pH and ionic strength in the crystals and n.m.r. samples. Further systematic comparisons of local conformational features indicated that there are correlations between amino acid types, local precision of the structure determination by both techniques and local differences between the structures in the crystals and in solution. Overall, hydrophobic residues are most precisely characterized and agree most closely in the two environments.     

The Wellcome Foundation lecture, 1984. Nuclear magnetic resonance imaging in medicine: medical and biological applications and problems

From early biological work and the first T1 nuclear magnetic resonance (n.m.r.) animal image in 1974, whole-body patient images, by using a two-dimensional Fourier transform method were achieved in Aberdeen in 1980 with a 0.04 T vertical resistive magnet. Different pulse sequences produce images dependent by different amounts on proton density, T1 and T2, and for clinical work it is advantageous to use more than one pulse sequence to image pathology. The slow improvement of spatial resolution with increasing standing magnetic field strength is discussed and information on the T1 and T2 contrast dependence is reviewed: it suggests that the gains from high fields may be less than believed hitherto. Electrocardiogram gating can be used to produce moving images of the beating heart; blood flow can be imaged and surface radiofrequency coils are used for improved detail. N.m.r. imaging has considerable potential for studying response to therapy; mental states and dementia; tissue generation; discriminating body fat and body fluids. Other nuclei such as 23Na can be imaged and the potential to image fluorine-labelled pharmaceuticals could be very exciting; n.m.r. contrast agents are now being developed. Images formed from T1 values measured for each pixel are very useful for diagnosis, but the numerical values themselves are less valuable for distinctive pathological identification. With 15 companies manufacturing n.m.r. imagers and over 200 in use in hospitals, the technique is rapidly becoming established in diagnostic clinical practice and some typical uses are presented.     

From fold to function

A number of recent advances have been made in deriving function information from protein structure. A fold relationship to an already characterized protein will often allow general information about function to be deduced. More detailed information can be obtained using sequence relationships to already studied proteins. Methods of deducing function directly from structure, without the use of evolutionary relationships, are developing rapidly. All such methods may be used with models of protein structure, rather than with experimentally determined ones, but model accuracy imposes limitations. The rapid expansion of the structural genomics field has created a new urgency for improved methods of structure-based annotation of function.     

Prediction of protein folding pathways.

Recent 1H nuclear magnetic resonance (n.m.r.) hydrogen exchange experiments on five different proteins have delineated the secondary structures formed in trapped, partially folded intermediates. The early forming structural elements are identifiable through a technique described in this work to predict folding pathways. The method assumes that the sequential selection of structural fragments such as alpha-helices and beta-strands involved in the folding process is founded upon the maximal burial of solvent accessible surface from both the formation of internal structure and substructure association. The substructural elements were defined objectively by major changes in main-chain direction. The predicted folding pathways are in complete correspondence with the n.m.r. results in that the formed structural fragments found in the folding intermediates are those predicted earliest in the pathways. The technique was also applied to proteins of known tertiary structure and with fold similar to one of the five proteins examined by 1H n.m.r. The pathways for these structures also showed general consistency with the n.m.r. observations, suggesting conservation of a secondary structural framework or molten globule about which folding nucleates and proceeds.