Structure determination of membrane-associated proteins from nuclear magnetic resonance data

This Review covers the delineation and optimization of protein-lipid systems for study using solution-state NMR spectroscopy. The first half presents the necessary background for a membrane protein biochemist to initiate collaboration with an NMR spectroscopist. The second half provides guidelines for the spectroscopist on data collection, analysis for obtaining conformational information, and structure generation and assessment. Although the emphasis is on the study of peptides in detergent micelles, methods are outlined for larger membrane-associated proteins and for use of other solubilizing agents.     

Elucidation of glycolipid structure by proton nuclear magnetic resonance spectroscopy

The primary structure of the oligosaccharide moiety of a glycosphingolipid can be elucidated by employing high-field proton nuclear magnetic resonance (NMR) spectroscopy. Information with respect to the composition and configuration of its sugar residues, and the sequence and linkage sites of the oligosaccharide chain can be obtained by employing a variety of one- and two-dimensional techniques. The latter include both scalar and dipolar correlated two-dimensional NMR spectroscopy. These techniques are also useful in establishing the solution conformation (secondary structure) of the oligosaccharide moiety. Examples in utilizing these techniques in elucidating the primary and secondary structures of glycolipids are presented.     

Mobile domains in ribosomes revealed by proton nuclear magnetic resonance

Ribosomes and subunits from eukaryotic and prokaryotic sources were studied by high-resolution proton magnetic-resonance spectroscopy. If all ribosomal components are firmly bound within the particle, then only broad spectra would be expected. However, relatively sharp resonances were found both in ribosomal subunits and in 70 or 80 S ribosomes. The regions of these mobile protein domains have been partially assigned in Escherichia coli ribosomes. Large and small ribosomal subunits were treated to remove selectively proteins L7/12 and S1, respectively. Sharp proton magnetic resonance spectra were not observed for the stripped large subunit showing that proteins L7/12 comprise the flexible protein region and that there is little other flexibility in the stripped subunit. Complete removal of S1 from the small subunit greatly reduced but did not abolish the sharp protein resonance peaks, indicating that protein S1 contains a substantial flexible component but that other flexible components remain in the stripped small subunit. Evidence for generality of these features of ribosome organization is provided by similar studies on ribosomes from eukaryotic sources.     

Biotransformations monitored in situ by proton nuclear magnetic resonance spectroscopy

One-dimensional Fourier-transform proton nuclear magnetic resonance (1H-NMR) spectroscopy can be used to study biotransformations in situ, in vivo and in aqua (1H2O). Although an insensitive method, it rapidly provides solution-structural information of mixtures of diverse compounds that are used and formed during enzymic reactions and culture fermentations; the samples do not require any physical or chemical processing for analysis. The absolute stereochemistry of some reactions can also be determined, and assessments of metabolic fluxes made. This technique, with appropriate modifications, is of obvious value for on-line assessments of industrial fermentation processes.     

Sequential resonance assignments as a basis for determination of spatial protein structures by high resolution proton nuclear magnetic resonance

A general scheme is proposed for the determination of spatial protein structures by proton nuclear magnetic resonance. The scheme relies on experimental observation by two-dimensional nuclear magnetic resonance techniques of complete throughbond and through-space proton-proton connectivity maps. These are used to obtain sequential resonance assignments for the individual residues in the amino acid sequence and to characterize the spatial polypeptide structure by a tight network of semi-quantitative, intramolecular distance constraints.     

Partial characterization of dermatan sulfate by proton nuclear magnetic resonance spectroscopy

The degree of galactosamine N-acetylation, iduronic acid composition, and total uronic acid/hexosamine ratios of the three dermatan sulfates of human skin, DS18, DS28, and DS35 (M. O. Longas et al. (1987) Carbohydr. Res. 159, 127-136), were determined by Fourier transform, proton nuclear magnetic resonance (FT 1H NMR) spectroscopy. Analysis of DS of varying ages was conducted at 400 MHz and 60 degrees C. Chemical shifts for H-1, H-2, H-4, and H-5 of L-IdUA were independent of those for the respective protons of D-GalNAc and D-GlcUA. The resonance intensities of H-1 and acetamido methyl protons of D-GalNac did not display the expected 1:3 ratios. Therefore, their integration values were employed to estimate the percentage N-acetylation (N-CH3/3 H-1) which was corroborated chemically. The L-IdUA content, relative to total uronic acid, was calculated from signal intensities of H-1 of L-IdUA and D-GlcUA and ascertained by quantitative chemical methods. Total uronic acid/hexosamine ratios were determined from both 1H NMR spectroscopy and chemical analyses. The data show the following N-acetylation (N-CH3/3 H-1) of galactosamine in DS:DS18, 61-72% between 17 and 60 years, unaffected by senescence; DS28, 78-86% with no age-related trend; DS35, 101% at 19 years. Furthermore, in all ages investigated, the percentage (wt/wt) L-IdUA relative to total uronic acid was 42-44% for DS18 and 37-40% for DS28. At age 19 years, DS35 had a 29% (wt/wt) L-IdUA. The total uronic acid/hexosamine ratios for DS18 and DS28 varied from 1.40:1.0 to 1.70:1.0 irrespective of age.     

A proton nuclear magnetic resonance assignment and secondary structure determination of recombinant human thioredoxin

Two-dimensional 1H NMR spectroscopy has been applied to a structural analysis of the reduced form of a recombinant human thioredoxin, a ubiquitous dithiol oxidoreductase recently isolated from an immunocompetent lymphoblastoid cell line. The sequential assignment of the spectrum, including all proline residues, has been accomplished by using experiments to demonstrate through-bond and through-space connectivities. The secondary structure has been determined by a qualitative interpretation of nuclear Overhauser effects, NH exchange data, and 3JHN alpha coupling constants. The secondary structure was found to be similar to that of the X-ray structure of Escherichia coli thioredoxin, consisting of a mixed five-stranded beta-sheet surrounded by four alpha-helices. The assignment and structural characterization of human thioredoxin was facilitated by the increased resolution and sensitivity afforded by a magnetic field strength of 600 MHz and required the use of two temperatures and two pH conditions to resolve ambiguities caused by a duplication of resonances. This duplication, extending from Phe-41 to Val-59, and including Lys-3-Ile-5, Val-24, Val-25, Asn-39, and Ile-101-Glu-103, appears to be due to heterogeneity arising from the presence or absence of the N-terminal methionine.     

Sugar ring conformations of guanine nucleosides by proton nuclear magnetic resonance

Proton NMR studies at 250 MHz showed that ribofuranosyl and 2-deoxyribofuranosyl derivatives of N2-(p-n-butylphenyl)guanine (BuPG) favored the C2'-endo (S) sugar pucker and the gg exocyclic group rotamer, although less so than guanosine and 2'-deoxyguanosine themselves. The correlation calculated between C3'-endo (N) and gg conformational states in these compounds may result from destabilization of syn glycosidic bond conformers by the bulky N2 substituent. Results for a bis(ribofuranosyl) derivative of BuPG showed a strong correlation between N and gg states in both sugar rings, suggesting that both rings are anti and are stabilized by intramolecular hydrogen bonding between C3'-O and H8.     

High-resolution nuclear magnetic resonance studies of proteins

The combination of advanced high-resolution nuclear magnetic resonance (NMR) techniques with high-pressure capability represents a powerful experimental tool in studies of protein folding. This review is organized as follows: after a general introduction of high-pressure, high-resolution NMR spectroscopy of proteins, the experimental part deals with instrumentation. The main section of the review is devoted to NMR studies of reversible pressure unfolding of proteins with special emphasis on pressure-assisted cold denaturation and the detection of folding intermediates. Recent studies investigating local perturbations in proteins and the experiments following the effects of point mutations on pressure stability of proteins are also discussed. Ribonuclease A, lysozyme, ubiquitin, apomyoglobin, alpha-lactalbumin and troponin C were the model proteins investigated.     

Structural investigations of chromatin core protein by nuclear magnetic resonance

A complex derived from chromatin containing one molecule of each of histones H2A, H2B, H3, and H4, termed core protein, was studied by 13C and 1H nuclear magnetic resonance. 13C line widths, when analyzed and compared with those of native and thermally unfolded representative globular proteins, showed that regions of the core protein possess considerable mobility. Studies of Calpha and Cbeta line widths, and Calpha spin-spin relaxation times, show that this mobility arises from sections of random-coil polypeptide. It is argued that these regions are N-terminal "tails", attached to C-terminal globular polypeptides. The 270-MHz 1H nuclear magnetic resonance spectrum shows numerous ring current shifted resonances, indicating that the C-terminal globular domain has a precise tertiary structure. The globular domain most likely forms the histone "core" of the chromatin monomer particle, whilst the basic tails probably wind around the grooves of the double helix, enabling the basic side chains to interact with the DNA phosphate groups. Some biological implications of this model are considered.     

High-resolution proton nuclear magnetic resonance studies of human gastrin

High-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz has been used to study the behavior of human gastrin in aqueous solution. A large number of resonances have been assigned by analysis of one- and two-dimensional NMR spectra and the effects of pH and by comparison with the spectrum of des-less than Glu1-gastrin. In gastrin, the ratio of cis to trans conformations around the Gly-2 to Pro-3 peptide bond is 3:7. This is reflected in splitting of the resonances of several neighboring residues and of a residue distant in the sequence, Tyr-12. The pKa of Tyr-12 is 10.7. Sulfation of this residue perturbs the resonances of Tyr-12 and Gly-13 but has very little effect on the rest of the spectrum. A study of the temperature dependence shows that several perturbed resonances move toward their expected positions as the temperature is raised but with a linear dependence on temperature, consistent with a redistribution of populations among accessible local conformations rather than a cooperative conformational change. Addition of Na+ or Ca2+ causes only minor changes in the spectrum. The paramagnetic metal ion Co2+ produces a number of spectral changes, reflecting strong binding to at least one site involving the Glu residues and weaker binding to Asp-16.     

A proton nuclear magnetic resonance study of sulfmyoglobin cyanide

The proton nuclear magnetic resonance spectrum of sulfmyoglobin cyanide was studied at 400 MHz. The position of a methyl-group resonance at low field is consistent with a chlorin-like structure for the prosthetic group. The proton NMR spectrum of the cyanide derivative of the purified prosthetic group which decomposes upon extraction from the protein was found to be the same as that of the cyanide derivative of the prosthetic group extracted from myoglobin and a sample prepared from hemin-Cl.     

High resolution proton nuclear magnetic resonance spectroscopy of lactoperoxidase

The first high resolution proton nuclear magnetic resonance spectra are reported for the native ferric and ferric cyano complexes of bovine lactoperoxidase. The spectrum of the native species exhibits broad heme signals in a far downfield region characteristic of the high-spin ferric state. The low-spin cyano complex yields a proton nuclear magnetic resonance spectrum with signals as far as 68.5 ppm downfield and as far as -28 ppm upfield of the tetramethylsilane reference. These peak positions are anomalous with respect to those seen only as far as 35 ppm downfield in other cyano hemoprotein complexes. An extreme asymmetry in the unpaired spin delocalization pattern of the iron porphyrin is suggested. The unusual proton nuclear magnetic resonance properties parallel distinctive optical spectral properties and the exceptional resistance to heme displacement from the enzyme. Lactoperoxidase utilized in these studies was isolated from raw milk and purified by an improved, rapid chromatographic procedure.     

Amino acid analysis of angiotensin I by proton nuclear magnetic resonance spectroscopy

The chemical shifts of the isoleucine and histidine protons of angiotensin I were assigned and the chemical shifts of the protons of the other amino acids in the peptide were confirmed at a field strength of 400 MHz. These chemical shift assignments were used to determine the amino acid composition of angiotensin I. These data were then compared to the amino acid composition which was determined by chromatographic analysis of the peptide hydrolysate. The results obtained by the chromatographic method were similar to those obtained by the NMR method. The standard deviations of the results were similar, indicating that these methods are equally precise. The major advantages of the NMR method are that it permits the recovery of the peptide after completion of the analysis and improves the quantitation of amino acids which are either partially destroyed by the hydrolysis procedure or require special derivatization methods for detection and quantitation.     

Epidermal growth factor from the mouse. Structural characterization by proton nuclear magnetic resonance and nuclear overhauser experiments at 500 MHz

Mouse epidermal growth factor (mEGF), a protein hormone effector molecule that regulates cellular development and division, has been investigated by using proton nuclear magnetic resonance techniques at 500 MHz. Well-resolved downfield aromatic and alpha-CH proton resonances (5.0-8.0 ppm) and an upfield ring current shifted isoleucine delta-methyl resonance (approximately 0.5 ppm) have been examined by using principally nuclear Overhauser methods. The data are analyzed in terms of model building based on the predictive Chou-Fasman secondary structure algorithm applied to mEGF [Holladay, L. A., Savage, C. R., Cohen, S., & Puett, D. (1976) Biochemistry 15, 2624-2633], which suggests the existence of some beta-structure and little or no alpha-helicity. Proximity relationships derived from nuclear Overhauser data among Tyr-3, -10, and -13, His-22, and Ile-23 allow refinement of some aspects of the predicted secondary structure and render additional information on how the protein backbone in mEGF is folded (i.e., tertiary structure). Nuclear Overhauser effects (NOEs) from irradiation of several alpha-CH proton resonances give evidence for tiered beta-sheet structure in mEGF. Such proximity relationships derived from NOE data place stringent limitations on possible models for the molecule. pH titration data demonstrate a His-22 pKa of 7.1, indicating either a salt bridge or hydrogen-bond formation between His-22 and another residue. The His-22 pKa is also reflected in the chemical shift changes of several other resonances as a function of pH. Nuclear Overhauser methods, used to differentiate direct (protonation) and indirect (conformation) effects on the chemical shift changes in the spectra of mEGF by varying the pH, yield evidence for a pH-induced conformational transition in the protein hormone associated with the breaking of the His-22 salt bridge or hydrogen bond.