Low-temperature solid-state 13C NMR studies of the retinal chromophore in rhodopsin

Magic angle sample spinning (MASS) 13C NMR spectra have been obtained of bovine rhodopsin regenerated with retinal prosthetic groups isotopically enriched with 13C at C-5 and C-14. In order to observe the 13C retinal chromophore resonances, it was necessary to employ low temperatures (-15-----35 degrees C) to restrict rotational diffusion of the protein. The isotropic chemical shift and principal values of the chemical shift tensor of the 13C-5 label indicate that the retinal chromophore is in the twisted 6-s-cis conformation in rhodopsin, in contrast to the planar 6-s-trans conformation found in bacteriorhodopsin. The 13C-14 isotropic shift and shift tensor principal values show that the Schiff base C = N bond is anti. Furthermore, the 13C-14 chemical shift (121.2 ppm) is within the range of values (120-123 ppm) exhibited by protonated (C = N anti) Schiff base model compounds, indicating that the C = N linkage is protonated. Our results are discussed with regard to the mechanism of wavelength regulation in rhodopsin.     

Detection of nucleotide binding to Na,K-ATPase in an aqueous membrane suspension by 13C cross-polarization magic-angle spinning NMR spectroscopy

Binding of uniformly (13)C labelled ATP to Na,K-ATPase was studied by (13)C cross-polarization magic-angle spinning (CP-MAS) NMR. In the presence of 30 mM Na(+) , and with sample- and time-averaging, NMR spectra obtained at 4 degrees C exhibited several resonances for the bound nucleotide. Chemical shifts suggested that site-specific changes in the micro-environment or conformation of the nucleotide occurred in the high affinity binding site. These experiments permit further studies of nucleotide dynamics, structure and binding under physiologically relevant conditions.     

Probing the gel to liquid-crystalline phase transition and relevant conformation changes in liposomes by 13C magic-angle spinning NMR spectroscopy

A straightforward way to visualize gel to liquid-crystalline phase transition in phospholipid membranes is presented by using ¹³C magic-angle spinning NMR. The changes in the ¹³C isotropic chemical shifts with increasing temperature are shown to be a sensitive probe of the main thermotropic phase transition related to lipid hydrocarbon chain dynamics and relevant conformational changes. The average value of the energy difference between trans and gauche states in the central C4–11 fragment of the DMPC acyl chain was estimated to be 4.02 ± 0.2 kJ mol^− 1 in the liquid crystalline phase. The reported spectral features will be useful in ¹³C solid state NMR studies for direct monitoring of the effective lipid chain melting allowing rapid uniaxial rotation of membrane proteins in the biologically relevant liquid-crystalline phase.     

Signal assignments and chemical-shift structural analysis of uniformly 13C, 15N-labeled peptide, mastoparan-X, by multidimensional solid-state NMR under magic-angle spinning

Carbon-13 and nitrogen-15 signals of fully isotope-labeled 15-residue peptide, glycinated mastoparan-X, in a solid state were assigned by two- and three-dimensional NMR experiments under magic-angle spinning conditions. Intra-residue spin connectivities were obtained with multidimensional correlation experiments for C'-C(alpha)-C(beta) and N-C(alpha)-C(beta). Sequence specific assignments were performed with inter-residue C(alpha)-C(alpha) and N-C(alpha)C(beta) correlation experiments. Pulse sequences for these experiments have mixing periods under recoupled zero- and double-quantum (13)C-(13)C and (15)N-(13)C dipolar interactions. These correlation spectra allowed the complete assignments of (13)C and (15)N backbone and (13)C(beta) signals. Chemical shift analysis of the (13)C and (15)N signals based on empirical and quantum chemical databases for proteins indicated that the backbone between residues 3 and 14 forms alpha-helix and residue 2 has extended conformation in the solid state. This structure was compared with the G-protein- and membrane-bound structures of mastoparan-X.     

13C NMR studies of bacterial fermentations.

We describe the experimental methods used and the constraints that apply in studies of anaerobic cell metabolism by 13C NMR. We review some of the results of our recent work in this area. Clostridium neopropionicum was shown to ferment ethanol into propionate by the acrylate, non-randomizing pathway. The same metabolic route accounts for 50% of the propionate formed in the complex ecosystem that inhabits the pig's large intestine. The rest is formed via the randomizing succinate pathway. Reductive, hydrogenotrophic acetogenesis was studied in several ecosystems. Although it is usually overshadowed by methanogenesis in the competition for hydrogen, it may become an efficient electron sink when methane biosynthesis is blocked by a specific inhibitor.     

Solid-state magic-angle spinning NMR of membrane proteins and protein-ligand interactions

Structural biology is developing into a universal tool for visualizing biological processes in space and time at atomic resolution. The field has been built by established methodology like X-ray crystallography, electron microscopy and solution NMR and is now incorporating new techniques, such as small-angle X-ray scattering, electron tomography, magic-angle-spinning solid-state NMR and femtosecond X-ray protein nanocrystallography. These new techniques all seek to investigate non-crystalline, native-like biological material. Solid-state NMR is a relatively young technique that has just proven its capabilities for de novo structure determination of model proteins. Further developments promise great potential for investigations on functional biological systems such as membrane-integrated receptors and channels, and macromolecular complexes attached to cytoskeletal proteins. Here, we review the development and applications of solid-state NMR from the first proof-of-principle investigations to mature structure determination projects, including membrane proteins. We describe the development of the methodology by looking at examples in detail and provide an outlook towards future 'big' projects.     

X-ray and 13C solid-state NMR studies of N-benzoyl-phenylalanine.

A crystalline sample of N-benzoyl-DL-phenylalanine 1 and a polycrystalline sample of N-benzoyl-L-phenylalanine 2 were studied using 13C high-resolution solid-state NMR spectroscopy. The X-ray structure of the DL form was established. Sample 1 crystallizes in a monoclinic form with a P21/c space group, a=11.338(1) A, b=9.185(1) A, c=14.096(2) A, beta=107.53(3) degrees, V=1400(3) A3, Z=4 and R=0.053. The principal elements of the 13C chemical shift tensors deltaii for 1 and 2, selectively 13C (99%) labeled at the carboxyl groups were calculated. On the basis of 13C (delta)ii analysis the hydrogen bonding pattern for sample 2 was deduced. Enriched samples were used to establish the intermolecular distance between chemically equivalent nuclei for 1 and spatial proximity in heterogeneous domain for 2, employing the ODESSA pulse sequence. The consistence of the complementary approach covering X-ray data, analysis of the 13C (delta)ii parameters and ODESSA results is revealed.     

Cytochrome c interactions with cardiolipin in bilayers: a multinuclear magic-angle spinning NMR study.

The influence of cytochrome c binding to cardiolipin bilayers on the motional characteristics of each component has been analyzed by magic-angle spinning (MAS) NMR. Observations were made by NMR of natural abundance 31P, 13C, and 1H nuclei in the lipid as well as sites enriched with 13C in the protein. Analysis of methyl carbons enriched in ([epsilon-13CH3]methionine)cytochrome c at residues 65 and 80 reveal quite different behavior for these sites when the protein was bound at a 1:15 molar ratio with hydrated cardiolipin. Cross-polarization (CP) shows a single broad resonance downfield in the methyl region which corresponds to the spectral characteristics of methionine 65 in the solution protein when subjected to moderate thermal perturbations. These observations suggest that although methionine 65 remains motionally restricted when the protein binds to the lipid bilayers, this residue becomes less shielded and exposed to more chemically distinct environments than in the native state of the protein. In contrast to its behavior in native oxidized protein, the methionine 80 methyl could be detected following direct pi/2 pulse excitation, and this residue is assumed to be released from the axial ligand site on the heme iron to become more exposed and highly mobile in the protein-lipid complex. An analysis of the CP response for natural abundance 13C nuclei in the lipid reveals a general increase in motions with slower rates (tens of kilohertz) on binding with cytochrome c, except for sites within the region of fatty acyl chain unsaturation which appear to be selectively mobilized in the complex with protein. It is concluded that, aside from effects on the unsaturated segments, the bound protein induces new modes of slow motions in the lipid assemblies rather than restricting the overall reorientation freedom of the lipid. The strong paramagnetic effects observed previously on the relaxation of phosphorus in protein-bound lipid [Spooner, P.J.R., & Watts, A. (1991) Biochemistry 30, 3880-3885] were not extended to any carbon and proton sites observable by MAS NMR in the lipid, and this infers a specific interaction of lipid phosphate groups with the heme. However, when protein was bound to cardiolipin mixed at a 1:4 mole ratio with dioleoylphosphatidylcholine in bilayers, no direct interaction with the heme was apparent from the phosphorus NMR relaxation behavior in this component, resolved by MAS. Instead, the spectral anisotropy of cardiolipin phosphorus was determined to be reduced, indicating that, on binding with cytochrome c, the headgroup organization was perturbed in this component.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spectral editing: selection of methyl groups in multidimensional solid-state magic-angle spinning NMR

A simple spectroscopic filtering technique is presented that may aid the assignment of ¹³C and ¹⁵N resonances of methyl-containing amino-acids in solid-state magic-angle spinning (MAS) NMR. A filtering block that selects methyl resonances is introduced in two-dimensional (2D) ¹³C-homonuclear and ¹⁵N–¹³C heteronuclear correlation experiments. The 2D ¹³C–¹³C correlation spectra are recorded with the methyl filter implemented prior to a ¹³C–¹³C mixing step. It is shown that these methyl-filtered ¹³C-homonuclear correlation spectra are instrumental in the assignment of C_δ resonances of leucines by suppression of C_γ–C_δ cross peaks. Further, a methyl filter is implemented prior to a ¹⁵N–¹³C transferred-echo double resonance (TEDOR) exchange scheme to obtain 2D ¹⁵N–¹³C heteronuclear correlation spectra. These experiments provide correlations between methyl groups and backbone amides. Some of the observed sequential ¹⁵N–¹³C correlations form the basis for initial sequence-specific assignments of backbone signals of the outer-membrane protein G.     

13C and 15N chemical shift assignments and secondary structure of the B3 immunoglobulin-binding domain of streptococcal protein G by magic-angle spinning solid-state NMR spectroscopy

Complete ¹³C and ¹⁵N assignments of the B3 IgG-binding domain of protein G (GB3) in the microcrystalline solid phase, obtained using 2D and 3D MAS NMR, are presented. The chemical shifts are used to predict the protein backbone conformation and compared with solution-state shifts. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Signal assignment and secondary structure analysis of a uniformly [13C, 15N]-labeled membrane protein, H+-ATP synthase subunit c, by magic-angle spinning solid-state NMR

Signal assignment and secondary structural analysis of uniformly [¹³C, ¹⁵N] labeled H⁺-ATP synthase subunit c from E. coli (79 residues) in the solid state were carried out by two- and three-dimensional solid-state NMR under magic-angle spinning. The protein took on a unique structure even in the solid state from the ¹³C linewidths of about 1.7 ppm. On the basis of several inter- and intra-residue ¹³C–¹³C and ¹³C–¹⁵N chemical shift correlations, 78% of , 72% of , 62% of C′ and 61% of N^H signals were assigned, which provided the secondary structure information for 84% of the 79 residues. Here, inter-residue correlations involving Gly, Ala, Pro and side-chains and a higher resolution in the 3D spectrum were significantly useful for the sequence specific assignment. On top of this, the ¹³C–¹³C correlation spectra of subunit c was analyzed by reproducing experimental cross peaks quantitatively with chemical shift prediction and signal-intensity calculation based on the structure. It revealed that the subunit c in the solid state could be specified by -helices with a loop structure in the middle (at sequence 41–45) as in the case of the solution structure in spite of additional extended conformations at 76–79 at the C-terminus.     

Metabonomic studies of human hepatocellular carcinoma using high-resolution magic-angle spinning 1H NMR spectroscopy in conjunction with multivariate data analysis

High-resolution magic-angle spinning (MAS) 1H nuclear magnetic resonance spectroscopy has been employed to characterize the metabolite composition (i.e., metabonome) of the human hepatocellular carcinoma (HCC) tumor in combination with principal component analysis (PCA). The results showed that (a) the metabonomes of both low-grade HCC and high-grade HCC tumors differ markedly from that of the adjacent non-involved tissues; and (b) low-grade HCC tumors have clear differences in metabonome from that of the high-grade HCC tumors. Compared with the non-involved adjacent liver tissues, HCC tumors had elevated levels of lactate, glutamate, glutamine, glycine, leucine, alanine, choline metabolites, and phosphorylethanolamine (PE), but declined levels of triglycerides, glucose, and glycogen. The levels of lactate, amino acids including glutamate, glutamine, glycine, leucine and alanine, choline and phosphorylethanolamine (PE) were higher but the levels of PC, GPC, triglycerides, glucose, and glycogen were lower in high-grade HCC than in low-grade HCC tumors. Compared with non-cirrhotic, low-grade HCC tumors, the cirrhotic, low-grade HCC tumors showed statistically significant increases in lactate, phosphocholine (PC), and glycerophosphocholine (GPC). The necrosis in HCC tumors resulted in a drastic increase in the levels of observable triglycerides, signals of which dominated their 1H NMR spectra. These results indicated that HRMAS combined with PCA offers a useful tool for understanding the tumor biochemistry and classification of liver tumor tissues; such tool may also have some potential for liver tumor diagnosis and prognosis even when some other disease processes are present.     

Complete assignment of the hydrogen out-of-plane wagging vibrations of bathorhodopsin: chromophore structure and energy storage in the primary photoproduct of vision

Resonance Raman vibrational spectra of the retinal chromophore in bathorhodopsin have been obtained after regenerating bovine visual pigments with an extensive series of 13C- and deuterium-labeled retinals. A low-temperature spinning cell technique was used to produce high-quality bathorhodopsin spectra exhibiting resolved hydrogen out-of-plane wagging vibrations at 838, 850, 858, 875, and 921 cm-1. The isotopic shifts and a normal coordinate analysis permit the assignment of these lines to the HC7 = C8H Bg, C14H, C12H, C10H, and C11H hydrogen out-of-plane wagging modes, respectively. The coupling constant between the C11H and C12H wags as well as the C12H wag force constant are unusually low compared to those of retinal model compounds. This quantitatively confirms the lack of coupling between the C11H and C12H wags and the low C12H wag vibrational frequency noted earlier by Eyring et al. [(1982) Biochemistry 21, 384]. The force constants for the C10H and C14H wags are also significantly below the values observed in model compounds. We suggest that the perturbed hydrogen out-of-plane wagging and C-C stretching force constants for the C10-C11 = C12-C13 region of the chromophore in bathorhodopsin result from electrostatic interactions with a charged protein residue. This interaction may also contribute to the 33 kcal/mol energy storage in bathorhodopsin.     

A study of a Calpha,beta-didehydroalanine homo-oligopeptide series in the solid-state by 13C cross-polarization magic angle spinning NMR.

The fully extended peptide conformation (2.0(5)-helix) has been investigated for the first time in the solid-state by 13C cross-polarization magic angle spinning NMR. The compounds examined are members of a terminally protected, homo-oligopeptide series (from monomer through hexamer) based on Calpha,beta-didehydroalanine.     

13C NMR studies of the binding of soybean trypsin inhibitor to trypsin.

NMR studies of the complex between trypsin and soybean trypsin inhibitor with 1-¹³C-arginine and modified inhibitor with 1-¹³C-lysine show that these complexes involve almost exclusively non-covalent binding of the inhibitor to the enzyme for trypsin/¹³C-Lys-inhibitor at pH 6.5 and 8.1 and for trypsin/¹³C-Arg-inhibitor at pH 5.0. At pH 7.1 for trypsin/¹³C-Arg-inhibitor both non-covalent and acyl enzyme forms are observed. Under no conditions did we observe evidence for a tetrahedral adduct between enzyme and inhibitor.     

13C]Methylated ribonuclease A. 13C NMR studies of the interaction of lysine 41 with active site ligands

A unique resonance in the 13C NMR spectrum of [13C]methylated ribonuclease A has been assigned to a N epsilon, N-dimethylated active site residue, lysine 41. The chemical shift of this resonance was studied over the pH range 3 to 11, and the titration curve showed two inflection points, at pH 5.7 and 9.0. The higher pKa, designated pKa1, was assigned to the ionization of the lysyl residue itself while the pKa of 5.7, designated pKa2, was assigned on the basis of its pKa to the ionization of a histidyl residue which is somehow coupled to lysine 41. Both pKa values are measurably perturbed by the binding of active site ligands including nucleotides, nucleosides, phosphate, and sulfate. In most cases, the alterations in pKa values induced by the ligands were larger for pKa2. The ligand-induced perturbations in pKa2 generally paralleled those reported for histidine 12, another active site residue (Griffin, J. H., Schechter, A. N., and Cohen, J. S. (1973) Ann. N. Y. Acad. Sci. 222, 693-708). The sensitivity of the N epsilon, N-dimethylated lysine 41 resonance to the histidyl ionization may result from a conformational change in the active site region of ribonuclease which is coupled to the histidyl ionization. This coupling between lysine 41 and another ribonuclease residue, which has not been documented previously, offers new insight into the interrelationship between residues in the active site of this well characterized enzyme.     

13C NMR studies of butyric fermentation in Clostridium kluyveri

The fermentation of 13C-labeled ethanol and acetate into butyrate and caproate by Clostridium kluyveri has been studied by using 13C NMR. The pathway involves the conversion of both ethanol and acetate into acetyl coenzymes A, two of which condense to form CoA-linked precursors of butyrate. If butyryl-CoA is involved in the condensation, caproate is the ultimate product. ATP is produced from acetyl-CoA via the reactions catalyzed by phosphotransacetylase and acetate kinase with acetate, a required carbon source, as a co-product. In spectra of whole cells incubated with the labeled carbon sources, label from ethanol appears rapidly in acetate, which then reaches a lower, steady-state concentration due to its re-entry into the pathway. The rapid initial production of acetate indicates equally rapid production of ATP. Label from acetate appears in ethanol only if ethanol is already present, indicating that this process is one of isotopic equilibration rather than net synthesis of ethanol from acetate. The ratio of butyrate to caproate produced depends strongly on the initial ratio of ethanol to acetate in the medium. The relative rates of utilization of ethanol and acetate vary as the fermentation proceeds. 13C-13C coupling in the butyrate and caproate produced from [1-13C]ethanol and [2-13C]acetate can be used to determine if the acetyl-CoA molecules arising from ethanol and acetate enter the same pool or if they remain separated. The data are consistent with random mixing of the acetyl-CoA produced from the two carbon sources.     

Efficient enzymatic synthesis of 13 C,15N-labeled DNA for NMR studies

The power of heteronuclear NMR spectroscopy to study macromoleculesand their complexes has been amply demonstrated over the last decade. Theobstacle to routinely applying these techniques to the study of DNA has beenthe synthesis of ¹³C,¹⁵N-labeled DNA. Here wepresent a simple and efficient method to generate isotope-labeled DNA forNMR studies that is as easy as that for isotope labeling of RNA. The methodwas used to synthesize a uniformly¹³ C,¹⁵N-labeled 32-nucleotide DNA that binds tohuman basic fibroblast growth factor with high affinity and specificity.Isotope-edited experiments were applied to the¹³ C,¹⁵N-labeled DNA bound to unlabeled protein,and the ¹³ C,¹⁵N-labeled DNA was also examined incomplex with ¹⁵N-labeled protein. The NMR experiments showthat the DNA adopts a well-defined stable structure when bound to theprotein, and illustrate the potential of¹³ C,¹⁵N-labeled DNA for structural studies ofDNA–protein complexes.     

A study of the Schiff base mode in bovine rhodopsin and bathorhodopsin

We have obtained the resonance Raman spectra of bovine rhodopsin, bathorhodopsin, and isorhodopsin for a series of isotopically labeled retinal chromophores. The specific substitutions are at retinal's protonated Schiff base moiety and include -HC = NH+-, -HC = ND+-, -H13C = NH+-, and -H13C = ND+-. Apart from the doubly labeled retinal, we find that the protonated Schiff base frequency is the same, within experimental error, for both rhodopsin and bathorhodopsin for all the substitutions measured here and elsewhere. We develop a force field that accurately fits the observed ethylenic (C = C) and protonated Schiff base stretching frequencies of rhodopsin and labeled derivatives. Using MINDO/3 quantum mechanical procedures, we investigate the response of this force field, and the ethylenic and Schiff base stretching frequencies, to the placement of charges close to retinal's Schiff base moiety. Specifically, we find that the Schiff base frequency should be measurably affected by a 3.0-4.5-A movement of a negatively charged counterion from the positively charged protonated Schiff base moiety. That there is no experimentally discernible difference in the Schiff base frequency between rhodopsin and bathorhodopsin suggests that models for the efficient conversion of light to chemical energy in the rhodopsin to bathorhodopsin photoconversion based solely on salt bridge separation of the protonated Schiff base and its counterion are probably incorrect. We discuss various alternative models and the role of electrostatics in the rhodopsin to bathorhodopsin primary process.