Hans Senn

Editing of 2D 1H NMR spectra using X half-filters. combined use with residue-selective 15N labeling of proteins

The introduction of efficient sequential assignment techniques for obtaining sequence-specific H NMR assignments in proteins (1, 2) has provided a basis for determination of the three-dimensional molecular structure in solution (3, 4), as well as for detailed studies of the molecular dynamics (e.g., (5)) and of intermolecular interactions (4). Limitations on the use of this approach arise if two or several cross peaks in the 2D H NMR spectra overlap. The degree of spectral overlap increases quite naturally with increasing molecular size , but if the H resonances are not well dispersed complete sequence -specific assignment of the spectrum may be difficult even for small proteins. As was also pointed out by others (6-9), the situation can be improved by residue-selective isotope labeling with15N or t3C. In such preparations the protons directly bound to 15N or 13C can be singled out on the basis of the large heteronuclear spin-spin coupling constants JXH. Simplified spectra have thus been obtained which correspond either to the difference between recordings with and without broadband decoupling of the X nucleus (6, 7), or with and without ir(15N) refocusing pulse (8), or were recorded with a heteronuclear zero-quantum filter (9). In this communication we describe the use of X(w,) and X(w2) half-filters for editing the 2D H NMR spectra of X-labeled molecules. Compared to difference spectroscopy techniques the half-filters have the advantage of providing two subspectra corresponding, respectively, to the X-labeled H peaks and to all other peaks. Compared to the heteronuclear zero-quantum filter (9) the sensitivity of the corresponding half-filter is improved by a factor 2. X half-filters can be employed with a wide variety of 2D NMR experiments, including 2D correlated spectroscopy (COSY), 2D total correlation spectroscopy (TOCSY), and 2D nuclear Overhauser enhancement spectroscopy (NOESY) (Fig. 1). Clearly, their use is not limited to proteins and there is a wide spectrum of potential applications with other macromolecules and with small molecules , in particular for studies of intermolecular interactions. In the experimental schemes for obtaining the desired simplified spectra with 15Nlabeled proteins, we inserted 15N half-filters consisting of a [-T/2-7r(H, 15N)-T/2r(15N)-] pulse sequence, either before the evolution period (w, half-filter) or immediately before detection (w2 half-filter) of the conventional 2D H NMR experiments

Determination of the coordination geometry at the heme iron in three cytochromes c from Saccharomyces cerevisiae and from Candida krusei based on individual 1H-NMR assignments for heme c and the axially coordinated amino acids

The 1H-NMR lines of heme c and the axial ligands in reduced and oxidized Iso-1 and Iso-2 cytochromes c from Saccharomyces cerevisiae and in cytochrome c from Candida krusei were individually assigned and the conformation of the coordination sphere of the heme iron was investigated with the use of proton-proton Overhauser enhancement measurements and circular dichroism spectroscopy. The coordination geometry of the axial methionine and the axial histidine and the electronic structure of the heme were found to be closely similar in these yeast cytochromes c and in mammalian cytochromes c. In particular, R chirality at the sulfur atom of the iron-bound methionine was observed in both groups of proteins. Additional nuclear Overhauser enhancement studies of the spatial arrangement relative to the heme group of amino acid side-chains in the heme crevice of yeast ferrocytochromes c showed that the conformational homologies extend beyond the immediate coordination sphere of the heme iron. These data provide a conformational basis for observations on the functional properties of cytochromes c from yeast and mammalian species, which were reported previously by other groups.

Conformation of the axially bound ligands of the heme iron and electronics structure of heme c in the cytochromes c-551 from pseudomonas mendocina and pseudomonas stutzeri and in cytochrome c2 from rhodospirillum rubrum

Abstract The cytochromes c -551 from Pseudomonas mendocina , c -551 from Pseudomonas stutzeri and c 2 from Rhodospirillum rubrum have been isolated and investigated by 1 H-nuclear magnetic resonance at 360 MHz and 500 MHz and by circular dichroism spectroscopy. For all three proteins individual 1 H-NMR assignments were obtained for heme c and the axial ligands of the heme iron. The spatial arrangement of the axial ligands in the reduced proteins was determined by nuclear Overhauser enhancement experiments and in the oxidized proteins by CD studies. For the two cytochromes c -551 the axial methionine spatial structure corresponds to S chirality at the iron-bound sulfur atom and coincides closely with the previously established structure of cytochrome c -551 from Pseudomonas aeruginosa . In the cytochrome c 2 , R chirality of the axial methionine was observed, and the structure corresponds to that found previously for several mammalian-type cytochromes c and for the cytochromes c -552 from Euglena gracilis and Spirulina maxima . Concomitantly with the different methionine chirality, characteristically different electronic structures were observed for the ferricytochromes c -551 and the ferricytochrome c 2 , which coincides with previous observations with cytochromes c from different species.

Individual 1H-NMR assignments for the heme groups and the axially bound amino acids and determination of the coordination geometry at the heme iron in a mixture of two isocytochromes c-551 from Rhodopseudomonas gelatinosa

This paper describes chemical and physicochemical studies of two small isocytochromes c-551 (approx. 9000 dalton) from Rhodopseudomonas gelatinosa. In spite of numerous amino acid substitutions in the N-terminal half of the sequence the two isoproteins could not be separated by the procedures used, presumably because they have identical size, charge and isoelectric points. Individual assignments of the 1H-NMR lines of heme c and the axial ligands to the heme iron were therefore obtained by nuclear Overhauser enhancement measurements and saturation transfer experiments in a mixed solution of the two isocytochromes c-551. The conformation of the coordination sphere was investigated by additional 1H-NMR and circular dichroism studies. For both isoproteins the electronic structure of the heme and the chirality of the methionine attachment to the iron were found to coincide with those in Pseudomonas cytochromes c-551, i.e., S chirality was observed for the axial methionine. The Rps. gelatinosa cytochromes c-551 thus differ from mammalian, yeast, Euglena gracilis and Rhodospirillum rubrum cytochromes c, which all have R chirality at the axial methionine and concomitantly a characteristically different electronic heme structure. This is the first observation of S chirality of the axially bound methionine in a species outside the Pseudomonas family. The redox potentials of the two isocytochromes c-551 of Rps. gelatinosa differ by approx. 120 mV, and there is no cross-exchange of electrons between the two species. The two isoproteins could thus function in two different, parallel electron-transfer chains or at two different locations in a single transfer sequence.

Coordination of the heme iron in the low-potential cytochromes c-553 from Desulfovibrio vulgaris and Desulfovibrio desulfuricans: Different chirality of the axially bound methionine in the oxidized and reduced states

The coordination geometry at the heme iron of the cytochromes c-553 from Desulfovibrio vulgaris and Desulfovibrio desulfuricans was investigated by 1H-nuclear magnetic resonance and circular dichroism spectroscopy. Individual assignments were obtained for heme c and the axial ligands. From studies of nuclear Overhauser enhancements the axial histidine imidazole ring orientation relative to the heme group was found to coincide with other c-type cytochromes. In contrast, a new structure was observed for the axial methionine in the reduced cytochromes c-553. This includes S chirality at the iron-bound sulfur atom, but compared to cytochromes c-551 from Pseudomonads and Rhodopseudomonas gelatinosa and cytochrome c5 from Pseudomonas mendocina, which also contain S-chiral methionine, a different spatial arrangement of the gamma- and beta-methylene groups and the alpha carbon of methionine prevails. For the ferricytochromes c-553 R chirality was found for the iron-bound sulfur. This is the first observation of different methionine chirality in different oxidation states of the same c-type cytochrome.

15N-labeled P22 c2 repressor for nuclear magnetic resonance studies of protein-DNA interactions

The salmonella phage P22 c2 repressor was produced with 90% 15N isotope labeling of all leucines, using the expression system E. coli W3110 lac IQ/pTP 125. The N-terminal DNA-binding domain 1–76 was obtained by chymotrypsin cleavage. Its characterization by biochemical techniques, mass spectrometry, and one- and two-dimensional nuclear magnetic resonance (NMR) showed that highly residue-selective isotope labeling was achieved with the minimal growth medium used. The ability to obtain such isotope labeling opens new avenues for NMR studies of protein-DNA interactions in the P22 operator system.

A new spatial structure for the axial methionine observed in cytochrome c5 from Pseudomonas mendocina: Correlations with the electronic structure of heme c

Cytochrome c5 from Pseudomonas mendocina has been isolated and the coordination geometry at the heme iron was investigated by 1H nuclear magnetic resonance and circular dichroism spectroscopy. Individual assignments were obtained for heme c and the axial ligands. From studies of nuclear Overhauser enhancements the axial histidine imidazole ring orientation relative to the heme group was found to coincide with that of other c-type cytochromes. In contrast, a new structure was observed for the axial methionine. This includes S chirality at the iron-bound sulfur atom, but compared to cytochromes c-551 from Pseudomonads and Rhodopseudomonas gelatinosa, which also contain S-chiral methionine, the spatial arrangement of the gamma- and beta-methylene groups and the alpha carbon of methionine is markedly different. Analysis of the electron spin density distribution in ferricytochrome c5 in the light of this new coordination geometry provides additional support for the hypothesis that the electronic structure of heme c is primarily governed by the orientation of the sp3 lone-pair orbital of the axial sulfur atom with respect to the heme plane.

Studies of the solution conformation of Spirulina platensis cytochrome c-553 by 1H-nuclear magnetic resonance and circular dichroism

Abstract The 1 H-NMR lines of heme c and the axial ligands of the heme iron in reduced cytochrome c -553 from the blue-green algae Spirulina platensis were individually assigned and the coordination geometry of the axial ligands was determined. In ferricytochrome c -553, corresponding resonance assignments were to a limited extent also obtained and the chirality of the axial methionine was determined with circular dichroism. The axial methionine chirality and the electronic structure in S. platensis cytochrome c -553 were found to be of the same type as in cytochrome c -552 from Euglena gracilis and in mammalian cytochromes c . Further studies, mainly by nuclear Overhauser enhancement experiments and by chemical determination of the amino-acid sequence for positions 1–44 in S. platensis cytochrome c -553, indicated that the spatial structure homologies between mitochondrial cytochrome c from horse, chloroplast cytochrome c -552 from E. gracilis and cyanobacterial cytochrome c -553 from S. platensis extend also to the polypeptide conformation of the heme pocket outside the first coordination sphere of the heme iron.

1H-NMR assignments for the heme group and electronic structure in Chlorobium thiosulfatophilum cytochrome c-555

Abstract Cytochrome c-555 from Chlorobium thiosulfatophilum was investigated by 1H nuclear magnetic resonance at 360 MHz. Individual 1H-NMR assignments were obtained for heme c, and the side-chain proton resonances of the axial methionine and the imidazole ring proton lines of the axial histidine were identified. The unpaired electron spin distribution in the heme of the oxidized protein is characterized by outstandingly large hyperfine shifts for all four ring methyl groups, with a particularly large shift for the ring methyl in position 8. Although the origin of these large hyperfine shifts has not been determined, they may reflect specific polypeptide-heme interactions which result in the comparatively low oxidation-reduction potential (145 mV) of cytochrome c-555.

Influence of vibration on NMR experiments

Highly sensitive NMR experiments require a short-term stability of the magnetic field corresponding to the measurement frequency of 10-i’ to lo-” s-‘. External influences which cannot be compensated by appropriate stabilization circuits impair the NMR spectrum. Vibrations of the building, which are discussed in this report, are one of these external influences. The central NMR laboratory in Sandoz Ltd., Basel, is situated in the 12th story of a multistory building ( 15 floors). The building is basically a skeletal structure with wide floor span constructions of horizontal dimensions 16 m (x) by 66 m ( y) and a height of 76 m. Until now this extreme location was justifiable for routine NMR spectroscopy. However, for the introduction of inverse spectroscopy and the new installation of a Bruker AM-500 spectrometer, the position was carefully evaluated with respect to vibrations, their effects on the experiment, and their elimination. The vibrations were measured directly at the floor level of the laboratory from the acceleration of a test body using a piezo-transducer (Accelerometer Type 83 18, Brtiel and Kjaer, Naerum, Denmark; high pass = 1 kHz, low pass = 0.2 Hz, 1 V = 1 m sP2). The signal from the measuring instrument was passed to the AD converter of the NMR spectrometer. The frequencies and subsequently the vibrational amplitudes were determined from the measured acceleration as a function of time, using a Fourier transformation. Figure 1 A shows typical lateral and vertical vibration spectra in the three spatial dimensions. In the transverse direction (x) of the building, there is a low-frequency vibration of 0.7 Hz. The vibrational amplitude is strongly influenced by the wind conditions and varies between 50 and 150 pm. The same vibration of 0.7 Hz, which is however less dependent on the weather conditions, is also present in the longitudinal direction ( y) with an amplitude of 30 to 50 pm. In both horizontal directions (x, y) numerous vibrations occur in the range 5 to 50 Hz, with a maximum amplitude of 5 Hz; more vibrations with a larger amplitude (up to 5 pm) are observed. The strongest vibration observed at 0.7 Hz in the horizontal directions does not appear in the vertical direction. The vibrations of the building have a devastating effect on the baseline of the resonance signal (hump test, Fig. 2Aa). Sidebands occur across the whole frequency