Refinement of the main chain directed assignment strategy for the analysis of 1H NMR spectra of proteins.

The underlying basis of the main chain directed (MCD) resonance assignment strategy for the analysis of 1H NMR spectra of proteins is reexamined. The criteria used in the construction of the patterns used in the MCD method have been extended to increase the robustness of the approach to the presence of variable protein secondary structure and significant spectral degeneracy. These criteria have led to the development of several dozen patterns exclusively involving the short distance relationships between main chain amide NH-C alpha-H-C beta H (NAB) J-coupled subspin systems of the amino acid residues. The MCD patterns have been examined for fidelity and frequency of occurrence in a database composed of the high resolution crystal structures of 39 proteins. The analysis has identified several extremely robust patterns, suitable for initiating a hierarchical construction of units of secondary structure based upon a systematic analysis of two-dimensional nuclear Overhauser effect spectra. A formal procedure, suitable for the computer assisted application of the MCD strategy, is developed. This procedure, termed MCDPAT, has been applied to the analysis of the crystal structures of human ubiquitin, T4 lysozyme, and ribonuclease A. It has been found that the MCDPAT procedure is conservative producing no significant errors and is globally successful in correctly identifying the appropriate units of secondary structure contained in these three proteins.     

Two-dimensional 1H NMR study of human ubiquitin: a main chain directed assignment and structure analysis

The 1H resonances of human ubiquitin were studied by two-dimensional nuclear magnetic resonance techniques. A recently introduced assignment algorithm termed the main chain directed (MCD) assignment [Englander, S. W., & Wand, A. J. (1987) Biochemistry 26, 5953-5958] was applied. This approach relies on an ordered series of searches for prescribed patterns of connectivities in two-dimensional J-correlated and nuclear Overhauser effect spectra and centers on the dipolar interactions involving main-chain amide NH, alpha-CH, and beta-CH. Unlike the sequential assignment procedure, the MCD approach does not rest upon definition of side-chain J-coupled networks and is generally not sequential with the primary sequence of the protein. The various MCD patterns and the general algorithm are reiterated and applied to the analysis of human ubiquitin. With this algorithm, the vast majority of amino acid residue amide NH-C alpha H-C beta H J-coupled subspin systems could be associated with and aligned within units of secondary structure without any knowledge of the identity of the side chains. This greatly simplified recognition of side-chain spin systems by restricting their identity. Essentially complete resonance assignments are presented. The MCD method is compared with the sequential assignment method in some detail. The MCD method is highly amenable to automation. Human ubiquitin is found, at pH 5.8 and 30 degrees C, to be composed of an extensive beta-sheet structure involving five strands. Three of these strands form an antiparallel set sharing a common strand and have a parallel orientation to two antiparallel strands. Two helical segments were also observed. The largest, spanning 13 residues, shows dipolar interactions consistent with an alpha-helix while the smaller 4-residue helical segment appears, on the basis of observed nuclear Overhauser effects, to be a 3(10) helix. Five classical tight turns could be demonstrated.     

A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin

A novel approach is described for obtaining sequential assignment of the backbone 1H, 13C, and 15N resonances of larger proteins. The approach is demonstrated for the protein calmodulin (16.7 kDa), uniformly (approximately 95%) labeled with 15N and 13C. Sequential assignment of the backbone residues by standard methods was not possible because of the very narrow chemical shift distribution range of both NH and C alpha H protons in this largely alpha-helical protein. We demonstrate that the combined use of four new types of heteronuclear 3D NMR spectra together with the previously described HOHAHA-HMQC 3D experiment [Marion, D., et al. (1989) Biochemistry 28, 6150-6156] can provide unambiguous sequential assignment of protein backbone resonances. Sequential connectivity is derived from one-bond J couplings and the procedure is therefore independent of the backbone conformation. All the new 3D NMR experiments use 1H detection and rely on multiple-step magnetization transfers via well-resolved one-bond J couplings, offering high sensitivity and requiring a total of only 9 days for the recording of all five 3D spectra. Because the combination of 3D spectra offers at least two and often three independent pathways for determining sequential connectivity, the new assignment procedure is easily automated. Complete assignments are reported for the proton, carbon, and nitrogen backbone resonances of calmodulin, complexed with calcium.     

Near-infrared magnetic circular dichroism of cytochrome c'.

The near-infrared magnetic circular dichroism (MCD) of Rhodospirillum rubrum, Chromatium vinosum, and Rhodopseudomonas palustris cytochromes c' are reported. The spectra of the reduced protein are very similar to those of deoxymyoglobin. The spectra of the oxidized proteins in the pD range 1-13 can be analyzed on the basis of four species A, B, C, and D. The existence of nine species, reported in a recent electron paramagnetic resonance study, is not substantiated. The MCD spectra support the assignment of B as high spin and C and D as low spin. The MCD of species A is close to that of high-spin proteins and does not support the recently proposed assignment of a mixed high- and intermediate-spin ground state for this species. The energies of the near-IR electronic transitions of all four oxidized species point to axial ligation via oxygen, assuming histidine to be the opposite axial ligand. Unfortunately, insufficient model compounds with ligation by carboxyl or hydroxyl moieties exist to enable more precise assignments.     

Circular dichroism and magnetic circular dichroism spectra of chlorophylls a and b in nematic liquid crystals. II. Magnetic circular dichroism spectra

Absorption and magnetic circular dichroism (MCD) spectra are reported for chlorophyll (Chl) a and Chl b dissolved in nematic liquid crystal solvents. The spectra were measured with the dye molecules oriented uniaxially along the direction of. the magnetic field and measuring light beam. It is significant that under such conditions the MCD spectra recorded in the wavelength region of the Q and Soret bands of the chlorophyll are essentially unchanged with respect to rotation of the sample cell around this axis, even though there is almost complete orientation of the chlorophyll molecules by the liquid crystals. The MCD spectra of Chl a and b in the nematic liquid crystal solvents used in this study are surprisingly similar to the spectra obtained under isotropic conditions. These results illustrate an important technique with which to examine the optical spectra of dyes oriented in liquid crystal matrices in which the anisotropic effects can be reduced the negligible proportions by the application of a strong magnetic field parallel to the direction of the measuring light beam. The first deconvolution calculations are reported that describe the deconvolution of pairs of absorption and MCD spectra, in the Q and B band regions, for both Chl a and b. The spectral analysis to obtain quantitative estimates of transition energies was accomplished by carrying out detailed deconvolution calculations in which the both the absorption and MCD spectral envelopes were fitted with the same number of components; each pair of components had the same hand centres and bandwidth values. This procedure resulted in an assignment of each of the main transitions in the absorption spectra of both Chl a and b. Chl a is clearly monomeric, with Qy, Qx, By and Bx located at 671, 582, 439 and 431 nm, respectively. Analysis of the spectral data for Chl b located Qy, By and Bx, at 662, 476 and 464 nm, respectively.     

A comparative study of the low-temperature magnetic circular dichroism spectra of horse heart metmyoglobin and bovine liver catalase derivatives

The magnetic circular dichroism (MCD) spectra of three horse heart metmyoglobin compounds, the cyanide, azide and hydroxide forms, have been measured in the visible and near infrared spectral regions at temperatures down to 1.5 K. The three compounds are all virtually completely low-spin at low temperatures with ground g factors of decreasing rhombicity in the order CN- greater than N3- greater than OH-. The MCD magnetization curves have been constructed at selected wavelengths throughout the visible and near infrared regions. The curves are independent of wavelength, showing that all the bands studied are x,y polarized and can, moreover, be satisfactorily fitted to the g factors determined by EPR spectroscopy with theoretical expressions (Thomson, A.J. and Johnson, M.K. (1980) Biochem. J. 191, 411-420). This confirms the assignment and polarizations of the near infrared region low-spin ferric haem charge-transfer bands. The energies of these transitions are markedly dependent upon the added axial ligand, ranging from 1595 to 1295, and 1050 nm for the compounds CN-, N3- and OH-. The MCD spectra of bovine liver catalase and its cyanide adduct have been recorded in the Soret, visible and near infrared regions. Catalase is know to have phenolate anion as the proximal ligand of the haem group. The forms of the spectra make an interesting comparison with those of the analogous metmyoglobin derivatives, in which histidine is the proximal ligand. The MCD spectra of catalase at 4.2 K is an example of a fully high-spin haemoprotein. The cyanide compound is completely low-spin at 4.2 K. The near infrared charge-transfer band is at 1300 nm, showing the effect on the energy of this band of changing from imidazole to phenolate ion as the proximal ligand to haem.     

SANE (Structure Assisted NOE Evaluation): An automated model-based approach for NOE assignment

A reliable automated approach for assignment of NOESY spectra would allow more rapid determination of protein structures by NMR. In this paper we describe a semi-automated procedure for complete NOESY assignment (SANE, Structure Assisted NOE Evaluation), coupled to an iterative procedure for NMR structure determination where the user is directly involved. Our method is similar to ARIA [Nilges et al. (1997) J. Mol. Biol., 269, 408–422], but is compatible with the molecular dynamics suites AMBER and DYANA. The method is ideal for systems where an initial model or crystal structure is available, but has also been used successfully for ab initio structure determination. Use of this semi-automated iterative approach assists in the identification of errors in the NOE assignments to short-cut the path to an NMR solution structure.     

AUTOBA: Automation of backbone assignment from HN(C)N suite of experiments

Development of efficient strategies and automation represent important milestones of progress in rapid structure determination efforts in proteomics research. In this context, we present here an efficient algorithm named as AUTOBA (Automatic Backbone Assignment) designed to automate the assignment protocol based on HN(C)N suite of experiments. Depending upon the spectral dispersion, the user can record 2D or 3D versions of the experiments for assignment. The algorithm uses as inputs: (i) protein primary sequence and (ii) peak-lists from user defined HN(C)N suite of experiments. In the end, one gets H^N, ¹⁵N, C^α and C′ assignments (in common BMRB format) for the individual residues along the polypeptide chain. The success of the algorithm has been demonstrated, not only with experimental spectra recorded on two small globular proteins: ubiquitin (76 aa) and M-crystallin (85 aa), but also with simulated spectra of 27 other proteins using assignment data from the BMRB.     

Infrared magnetic circular dichroism of myoglobin derivatives.

By use of a newly constructed CD instrument, infrared magnetic circular dichroism (MCD) spectra were observed for various myoglobin derivatives. The ferric high spin myoglobin derivatives such as fluoride, water and hydroxide complexes, commonly exhibited the MCD spectra consisting of positive A terms. Therefore, the results reinforced the assignment that the infrared band is the charge transfer transition to the degenerate excited state (eg (dpi)). Since the fraction of A term estimated was approximately 80% for myoglobin fluoride and approximately 35% for myoglobin water, the effective symmetry for myoglobin fluoride is determined to be as close as D4h, while that for myoglobin water seems to have lower symmetry components. The ferric low spin derivatives such as myoglobin cyanide, myoglobin imidazole and myoglobin azide showed positive MCD spectra which are very similar to the electronic absorption spectra. These MCD spectra were assigned to the charge transfer transitions from porphyrin pi to iron d orbitals on the ground that they were observed only for the ferric low spin groups and insensitive to the axial ligands. The lack of temperature dependence in the MCD magnitude indicated that the MCD spectra are attributable to the Faraday B terms. Deoxymyoglobin, the ferrous high spin derivative, had fairly strong positive MCD around 760 nm with an anisotropy factor (delta epsilon/epsilon) of 1.4-10(-4). It shows some small MCD bands from 800 to 1800 nm. Among the ferrous low spin derivatives, carbonmonoxymyoglobin did not give any observable MCD in the infrared region while oxymyoglobin seemed to have significant MCD in the range from 700 to 1000 nm.     

Electronic state of heme in cytochrome oxidase. I. Magnetic circular dichroism of the isolated enzyme and its derivatives.

Magnetic circular dichroism (MCD) spectra have been recorded for beef heart cytochrome oxidase and a number of its inhibitor complexes. The resting enzyme exhibits a derivate shape Faraday C term in the Soret region, characteristic of low spin ferric heme, which accounts for 50% of the total oxidase heme a. The remaining heme a (50%) is assigned to the high spin state. MCD temperature studies, comparison with the MCD spectra of heme a-imidazole model compounds, and ligand binding (cyanide, formate) studies are consistent with these spin state assignments in the oxidized enzyme. Furthermore, the ligand binding properties and correlations between optical and MCD parameters indicate that in the resting enzyme the low spin heme a is due solely to cytochrome a3+ and the high spin heme a to cytochrome a33+. The Soret MCD of the reduced protein is interpreted as th sum of two MCD curves: an intense, asymmetric MCD band very similar to that exhibited by deoxymyoglobin which we assign to paramagnetic high spin cytochrome a3(2+) and a weaker, more symmetric MCD contribution, which is attributed to diamagnetic low spin cytochrome a2+. Temperature studies of the Soret MCD intensity support this proposed spin state heterogeneity. Ligand binding (CO, CN-) to the reduced protein eliminates the intense MCD associated with high spin cytochrome a3(2+); however, the band associated with cytochrome a2+ is observed under these conditions as well as in a number of inhibitor complexes (cyanide, formate, sulfide, azide) of the partially reduced protein. The MCD spectra of oxidized, reduced, and inhibitor-complexed cytochrome oxidase show no evidence for heme-heme interaction via spectral parameters. This conclusion is used in conjunction with the fact that ferric, high spin heme exhibits weak MCD intensity to calculate the MCD spectra for the individual cytochromes of the oxidase as well as the spectra for some inhibitor complexes of cytochrome a3. The results are most simply interpreted using the model we have recently proposed to account for the electronic and magnetic properties of cytochrome (Palmer, G., Babcock, F.T., and Vcikery, L.E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210).     

Assignment of protein NMR spectra based on projections,multi-way decomposition and a fast correlation approach

We present an approach for the assignment of protein NMR resonances that combines established and new concepts: (a) Based on published reduced dimensionality methods, two 5-dimensional experiments are proposed. (b) Multi-way decomposition (PRODECOMP) applied simultaneously to all acquired NMR spectra provides the assignment of resonance frequencies under conditions of very low signal-to-noise. (c) Each resulting component characterizes all spin (1/2) nuclei in a (doubly-labeled) CbetaH(n)-CalphaH-C'-NH-CalphaH-CbetaH(n) fragment in an unambiguous manner, such that sequentially neighboring components have about four atoms in common. (d) A new routine (SHABBA) determines correlations for all component pairs based on the common nuclei; high correlation values yield sequential chains of a dozen or more components. (e) The potentially error-prone peak picking is delayed to the last step, where it helps to place the component chains within the protein sequence, and thus to achieve the final backbone assignment. The approach was validated by achieving complete backbone resonance assignments for ubiquitin.     

Automated combined assignment of NOESY spectra and three-dimensional protein structure determination

A procedure for automated protein structure determination is presented that is based on an iterative procedure during which the NOESY peak list assignment and the structure calculation are performed simultaneously. The input consists of a list of NOESY peak positions and a list of chemical shifts as obtained from sequence-specific resonance assignment. For the present applications of this approach the previously introduced NOAH routine was implemented in the distance geometry program DIANA. As an illustration, experimental 2D and 3D NOESY cross-peak lists of six proteins have been analyzed, for which complete sequence-specific ¹H assignments are available for the polypeptide backbone and the amino acid side chains. The automated method assigned 70–90% of all NOESY cross peaks, which is on average 10% less than with the interactive approach, and only between 0.8% and 2.4% of the automatically assigned peaks had a different assignment than in the corresponding manually assigned peak lists. The structures obtained with NOAH/DIANA are in close agreement with those from manually assigned peak lists, and with both approaches the residual constraint violations correspond to high-quality NMR structure determinations. Systematic comparisons of the bundles of conformers that represent corresponding automatically and interactively determined structures document the absence of significant bias in either approach, indicating that an important step has been made towards automation of structure determination from NMR spectra.     

Magnetic circular dichroism study of cytochrome ba3 from Thermus thermophilus: spectral contributions from cytochromes b and a3 and nanosecond spectroscopy of CO photodissociation intermediates.

Near-UV-vis magnetic and natural circular dichroism (MCD and CD) spectra of oxidized, reduced, and carbonmonoxy-complexed cytochrome ba3, a terminal oxidase from the bacterium Thermus thermophilus, and nanosecond time-resolved MCD (TRMCD) and CD (TRCD) spectra of the unligated species formed after photodissociation of the CO complex are presented. The spectral contributions of individual cytochromes b and a3 to the Soret region MCD are identified. TRMCD spectroscopy is used to follow the spin state change (S = 0 to S = 2) in cytochrome a3(2+) following photodissociation of the CO complex. There is prompt appearance of the high-spin state after photolysis, as found previously in mammalian cytochrome oxidase [Goldbeck, R. A., Dawes, T. D., Einarsdóttir, O., Woodruff, W. H., & Kliger, D. S. (1991) Biophys. J. 60, 125-134]. Peak shifts of 1-10 nm appear in the TRMCD, TRCD, and time-resolved UV-vis absorption spectra of the photolyzed enzyme throughout its observable lifetime, indicating that the photolyzed enzyme does not relax to its equilibrium deliganded form before recombination with CO occurs hundreds of milliseconds later. Direct heme-heme interaction is not found in cytochrome ba3, but red-shifts in the MCD and absorption spectra of both cytochromes b and (photolyzed) a3 are correlated with a CO-liganded form of the protein. The long time (tau approximately greater than 1 s) needed for relaxation of the cytochrome b and a3 peaks to their static positions suggests that CO binding to a3 induces a global conformational change in the protein that weakly perturbs the MCD and absorption spectra of b and photolyzed a3. Fea3 binds CO more weakly in cytochrome ba3 than in cytochrome aa3. The MCD spectrum of reduced enzyme solution placed under 1 atm of CO contains a peak at 446 nm that shows approximately 30% of total cytochrome a3 remains pentacoordinate, high-spin.     

Magnetic circular dichroism of non-heme iron proteins

The magnetic circular dichroism (MCD) at 45 kgauss has been determined for a group of non-heme iron proteins. Both transferrin and conalbumin exhibit a single, positive ellipticity band at 330 nm ([θ]_M = 560). Oxy- and methemerythrin, spinach and clostridial ferredoxins and rubredoxin all display distinctive multibanded spectra which may reflect such factors as coordination of the metal, its ligands, metal bridging by other atoms, and varying degrees of metalmetal coupling. The MCD spectra of both ferredoxins and rubredoxin undergo dramatic change upon oxidoreduction providing a potential means for relating the electronic structure of the iron to protein function. In contrast to the plant ferredoxins, the magnetic field does not significantly affect the CD spectra of adrenodoxin and putidaredoxin.     

Magnetic circular dichroism spectroscopy as a probe of axial heme ligand replacement in semisynthetic mutants of cytochrome c.

Horse heart cytochrome c with either histidine or cysteine replacing the endogenous axial methionine ligand at position 80 has been characterized with magnetic circular dichroism (MCD) spectroscopy in the UV-visible region. Comparison of the MCD spectra of the mutant proteins in the ferric state to those of authentic bis-imidazole- and imidazole/thiolate-ligated ferric heme proteins clearly shows that the histidine-imidazole and cysteine-thiolate groups of the replacement amino acids at position 80 are coordinated to the heme iron in the mutant proteins. This study demonstrates the power of MCD spectroscopy in identifying axial ligands in mutant heme proteins. Accurate axial ligand assignment is essential for proper interpretation of the altered properties of such novel proteins.     

Low-temperature magnetic circular dichroism spectra and magnetisation curves of 4Fe clusters in iron-sulphur proteins from Chromatium and Clostridium pasteurianum

The magnetic circular dichroism (MCD) spectra of the 4Fe clusters in the iron-sulphur proteins high-potential iron protein from Chromatium and the 8Fe ferredoxin from Clostridium pasteurianum have been measured over the wavelength range 300-800 nm at temperatures between approx. 1.5 and 50 K and at magnetic fields up to 5 tesla. In both cases the proteins have been studied in the oxidized and reduced states. The reduced state of high-potential iron protein gives a temperature-independent MCD spectrum up to 20 K, confirming the diamagetism of this state at low temperature. The MCD spectrum of samples of oxidized ferredoxin invariably show the presence of a low concentration of a paramagnetic species, in agreement with the observation that the EPR spectrum always shows a signal at g = 2.01. The paramagnetic MCD spectrum runs across the whole of the wavelength range studied and therefore most probably originates from an iron-sulphur centre. The diamagnetic component of the MCD spectrum of oxidized ferredoxin is very similar to that of reduced high-potential iron protein. The low-temperature MCD spectra of oxidized high-potential iron protein and reduced ferredoxin reveal intense, temperature-dependent bands. The spectra are highly structured with that of high-potential iron protein showing a large number of electronic transitions across the visible region. The MCD spectra of the two different oxidation levels are quite distinctive and should provide a means of establishing the identity of these state of 4Fe clusters in more complex proteins. MCD magnetisation curves have been constructed from detailed studies of the field and temperature dependence of the MCD spectra of the two paramagnetic oxidation states. These plots can be satisfactorily fitted to the theoretically computed curves for an S = 1/2 ground state with the g factors experimentally determined by EPR spectroscopy. The low-temperature MCD spectra of the reduced 2Fe-2S ferredoxin from Spirulina maxima are also presented and MCD magnetisation curves plotted and fitted to the experimentally determined g factors.     

Effects of cyanogen bromide modification of the distal histidine on the spectroscopic and ligand binding properties of myoglobin: magnetic circular dichroism spectroscopy as a probe of distal water ligation in ferric high-spin histidine-bound heme proteins

Magnetic circular dichroism (MCD) spectroscopy has been utilized to characterize the change in coordination structure in native ferric sperm whale myoglobin upon cyanogen bromide-modification. Comparison of the MCD properties of the ferric high-spin state of cyanogen bromide-modified myoglobin (BrCN-Mb) with those of native ferric horseradish peroxidase and Aplysia myoglobin suggests that ferric BrCN-Mb is a potential MCD model for the pentacoordinate state of ferric high-spin histidine-ligated heme proteins. These five-coordinate heme proteins afford a relatively weak and unsymmetric signal in the Soret region of the MCD spectrum. In contrast, native ferric myoglobin and the benzohydroxamic acid adduct of ferric horseradish peroxidase show a strong and symmetric derivative-shaped Soret MCD signal which is indicative of hexacoordination with water and histidine axial ligands. Therefore it seems that MCD spectroscopy could be used to probe the presence of water ligated to the distal side of ferric high-spin heme proteins. The MCD spectra of the ferric-azide, ferrous-deoxy and ferrous-CO forms of BrCN-Mb have also been measured and compared to those of analogous native myoglobin complexes. The present MCD study has been extended to include new ligands, NO, thiocyanate and cyanate, which bind to ferric BrCN-Mb. With exogenous ligands such as CO, NO and thiocyanate, the coordination structures of the BrCN-Mb complexes are similar to those of the respective native myoglobin adducts. In the case of ferrous-deoxy and ferric-cyanate BrCN-Mb, however, the altered MCD spectra (and EPR for the latter) reveal changes in electronic structure which likely correlate with alterations of the coordination environment of these BrCN-Mb derivatives. Data are also presented which support the proposed tetrazole-bound structure for azide-treated BrCN-Mb (Hori, H., Fujii, M., Shiro, Y., Iizuka, T., Adachi, S. and Morishima, I. (1989) J. Biol. Chem. 264, 5715-5719) and the inability of the distal histidine of BrCN-Mb to stabilize the ferric ligand-bound state.     

Proton resonance assignments of horse ferrocytochrome c

Two-dimensional nuclear magnetic resonance (NMR) spectroscopy was used to assign the proton resonances of horse ferrocytochrome c. Assignments were based on the main chain directed (MCD) and sequential assignment procedures. The fundamental units of the MCD approach, the main-chain NH-C alpha H-C beta H J-coupled subspin systems of each amino acid residue (NAB sets), were defined by analysis of direct and relayed coherence transfer spectra. Recognition of main-chain NOE connectivity patterns specified in the MCD algorithm then allowed NAB sets to be aligned in their proper juxtaposition within secondary structural units. The units of secondary structure were placed within the polypeptide sequence of identification of a small number of side-chain J-coupled spin systems, found by direct recognition in 2D spectra of some J-coupled spin systems and by pairwise comparisons of the J-correlated spectra of six homologous cytochromes c having a small number of known amino acid differences. The placement of a given segment in this way defines the amino acid identity of all its NAB sets. This foreknowledge allowed the vast majority of the side-chain resonances to be discerned in J-correlated spectra. Extensive confirmation of the assignments derives internally from multiple main-chain NOE connectivities and their consistency following temperature-induced changes of the chemical shifts of NOE-correlated protons. The observed patterns of main-chain NOEs provide some structural information and suggest small but potentially significant differences between the solution structure observed by NMR and that defined earlier in crystallographic studies at 2.8-A resolution.     

Use of graph theory for secondary structure recognition and sequential assignment in heteronuclear (13C, 15N) NMR spectra: Application to HU protein from Bacillus stearothermophilus

A computer-assisted procedure, based upon a branch of mathematics known as graph theory, has been developed to recognize secondary structure elements in proteins from their corresponding nuclear Overhauser effect spectroscopy (NOESY)-type spectra and to carry out their sequential assignment. In the method, NOE connectivity templates characteristic of regular secondary structures are identified in the spectra. Resonance assignment is then achieved by connecting these NOE patterns of secondary structure together, and thereby matching connected spin systems to specific parts of the primary sequence. The range of NOE-graph templates of secondary structure motifs, incorporating α-helices and β-strand motifs, has been examined for reliability and extent of secondary structure identification in a data base composed of the high resolution structures of 20 proteins. The analysis identified several robust NOE-graph templates and supports the implementation of an ordered search strategy. The method, known as SERENDIPITY, has been applied to the analysis of nuclear Overhauser effect data from a three-dimensional time-shared nuclear Overhauser effect spectroscopy (¹³C, ¹⁵N) heteronuclear single quantum correlation spectrum of the (α + β) type protein HU from Bacillus stearothermophilus. The arrangement of the elucidated elements of secondary structure is very similar to that of the x-ray and nmr structures of HU. In addition, our analysis revealed a pattern of interstrand nuclear Overhauser effect in the β-arm region (residues 53–76) of HU, which suggest irregularities, not reported in the x-ray structure, in both strands of the β-arm at Ala57 and Pro72, respectively. At these residues, both strands of the β-arm appear to flip inside out before continuing as a regular antiparallel β-sheet. © 1996 John Wiley & Sons, Inc.     

Magnetic circular dichroic spectra of cobalt(II) substituted metalloenzymes.

The magnetic circular dichroic (MCD) spectra of cobalt(II) sugstituted metalloenzymes have been studied and compared to a series of four-, five-, and six-coordinate cobalt(II) model complexes previously examined (T. A. Kaden et al. (1974), Inorg. Chem. 13, 2582). The MCD spectra of cobalt substituted carboxypeptidase A, procarboxypeptidase ta, and thermolysin are consistent with earlier deductions of tetrahedral coordination from absorption spectra and also with X-ray structure analysis. Inhibitors fail to alter their MCD spectra significantly. The MCD spectra of cobalt alkaline phosphatase and carbonic anhydrase are more complex and their pH dependence and alteration by inhibitors are discussed in terms of known cobalt(II) models.