NMR studies on Cu(II)-peptide complexes: exchange kinetics and determination of structures in solution

The interaction of copper(II) with histidine containing peptides has recently acquired renewed interest following the established link between abnormal protein behaviour in neurodegenerative processes and unpaired copper homeostasis. Five peptide sequences taken from the amyloid precursor protein and the prion protein were considered. Addition of paramagnetic Cu(II) ions to solutions of such peptides was not found to severely affect the appearance of NMR spectra, thus limiting the usual approach for structural determination. Exchange kinetics was shown to play a major role in determining the observed paramagnetic spin-lattice relaxation rates. Two independent methods were suggested for evaluating the exchange rates of His-containing peptides from the copper-coordination sphere and to calculate copper-proton distances. In such a way NMR was demonstrated to have the potential of providing detailed structures of the Cu(II)-peptide complexes in solution.     

Application of iterative soft thresholding for fast reconstruction of NMR data non-uniformly sampled with multidimensional Poisson Gap scheduling

The fast Fourier transformation has been the gold standard for transforming data from time to frequency domain in many spectroscopic methods, including NMR. While reliable, it has as a drawback that it requires a grid of uniformly sampled data points. This needs very long measuring times for sampling in multidimensional experiments in all indirect dimensions uniformly and even does not allow reaching optimal evolution times that would match the resolution power of modern high-field instruments. Thus, many alternative sampling and transformation schemes have been proposed. Their common challenges are the suppression of the artifacts due to the non-uniformity of the sampling schedules, the preservation of the relative signal amplitudes, and the computing time needed for spectra reconstruction. Here we present a fast implementation of the Iterative Soft Thresholding approach (istHMS) that can reconstruct high-resolution non-uniformly sampled NMR data up to four dimensions within a few hours and make routine reconstruction of high-resolution NUS 3D and 4D spectra convenient. We include a graphical user interface for generating sampling schedules with the Poisson-Gap method and an estimation of optimal evolution times based on molecular properties. The performance of the approach is demonstrated with the reconstruction of non-uniformly sampled medium and high-resolution 3D and 4D protein spectra acquired with sampling densities as low as 0.8%. The method presented here facilitates acquisition, reconstruction and use of multidimensional NMR spectra at otherwise unreachable spectral resolution in indirect dimensions.     

Proton resonance assignments of horse ferricytochrome c

Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) was used to obtain extensive resonance assignments in the 1H NMR spectrum of horse ferricytochrome c. Assignments were made for the main-chain and C beta protons of 102 residues (all except Pro-44 and Gly-84) and the majority of side-chain protons. As starting points for the assignment of the oxidized protein, a limited set of protons was initially assigned by use of 2D NMR magnetization transfer methods to correlate resonances in the oxidized form with assigned resonances in the reduced form [Wand, A. J., Di Stefano, D. L., Feng, Y., Roder, H., & Englander, S. W. (1989) Biochemistry (preceding paper in this issue)]. Given the complexity of the spectrum due to the size of this protein (104 residues) and its paramagnetic center, the initial search for side-chain spin systems in J-correlated spectra was successful only for the simplest side chains, but the majority of NH-C alpha H-C beta H subspin systems (NAB sets) could be identified at this stage. The subsequent search for sequential NOE connectivities focused on NAB sets, with use of previously assigned residues to place NOE-connected segments within the amino acid sequence. Selective proton labeling of either the slowly or the rapidly exchanging amide sites was used to simplify the spectra, and systematic work at two temperatures was used to resolve ambiguities in the 2D NMR spectra. These approaches, together with the use of magnetization transfer methods to correlate reduced and oxidized cytochrome c spectra, provide multiple cross-checks to verify assignments.     

1H, 13C, and 15N NMR backbone assignments and secondary structure of human interferon-gamma.

1H, 13C, and 15N NMR assignments of the protein backbone of human interferon-gamma, a homodimer of 31.4 kDa, have been made using the recently introduced three-dimensional (3D) triple-resonance NMR techniques. It is shown that, despite the approximately 40-50-Hz 13C alpha and 1H alpha line widths of this high molecular weight dimer and the extensive overlap in the 1H alpha and 13C alpha spectral regions, unique sequential assignments can be made on the basis of combined use of the 3D HNCO, HNCA, HN(CO)CA, and HCACO constant-time experiments, the 15N-separated 3D NOESY-HMQC, and the 3D HOHAHA-HMQC experiments. Analysis of the 15N-separated 3D NOESY-HMQC and 13C/15N-separated four-dimensional (4D) NOESY-HMQC spectra together with the secondary C alpha and C beta chemical shifts yielded extensive secondary structure information. The NMR-derived secondary structure essentially confirms results of a recently published low-resolution crystal structure [Ealick et al. (1991) Science 252, 698-702], i.e., six helices in the monomer which are mostly alpha-helical in nature, no beta-sheets, a long flexible loop between helices A and B, and a very hydrophobic helix C. The functionally important carboxy terminus, which was not observed in the X-ray study, does not adopt a rigid conformation in solution. A high degree of internal mobility, starting at Pro-123, gives rise to significantly narrower resonance line widths for these carboxy-terminal residues compared to the rest of the protein.     

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.     

Determinants of protein hyperthermostability: purification and amino acid sequence of rubredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus and secondary structure of the zinc adduct by NMR

The purification, amino acid sequence, and two-dimensional 1H NMR results are reported for the rubredoxin (Rd) from the hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C. The molecular mass (5397 Da), iron content (1.2 +/- 0.2 g-atom of Fe/mol), UV-vis spectrophotometric properties, and amino acid sequence (60% sequence identity with Clostridium pasteurianum Rd) are found to be typical of this class of redox protein. However, P. furiosus Rd is remarkably thermostable, being unaffected after incubation for 24 h at 95 degrees C. One- and two-dimensional 1H nuclear magnetic resonance spectra of the oxidized [Fe(III)Rd] and reduced [Fe(II)Rd] forms of P. furiosus Rd exhibited substantial paramagnetic line broadening, and this precluded detailed 3D structural studies. The apoprotein was not readily amenable to NMR studies due to apparent protein oxidation involving the free cysteine sulfhydryls. However, high-quality NMR spectra were obtained for the Zn-substituted protein, Zn(Rd), enabling detailed NMR signal assignment for all backbone amide and alpha and most side-chain protons. Secondary structural elements were determined from qualitative analysis of 2D Overhauser effect spectra. Residues A1-K6, Y10-E14, and F48-E51 form a three-strand antiparallel beta-sheet, which comprises ca. 30% of the primary sequence. Residues C5-Y10 and C38-A43 form types I and II amide-sulfur tight turns common to iron-sulfur proteins. These structural elements are similar to those observed by X-ray crystallography for native Rd from the mesophile C. pasteurianum. However, the beta-sheet domain in P. furiosus Rd is larger than that in C. pasteurianum Rd and appears to begin at the N-terminal residue. From analysis of the secondary structure, potentially stabilizing electrostatic interactions involving the charged groups of residues Ala(1), Glu(14), and Glu(52) are proposed. These interactions, which are not present in rubredoxins from mesophilic organisms, may prevent the beta-sheet from "unzipping" at elevated temperatures.     

High-resolution pyrimidine- and ribose-specific 4D HCCH-COSY spectra of RNA using the filter diagonalization method

The NMR spectra of nucleic acids suffer from severe peak overlap, which complicates resonance assignments. 4D NMR experiments can overcome much of the degeneracy in 2D and 3D spectra; however, the linear increase in acquisition time with each new dimension makes it impractical to acquire high-resolution 4D spectra using standard Fourier transform (FT) techniques. The filter diagonalization method (FDM) is a numerically efficient algorithm that fits the entire multi-dimensional time-domain data to a set of multi-dimensional oscillators. Selective 4D constant-time HCCH-COSY experiments that correlate the H5-C5-C6-H6 base spin systems of pyrimidines or the H1'-C1'-C2'-H2' spin systems of ribose sugars were acquired on the (13)C-labeled iron responsive element (IRE) RNA. FDM-processing of these 4D experiments recorded with only 8 complex points in the indirect dimensions showed superior spectral resolution than FT-processed spectra. Practical aspects of obtaining optimal FDM-processed spectra are discussed. The results here demonstrate that FDM-processing can be used to obtain high-resolution 4D spectra on a medium sized RNA in a fraction of the acquisition time normally required for high-resolution, high-dimensional spectra.     

The program XEASY for computer-supported NMR spectral analysis of biological macromolecules

Summary A new program package, XEASY, was written for interactive computer support of the analysis of NMR spectra for three-dimensional structure determination of biological macromolecules. XEASY was developed for work with 2D, 3D and 4D NMR data sets. It includes all the functions performed by the precursor program EASY, which was designed for the analysis of 2D NMR spectra, i.e., peak picking and support of sequence-specific resonance assignments, cross-peak assignments, cross-peak integration and rate constant determination for dynamic processes. Since the program utilizes the X-window system and the Motif widget set, it is portable on a wide range of UNIX workstations. The design objective was to provide maximal computer support for the analysis of spectra, while providing the user with complete control over the final resonance assignments. Technically important features of XEASY are the use and flexible visual display of strips, i.e., two-dimensional spectral regions that contain the relevant parts of 3D or 4D NMR spectra, automated sorting routines to narrow down the selection of strips that need to be interactively considered in a particular assignment step, a protocol of resonance assignments that can be used for reliable bookkeeping, independent of the assignment strategy used, and capabilities for proper treatment of spectral folding and efficient transfer of resonance assignments between spectra of different types and different dimensionality, including projected, reduced-dimensionality triple-resonance experiments.Abbreviations 1D, 2D, 3D, 4D one-, two-, three-, four-dimensional - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy - COSY correlation spectroscopy - TPPI time-proportional phase incrementation     

Determination of the functionally important heme peripheral vinyl group orientation in paramagnetic hemoprotein by 2D NMR

2D NMR spectroscopies have been successfully used to characterize the heme peripheral vinyl groups in paramagnetic hemoprotein in spite of the difficulties from the rapid paramagnetic relaxation and the low digital resolution of the 2D NMR map. The scalar coupling network system among the vinyl protons is clearly identified in the COSY spectra from its characteristic cross-peak pattern and the dipolar coupling connectivities of the vinyl proton resonances with other heme side-chain proton resonances not only provide the specific assignment of vinyl beta-proton resonances but also allow the determination of the vinyl group orientation with respect to the heme plane.     

The complexation of metal cations by D-galacturonic acid: a spectroscopic study

Solid complexes of D-galacturonic acid (GalA) with cobalt(II), copper(II), nickel(II) and oxovanadium(IV) (1-4) were prepared and characterised. The metal-to-ligand molar ratio was 1:2 for complexes 1-3 and 1:1 for complex 4. The alpha- and beta-anomers of GalA were detected in all the complexes in solid state and in solutions. An addition of small amounts of the paramagnetic complexes to the D2O solution of pure ligand led to NMR line broadening of some 1H and 13C nuclei. This broadening was sensitive to the anomeric state of GalA in the case of complexes 1 and 4. NMR and vibrational spectroscopic data indicate the formation of carboxylate complexes of all the cations, while noncarboxylic oxygens are also involved into the metal bonding in some cases. VCD spectra of complexes 1-4 in D2O and Me2SO-d6 solutions confirm that GalA carboxylic group may participate in the formation of optically active species around the metal cation. Possible ways of GalA coordination by metal cations of this study were proposed and discussed.     

Chemical shift assignment of the transmembrane helices of DsbB, a 20-kDa integral membrane enzyme, by 3D magic-angle spinning NMR spectroscopy

The Escherichia coli inner membrane enzyme DsbB catalyzes disulfide bond formation in periplasmic proteins, by transferring electrons to ubiquinone from DsbA, which in turn directly oxidizes cysteines in substrate proteins. We have previously shown that DsbB can be prepared in a state that gives highly resolved magic-angle spinning (MAS) NMR spectra. Here we report sequential 13C and 15N chemical shift assignments for the majority of the residues in the transmembrane helices, achieved by three-dimensional (3D) correlation experiments on a uniformly 13C, 15N-labeled sample at 750-MHz 1H frequency. We also present a four-dimensional (4D) correlation spectrum, which confirms assignments in some highly congested regions of the 3D spectra. Overall, our results show the potential to assign larger membrane proteins using 3D and 4D correlation experiments and form the basis of further structural and dynamical studies of DsbB by MAS NMR.     

Cobalt bovine superoxide dismutase. Reactivity of the cobalt chromophore in the copper-containing and in the copper-free enzyme.

1. The reactivity of the zinc site of bovine superoxide dismutase has been probed by observing optical and electron paramagnetic resonance changes, under several conditions, of the Co(II)-substituted protein. 2. Only in the absence of copper are the optical and electron paramagnetic resonance spectra of the cobalt chromophore appreciably affected by alkaline pH or by cyanide. With both reagents the reaction with the copper-containing protein appears to involve the water molecule bound to the copper and does not affect the magnetic coupling between copper and cobalt. 3. The reaction of cyanide with the copper-free Co(II) protein leads to a slow detachment of cobalt from the protein as pentacyanocobalt. An oxygen adduct forms in air, analogous to that described in Co(II) carbonic anhydrase (Haffner, P. H. and Coleman, J. E. (1975) J. Biol. Chem. 250, 996--1005.) 4. Acid titration modifies the Co(II) spectra in the same way in the Cu-containing and in the Cu-free protein and brings about uncoupling of the Co(II)--Cu(II) system. Protonation of histidine-61 on the zinc facing nitrogen is suggested. 5. H2O2 modifies the cobalt chromophore only in the presence of copper. Magnetic coupling between Cu(II) and Co(II) seems to be still present after H2O2 inactivation of the enzyme.     

Determining the three-dimensional fold of a protein from approximate constraints

We propose a new approach for calculating the three-dimensional (3D) structure of a protein from distance and dihedral angle constraints derived from experimental data. We suggest that such constraints can be obtained from experiments such as tritium planigraphy, chemical or enzymatic cleavage of the polypeptide chain, paramagnetic perturbation of nuclear magnetic resonance (NMR) spectra, measurement of hydrogen-exchange rates, mutational studies, mass spectrometry, and electron paramagnetic resonance. These can be supplemented with constraints from theoretical prediction of secondary structures and of buried/exposed residues. We report here distance geometry calculations to generate the structures of a test protein Staphylococcal nuclease (STN), and the HIV-1 rev protein (REV) of unknown structure. From the available 3D atomic coordinates of STN, we set up simulated data sets consisting of varying number and quality of constraints, and used our group's Self Correcting Distance Geometry (SECODG) program DIAMOD to generate structures. We could generate the correct tertiary fold from qualitative (approximate) as well as precise distance constraints. The root mean square deviations of backbone atoms from the native structure were in the range of 2.0 A to 8.3 A, depending on the number of constraints used. We could also generate the correct fold starting from a subset of atoms that are on the surface and those that are buried. When we used data sets containing a small fraction of incorrect distance constraints, the SECODG technique was able to detect and correct them. In the case of REV, we used a combination of constraints obtained from mutagenic data and structure predictions. DIAMOD generated helix-loop-helix models, which, after four self-correcting cycles, populated one family exclusively. The features of the energy-minimized model are consistent with the available data on REV-RNA interaction. Our method could thus be an attractive alternative for calculating protein 3D structures, especially in cases where the traditional methods of X-ray crystallography and multidimensional NMR spectroscopy have been unsuccessful.     

The solution structure of desulforedoxin,a simple iron-sulfur protein

Desulforedoxin is a simple dimeric protein isolated from Desulfovibrio gigas containing a distorted rubredoxin-like center with one iron coordinated by four cysteinyl residues (7.9?kDa with a 36-amino-acid monomer). ¹H NMR spectra of the oxidized Dx(Fe³⁺) and reduced Dx(Fe²⁺) forms were analyzed. The spectra show substantial line broadening due to the paramagnetism of iron. However, very low-field-shifted resonances, assigned to Hβ protons, were observed in the reduced state and their temperature dependence analyzed. The active site of Dx was reconstituted with zinc, and its solution structure was determined using 2D NMR methods. This diamagnetic form gave high-resolution NMR data enabling the identification of all the amino acid spin systems. Sequential assignment and the determination of secondary structural elements was attempted using 2D NOESY experiments. However, because of the symmetrical dimer nature of the protein standard, NMR sequential assignment methods could not resolve all cross peaks due to inter- and intra-chain effects. The X-ray structure enabled the spatial relationship between the monomers to be obtained, and resolved the assignment problems. Secondary structural features could be identified from the NMR data; an antiparallel β-sheet running from D5 to V18 with a well-defined β-turn around cysteines C9 and C12. The section G22 to T25 is poorly defined by the NMR data and is followed by a turn around V27-C29. The C-terminus ends up near residues V6 and Y7. Distance geometry (DG) calculations allowed families of structures to be generated from the NMR data. A family of structures with a low target function violation for the Dx monomer and dimer were found to have secondary structural elements identical to those seen in the X-ray structure. The amide protons for G4, D5, G13, L11 NH and Q14 NHε amide protons, H-bonded in the X-ray structure, were not seen by NMR as slowly exchanging, while structural disorder at the N-terminus, for the backbone at E10 and for the section G22–T25, was observed. Comparison between the Fe and Zn forms of Dx suggests that metal substitution does not have an effect on the structure of the protein.     

The solution structure of a [3Fe-4S] ferredoxin: oxidised ferredoxin II from Desulfovibrio gigas

The use of standard 2D NMR experiments in combination with 1D NOE experiments allowed the assignment of 51 of the 58 spin systems of oxidised [3Fe-4S] ferredoxin isolated from Desulfovibrio gigas. The NMR solution structure was determined using data from 1D NOE and 2D NOESY spectra, as distance constraints, and information from the X-ray structure for the spin systems not detected by NMR in torsion angle dynamics calculations to produce a family of 15 low target function structures. The quality of the NMR family, as judged by the backbone r.m.s.d. values, was good (0.80?Å), with the majority of φ/ψ angles falling within the allowed region of the Ramachandran plot. A comparison with the X-ray structure indicated that the overall global fold is very similar in solution and in the solid state. The determination of the solution structure of ferredoxin II (FdII) in the oxidised state (FdII_ox) opens the way for the determination of the solution structure of the redox intermediate state of FdII (FdII_int), for which no X-ray structure is available.     

2D NMR studies of aminoglycoside antibiotics. Use of relayed coherence transfer for 1H resonance assignment and in situ structure elucidation of amikacin derivatives in reaction mixtures

Phase-sensitive 2D 1H/1H COSY spectra can be used to identify the structures of individual pure specimens of the aminoglycoside antibiotic amikacin and its N-hemisuccinyl derivatives. However, even at 500 MHz the 2D chemical shift dispersion does not allow for unambiguous assignment of all cross-peaks. By use of 2D relayed coherence transfer experiments (RELAY) optimized to detect two-step 1H/1H scalar interactions in which one of the J-values is small, sufficient additional correlations can be obtained from the frequency-isolated resonances to allow facile tracing of all scalar connectivities. Complete assignments of the 1H NMR spectra of amikacin, its 6'-N-hemisuccinamide, and a novel bis(acylate) [gamma-N-(p-vinylbenzoyl)amikacin 6'-N-hemisuccinamide] were obtained for aqueous media. The NMR spectrum of amikacin free base was also assigned in dimethyl sulfoxide solution. The RELAY experiment can be extended to the analysis of reaction mixtures, which allows for the identification and resonance assignment of regioisomeric amikacin haptens in the mixture state. All of the N-monohemisuccinyl isomers of amikacin have been identified in reaction mixtures through the RELAY experiment. The relative reactivities of the amino functions of amikacin toward acylating agents were found to be 6'-N greater than 3-N equal to or greater than 3"-N equal to or greater than gamma-N. However, this reactivity order is altered after the initial acylation event.     

Spatially encoded strategies in the execution of biomolecular-oriented 3D NMR experiments

Three-dimensional nuclear magnetic resonance (3D NMR) provides one of the foremost analytical tools available for the elucidation of biomolecular structure, function and dynamics. Executing a 3D NMR experiment generally involves scanning a series of time-domain signals S(t ₃), as a function of two time variables (t ₁, t ₂) which need to undergo parametric incrementations throughout independent experiments. Recent years have witnessed extensive efforts towards the acceleration of this kind of experiments. Among the different approaches that have been proposed counts an “ultrafast” scheme, which distinguishes itself from other propositions by enabling—at least in principle—the acquisition of the complete multidimensional NMR data set within a single transient. 2D protein NMR implementations of this single-scan method have been demonstrated, yet its potential for 3D acquisitions has only been exemplified on model organic compounds. This publication discusses a number of strategies that could make these spatial encoding protocols compatible with 3D biomolecular NMR applications. These include a merging of 2D ultrafast NMR principles with temporal 2D encoding schemes, which can yield 3D HNCO spectra from peptides and proteins within ≈100 s timescales. New processing issues that facilitate the collection of 3D NMR spectra by relying fully on spatial encoding principles are also assessed, and shown capable of delivering HNCO spectra within 1 s timescales. Limitations and prospects of these various schemes are briefly addressed.     

Morpholino spin-labeling for base-pair sequencing of a 3'-terminal RNA stem by proton homonuclear Overhauser enhancements: yeast ribosomal 5S RNA

Base-pair sequences for 5S and 5.8S RNAs are not readily extracted from proton homonuclear nuclear Overhauser enhancement (NOE) connectivity experiments alone, due to extensive peak overlap in the downfield (11-15 ppm) proton NMR spectrum. In this paper, we introduce a new method for base-pair proton peak assignment for ribosomal RNAs, based upon the distance-dependent broadening of the resonances of base-pair protons spatially proximal to a paramagnetic group. Introduction of a nitroxide spin-label covalently attached to the 3'-terminal ribose provides an unequivocal starting point for base-pair hydrogen-bond proton NMR assignment. Subsequent NOE connectivities then establish the base-pair sequence for the terminal stem of a 5S RNA. Periodate oxidation of yeast 5S RNA, followed by reaction with 4-amino-2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO-NH2) and sodium borohydride reduction, produces yeast 5S RNA specifically labeled with a paramagnetic nitroxide group at the 3'-terminal ribose. Comparison of the 500-MHz 1H NMR spectra of native and 3'-terminal spin-labeled yeast 5S RNA serves to identify the terminal base pair (G1 . C120) and its adjacent base pair (G2 . U119) on the basis of their proximity to the 3'-terminal spin-label. From that starting point, we have then identified (G . C, A . U, or G . U) and sequenced eight of the nine base pairs in the terminal helix via primary and secondary NOE's.     

New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA-protein complexes

NMR spectra of large RNAs are difficult to assign because of extensive spectral overlap and unfavorable relaxation properties. Here we present a new approach to facilitate assignment of RNA spectra using a suite of four 2D-filtered/edited NOESY experiments in combination with base-type-specific isotopically labeled RNA. The filtering method was developed for use in 3D filtered NOESY experiments (Zwahlen et al., 1997), but the 2D versions are both more sensitive and easier to interpret for larger RNAs than their 3D counterparts. These experiments are also useful for identifying intermolecular NOEs in RNA-protein complexes. Applications to NOE assignment of larger RNAs and an RNA-protein complex are presented.     

Optical spectroscopic investigation of the alkaline transition in umecyanin from horseradish root

Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a family of plant cupredoxins. The protein possesses the typical type-1 His(2)Cys equatorial ligand set at its mononuclear copper site but has an axial Gln ligand in place of the usual weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. UMC exhibits, like other phytocyanins, altered visible, EPR, and paramagnetic (1)H NMR spectra at elevated pH values and also a modified reduction potential. This alkaline transition occurs with a pK(a) of approximately 10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study, we investigate the alkaline transition by complementary optical spectroscopic techniques. The contemporary use of absorption, fluorescence, dynamic light scattering, and resonance Raman spectroscopy allows us to demonstrate that the alkaline transition induces a reorganization of the protein and that the overall size of UMC increases, but protein aggregation does not occur. The transition does not have a dramatic influence on the active-site environment of UMC, but there are subtle alterations in the Cu site geometry. Direct evidence for the strengthening of a Cu-N(His) bond is presented, which is in agreement with the hypothesis that the deprotonation of the N(epsilon2)H moiety of one of the His ligands is the cause of the alkaline transition. A weakening of the Cu-S(Cys) bond is also observed which, along with a weakened axial interaction, must be due to the enhanced Cu-N(His) interaction.