1H NMR studies of lambda cro repressor. 2. Sequential resonance assignments of the 1H NMR spectrum

The cro repressor protein from bacteriophage lambda has been studied in solution by two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). Following the approach of Wüthrich and co-workers [Wüthrich, K., Wider, G., Wagner, G., & Braun, W. (1982) J. Mol. Biol. 155, 311-319], individual spin systems were identified by J-correlated spectroscopy (COSY) supplemented, where necessary, by relayed coherence transfer spectroscopy (RELAY). Nuclear Overhauser effect spectroscopy (NOESY) was used to obtain sequence-specific assignments. From the two-dimensional spectra, the peptide backbone resonances (NH and C alpha H) for 65 of the 66 amino acids were assigned, as well as most of the side chain resonances. The chemical shifts for the assigned protons are reported at 35 degrees C in 10 mM potassium phosphate, pH 6.8, and in 10 mM potassium phosphate, pH 4.6, 0.2 M KCl, and 0.1 mM EDTA. Small shifts were observed for some resonances upon addition of salt, but no major changes in the spectrum were seen, indicating that no global structural change occurs between these ionic strengths. NOE patterns characteristic of alpha-helices, beta-strands, and turns are seen in various regions of the primary sequence. From the location of these regions the secondary structure of cro in solution appears to be virtually identical with the crystal structure [Anderson, W. F., Ohlendorf, D. H., Takeda, Y., & Matthews, B. W. (1981) Nature (London) 290, 754-758]. Missing assignments include the Pro-59 resonances and the peripheral protons of the eight lysine, the three arginine, and three of the five isoleucine residues.     

Sequence-specific 1H-NMR assignments and determination of the secondary structure in aqueous solution of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica

Sequence-specific assignments of the 1H-nuclear magnetic resonance (NMR) spectra of the cardiotoxins CTXIIa and CTXIIb from Naja mossambica mossambica were obtained using two-dimensional NMR experiments at 500 MHz and the independently determined amino acid sequences. Assignments were obtained from data at 25 degrees C and 45 degrees C for all but one backbone proton of the 60 residues in each protein. Complete or partial assignments are also reported for the side-chain protons. These assignments supercede those published previously for the toxin preparation VII2 [Hosur, R. V., Wider, G. & Wüthrich K. (1983) Eur. J. Biochem. 130, 497-508]. The 1H/2H-exchange kinetics were measured in 2H2O at 20 degrees C for the amide protons and the N-terminal amino group. These and additional NMR data enabled the determination of the secondary structure in aqueous solution, which is virtually identical in CTXIIa and CTXIIb. Both proteins contain a short double-stranded antiparallel beta-sheet comprising the residues 2-4 and 11-13, and a triple-stranded antiparallel beta-sheet consisting of the residues 20-26, 35-39, and 49-55. The two peripheral strands of the triple-stranded beta-structure were found to be connected by a right-handed cross-over, and the locations of several tight turns were also identified.     

Amide proton exchange in proteins by EX1 kinetics: studies of the basic pancreatic trypsin inhibitor at variable p2H and temperature

With the use of one-dimensional 1H nuclear magnetic resonance, two-dimensional correlated spectroscopy, and two-dimensional nuclear Overhauser enhancement spectroscopy, the exchange mechanisms for numerous individual amide protons in the basic pancreatic trypsin inhibitor (BPTI) were investigated over a wide range of p2H and temperature. Correlated exchange under an EX1 regime was observed only for the most slowly exchanging protons in the central hydrogen bonds of the antiparallel beta-sheet and only over a narrow range of temperature and p2H, i.e., above ca. 55 degrees C and between p2H 7 and 9, where the opening rates of the structure fluctuations which promote the exchange of these protons are of the order 0.1 min-1. At p2H below 7, the exchange of this most stable group of protons is uncorrelated and is governed by an EX2 mechanism. At p2H above 9, the exchange is also uncorrelated and occurs via either EX2 or EX1 processes promoted by strictly local structure fluctuations. For all other backbone amide protons in BPTI, the exchange was found to be uncorrelated and by an EX2 mechanism under all conditions of p2H and temperature where quantitative measurements could be obtained with the methods used, i.e., for kex approximately less than 5 min-1. From these observations with BPTI it can be concluded that the amide proton exchange in globular proteins is quite generally via EX2 processes, with rare exceptions for measurements with extremely stable protons at high temperature and basic p2H. This emphasizes the need for further development of suitable concepts for the structural interpretation of EX2 amide proton exchange [Wagner, G. (1983) Q. Rev. Biophys. 16, 1-57; Wagner, G., Stassinopoulou, C. I., & Wüthrich, K. (1984) Eur. J. Biochem. 145, 431-436] and for more detailed investigations of the intrinsic exchange rates for solvent-exposed amide protons in the "open" states of a protein [Roder, H., Wagner, G., & Wüthrich, K. (1985) Biochemistry (following paper in this issue)].     

‘Random coil’ 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG

Summary Proton chemical shifts of a series of disordered linear peptides (H-Gly-Gly-X-Gly-Gly-OH, with X being one of the 20 naturally occurring amino acids) have been obtained using 1D and 2D ¹H NMR at pH 5.0 as a function of temperature and solvent composition. The use of 2D methods has allowed some ambiguities in side-chain assignments in previous studies to be resolved. An additional benefit of the temperature data is that they can be used to obtain ‘random coil’ amide proton chemical shifts at any temperature between 278 and 318 K by interpolation. Changes of chemical shift as a function of trifluoroethanol concentration have also been determined at a variety of temperatures for a subset of peptides. Significant changes are found in backbone and side-chain amide proton chemical shifts in these ‘random coil’ peptides with increasing amounts of trifluoroethanol, suggesting that caution is required when interpreting chemical shift changes as a measure of helix formation in peptides in the presence of this solvent. Comparison of the proton chemical shifts obtained here for H-Gly-Gly-X-Gly-Gly-OH with those for H-Gly-Gly-X-Ala-OH [Bundi, A. and Wüthrich, K. (1979) Biopolymers, 18, 285–297] and for Ac-Gly-Gly-X-Ala-Gly-Gly-NH₂ [Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S. and Sykes, B.D. (1995) J. Biomol. NMR, 5, 67–81] generally shows good agreement for CH protons, but reveals significant variability for NH protons. Amide proton chemical shifts appear to be highly sensitive to local sequence variations and probably also to solution conditions. Caution must therefore be exercised in any structural interpretation based on amide proton chemical shifts.     

Assignment of the 13C-NMR spectra of virgin and reactive-site modified turkey ovomucoid third domain

The virgin (reactive-site Leu18-Glu19 peptide bond intact) and modified (reactive-site Leu18-Glu19 peptide bond hydrolyzed) forms of turkey ovomucoid third domain (OMTKY3 and OMTKY3*, respectively) have been analyzed by proton-detected 1H(13C) two-dimensional single-bond correlation (1H[13C]SBC) spectroscopy. Previous 1H-nmr assignments of these proteins [A.D. Robertson, W.M. Westler, and J.L Markley (1988) Biochemistry, 27, 2519-2529; G. I. Rhyu and J. L. Markley (1988) Biochemistry, 27, 2529-2539] have been extended to directly bonded 13C atoms. Assignments have been made to 52 of the 56 backbone 13C alpha-1H units and numerous side-chain 13C-1H groups in both OMTKY3 and OMTKY3*. The largest changes in the 13C chemical shift upon conversion of OMTKY3 to OMTKY3* occur at or near the reactive site, and tend toward values observed in small peptides. Moreover, the side-chain prochiral methylene protons attached to the C gamma of Glu19 and C delta of Arg21 show nonequivalent chemical shifts in OMTKY3 but more equivalent chemical shifts in OMTKY3*. These results suggest that the reactive site region becomes less ordered upon hydrolysis of the Leu18-Glu19 peptide bond. Comparison of 13C alpha chemical shifts of OMTKY3 and bovine pancreatic trypsin inhibitor [D. Brühuiler and G. Wagner (1986) Biochemistry 25, 5839-5843; N. R. Nirmala and G. Wagner (1988) Journal of the American Chemical Society, 110, 7557-7558] with small peptide values [R. Richarz and K. Wüthrich (1978) Biopolymers, 17, 2133-2141] suggests that 13C alpha chemical shifts of residues residing in helices are generally about 2 ppm downfield of resonances from nonhelical residues.     

Kurt Wüthrich and NMR of biological macromolecules

Nuclear magnetic resonance (NMR) spectroscopy is the only experimental technique that can determine the structures and dynamics of biological macromolecules and their complexes in solution and with atomic resolution. The award of the 2002 Nobel Prize in Chemistry to Kurt Wüthrich of the Swiss Federal Institute of Technology and The Scripps Research Institute honors his pioneering efforts in developing and applying this technique. Wüthrich shared the prize with John B. Fenn and Koichi Tanaka, who were recognized for the development of ionization methods for the analysis of proteins using mass spectrometry.     

NMR studies of toxin III from the sea anemone Radianthus paumotensis and comparison of its secondary structure with related toxins

Nearly complete assignments of the proton nuclear magnetic resonance (NMR) spectrum of the polypeptide toxin III from the sea anemone Radianthus paumotensis (RP) are presented. The secondary structures of the related toxins RP II and RP III are described and are compared with each other and with another related toxin ATX Ia from Anemonia sulcata [Widmer, H., Wagner, G., Schweitz, H., Lazdunski, M., & Wüthrich, K. (1988) Eur. J. Biochem. 171, 177-192]. All of these proteins contain a highly twisted four-strand antiparallel beta-sheet core connected by loops of irregular structure. From the work done with AP-A from Anthopleura xanthogrammica [Gooley, P. R., & Norton, R. S. (1986) Biochemistry 25, 2349-2356], it is clear that this homologous toxin also has the same basic core. Some small differences are seen in the structures of these toxins, particularly in the position of the N-terminal residues that form one of the outside strands of the beta-sheet. In addition, the R. paumotensis toxins are two residues longer, extending the third strand of sheet containing the C-terminal residues. A comparison of chemical shifts for assigned residues is also presented, in general supporting the similarity of structure among these proteins.     

Hydrogen exchange in thermally denatured ribonuclease A

Hydrogen exchange has been used to test for the presence of nonrandom structure in thermally denatured ribonuclease A (RNase A). Quenched-flow methods and 2D 1H NMR spectroscopy were used to measure exchange rates for 36 backbone amide protons (NHs) at 65 degrees C and at pH* (uncorrected pH measured in D2O) values ranging from 1.5 to 3.8. The results show that exchange is approximately that predicted for a disordered polypeptide [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158]; we thus are unable to detect any stable hydrogen-bonded structure in thermally denatured RNase A. Two observations suggest, however, that the predicted rates should be viewed with some caution. First, we discovered that one of the approximations made by Molday et al. (1972), that exchange for valine NHs is similar to that for alanine NHs, had to be modified; the exchange rates for valine NHs are about 4-fold slower. Second, the pH minima for exchange tend to fall at lower pH values than predicted, by as much as 0.45 pH units. These results are in accord with those of Roder and co-workers for bovine pancreatic trypsin inhibitor [see Table I in Roder, H., Wagner, G., & Wüthrich, K. (1985) Biochemistry 24, 7407-7411]. The origin of the disagreement between predicted and observed pH minima is unknown but may be the high net positive charge on these proteins at low pH. In common with some other thermally unfolded proteins, heat-denatured ribonuclease A shows a significant circular dichroism spectrum in the far-ultraviolet region [Labhardt, A. M. (1982) J. Mol. Biol. 157, 331-355].(ABSTRACT TRUNCATED AT 250 WORDS)     

Tryptophan residues of creatine kinase: a fluorescence study

Spectroscopic studies of rabbit skeletal muscle creatine kinase (CPK) and its complexes with adenosine phosphates have long suggested the occurrence of a tryptophan residue at or near the coenzyme binding sites [K?gi, J. H. R., Li, T.-K., & Vallee, B. L. (1971) Biochemistry 10, 1007-1015; Price, N. C. (1972) FEBS Lett. 24, 21-23]. This conjecture was further supported by nuclear Overhauser effect (NOE) 1H NMR studies indicating through-space interactions between protons of the adenine ring of bound ADP and one or more aromatic side chains of the proteins [Vasák, M., Nagayama, K., Wüthrich, K., Mertens, M. L., & K?gi, J. H. R. (1979) Biochemistry 18, 5050-5055]. Further evidence for a tryptophan residue in the environment of the active site has now been obtained by fluorescence-quenching studies using iodide and acrylamide as external quenchers. Thus, while by the addition of iodide the tryptophan fluorescence of unliganded CPK is reduced to about 75% of the unquenched control, no such effect is manifested upon addition of this quencher to the CPK.ADP and CPK.ATP complexes. Similarly, the relative effectiveness of quenching of the CPK-coenzyme complexes by acrylamide is only about 60% of that measured in the unliganded enzyme. Both these data and the spectral characteristics of the quenched fluorescence suggest that coenzyme binding perturbs a tryptophan residue that is close to the active site and that is partially exposed to the solvent. The differential effectiveness of external quenchers on unliganded and liganded CPK allows the determination of the ligand binding equilibria by fluorescence-quenchability titration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-dimensional NMR approaches to the study of protein structure and function

Two-dimensional Fourier transform methods for homonuclear proton NMR spectroscopy have been introduced by Wüthrich and Ernst as a means of extending assignments in spectra of proteins. Multinuclear two-dimensional approaches also appear promising. We are applying current one- and two-dimensional NMR methods to protein family members that differ from one another by one or more amino acid substitutions. The overall goal is to elucidate relationships among the sequences, structures, and functions of these proteins: for example, to delineate primary structural requirements for changes in observable properties such as conformation, amino acid side chain dynamics, hydrogen exchange dynamics, pK'a values, and oxidation-reduction potentials. The ovomucoids from a variety of species of birds, which include a single set of 12 pairs of third-domain proteins (Mr = 6062 for turkey third domain, similar for others) that differ by single amino acid substitutions, provide a favorable system for the study of the structural and dynamic effects of single amino acid replacements. X-ray crystallographic structures of two ovomucoid third domains are available. Other series of proteins being studied by these methods include the photosynthetic electron transport proteins ferredoxin and plastocyanin.     

Cytogenetic characteristics of species of the Chironomus plumosus group (Chironomidae, Diptera) in Finland

The cytogenetic characteristics of species of the Chironomus plumosus group in Finland were examined. The species included C. balatonicus Devai, Wülker, Scholl, C. entis Shobanov, C. plumosus L., C. muratensis Ryser, Scholl, Wülker, and two karyotypes of unknown species. All belonged to the thummi complex having 2n = 8 chromosomes, with arm combinations of AB, CD, EF, G. In C. balatonicus arms A to G the band sequences corresponded with those of other populations. A new band sequence was found in arm B. In C. entis an arm A had a band sequence similar to those in C. agilis. A large pericentric inversion was observed in chromosome AB. The arms B to G had band sequences typical for C. entis. The chromosome arms A and B in C. plumosus from Lake Marsjon had band sequences corresponding to those of C. agilis and C. entis, respectively. C. plumosus from Helsinki may be a more divergent population with a large amount of centromere heterochromatin. C. muratensis was not distinguishable by band sequences from those of other Palaearctic populations. Two new karyotypes similar to those of species of the plumosus group have been described.     

Structural studies of cytochrome b5: complete sequence-specific resonance assignments for the trypsin-solubilized microsomal ferrocytochrome b5 obtained from pig and calf

We report complete sequence-specific proton resonance assignments for the trypsin-solubilized microsomal ferrocytochrome b5 obtained from calf liver. In addition, sequence-specific resonance assignments for the main-chain amino acid protons (i.e., C alpha, C beta, and amide protons) are also reported for the porcine cytochrome b5. Assignment of the majority of the main-chain resonances was rapidly accomplished by automated procedures that used COSY and HOHAHA peak coordinates as input. Long side chain amino acid spin system identification was facilitated by long-range coherence-transfer experiments (HOHAHA). Problems with resonance overlap were resolved by examining differences between the two-dimensional 500-MHz NMR spectra of rabbit, pig, and calf proteins and by examining the temperature-dependent variation of amide proton resonances. Calculations of the aromatic ring-current shifts for protons that the X-ray crystal structure indicated were proximal to aromatic residues were found to be useful in corroborating assignments, especially those due to the large shifts induced by the heme. Assignment of NOESY cross peaks was greatly facilitated by a prediction of intensities using a complete relaxation matrix analysis based on the crystal structure. These results suggest that the single-crystal X-ray structure closely resembles that of the solution structure although there is evidence that the solution structure has a more dynamic character.     

Structural studies of Desulfovibrio vulgaris ferrocytochrome c3 by two-dimensional NMR.

Two-dimensional NMR has been used to make specific assignments for the four haems in Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c3 and to determine their haem core architecture. The NMR signals from the haem protons were assigned according to type using two-dimensional NMR experiments which led to four sets of signals, one for each of the haems. Specific assignments were obtained by calculating the ring current shifts which arise from other haems and aromatic residues. Observation of interhaem NOEs confirmed the assignments and established that the relative orientation of the haems is identical to that found in the crystal structure of D. vulgaris (Miyazaki F.) ferricytochrome c3. Assignments were also made for all the aromatic residues except for the haem ligands and F20, which is shifted under the main envelope of signals. The NOEs observed between these aromatic protons and haem protons confirm the similarity between the structures in solution and in the crystal. The assignments reported here are the basis for the cross-assignments of the four microscopic haem redox potentials to specific haems in the protein structure [Salgueiro, C. A., Turner, D. L., Santos, H., LeGall, J. and Xavier, A. V. (1992) FEBS Lett., in the press]     

A 13C NMR spin-lattice relaxation study of the interaction of myelin proteins with lipid vesicles

Natural abundance 13C nuclear magnetic spin-lattice relaxation times have been measured for bovine brain phosphatidylserine vesicles with and without bound proteins. The relaxation times were lower than published values for the corresponding nuclei in egg phosphatidylcholine, but showed the same trend, with relaxation times increasing along the acyl chains away from the polar headgroup. These times were inversely related to the degree of saturation of the lipid. Cytochrome c caused insignificant changes in the lipid acyl chain relaxation rates but reduced the resonance intensities, in agreement with Brown and Wüthrich (Biochem. Biophys. Acta 468 (1977) 389). In contrast, the basic protein from bovine myelin did not affect the intensities but reduced the relaxation times for 13C nuclei near the bilayer centre, and for nuclei near carbon-carbon double bonds. These proteins also dramatically broadened the serine headgroup carboxyl resonance. It appears, in accord with other recent evidence, that the basic protein does penetrate the hydrophobic region of the bilayer (possibly to the centre), producing quantitatively similar changes in the relaxation rates to proteolipid protein, an integral membrane protein.     

Computer-assisted assignment of 2D1H NMR spectra of proteins: Basic algorithms and application to phoratoxin B

Summary A suite of computer programs (CLAIRE) is described which can be of assistance in the process of assigning 2D¹H NMR spectra of proteins. The programs embody a software implementation of the sequential assignment approach first developed by Wüthrich and co-workers (K. Wüthrich. G. Wider, G. Wagner and W. Braun (1982)J. Mol. Biol. 155, 311). After data-abstraction (peakpicking), the software can be used to detect patterns (spin systems), to find cross peaks between patterns in 2D NOE data sets and to generate assignments that are consistent with all available data and which satisfy a number of constraints imposed by the user. An interactive graphics program calledCONPAT is used to control the entire assignment process as well as to provide the essential feedback from the experimental NMR spectra. The algorithms are described in detail and the approach is demonstrated on a set of spectra from the mistletoe protein phoratoxin B, a homolog of crambin. The results obtained compare well with those reported earlier based entirely on a manual assignment process.     

Assignment of the 1H nuclear magnetic resonance spectrum of the trypsin inhibitor homologue K from Dendroaspis polylepis polylepis. Two-dimensional nuclear magnetic resonance at 360 and 500 MHz

The assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the trypsin inhibitor homologue K from the venom of Dendroaspis polylepis polylepis is described and documented. The assignments are based entirely on the amino acid sequence and on 2-dimensional n.m.r. experiments at 360 and 500 M Hz. Individual assignments were obtained for the backbone and C beta protons of all 57 residues of the inhibitor homologue K, with the exceptions of the N-terminal amino group, the amide protons of Arg16, Gly37 and Gly40 and the C beta protons of Arg16 and Pro19. The assignments for the non-labile protons of the amino acid side-chains are complete, with the exception of Gln29, Glu49 and all the proline, lysine and arginine residues. For Asn and Trp the labile side-chain protons have also been assigned. The chemical shifts for the assigned resonances are listed for an aqueous solution at 50 degrees C and pH 3.4.     

Besprechungen/Reviews

Book reviewed in this article: Zeitschriften: Maynard Smith, J. (1977): Parental investment: a prospective analysis Zeitschriften: Goss-Custard, J. D. (1977): Optimal foraging and the size selection of worms by Redshank Zeitschriften: Halliday, T. R. (1977): The effect of experimental manipulation of breathing behaviour on the sexual behaviour of the Smooth Newt Zeitschriften: Young, G. , und Th. G. Décarie (1977): An ethology-based catalogue of facial/vocal behaviour in infancy Zeitschriften: Sternglanz, S. H., J. L. Grey und M. Murakami (1977): Adult preferences for infantile facial features Zeitschriften: Bertram, B. C. R. (1977): Variation in the wing-song of the flappet lark Zeitschriften: McFarland, D. J. (1977): Decision making in animals Bücher: Heiligenberg, W. (1977): Principles of Electrolocation and Jamming Avoidance in Electric Fish Bücher: Löhrl, H. (1976): Der Mauerläufer. A. Ziemsen-Verlag, Wittenberg/Luther-stadt Bücher: Meyer, P. (1976): Taschenlexikon der Verhaltenskunde Bücher: Tembrock, G., ed. (1978): Wörterbücher der Biologie: Verhaltensbiologie, unter besonderer Berücksichtigung der Physiologie des Verhaltens Bücher: Heymer, A. (1977): Ethologisches WoUrterbuch — Ethological Dictionary — Vocabulaire Ethologique     

Secondary structure of acyl carrier protein as derived from two-dimensional 1H NMR spectroscopy

Sequence-specific assignments of 1H NMR resonances were obtained for the backbone protons in acyl carrier protein (ACP) from Escherichia coli, a protein of 77 residues. The observations, in the NOESY spectra, of 1H-1H sequential and medium-range connectivities indicate the presence of three or four alpha-helical segments joined by short sequences of mixed conformations. The observations are used to refine a secondary structure model previously proposed on the basis of a Chou-Fasman algorithm [Rock, C. O., & Cronan, J. E., Jr. (1979) J. Biol. Chem. 254, 9778-9785].