Conformational analysis of a cyclic thymopoietin-analogue by 1H n.m.r. spectroscopy and restrained molecular dynamics simulations

The internuclear distances of the cyclic thymopoietin derivative c[D-Val-Tyr-Arg-Lys-Glu] have been determined using two-dimensional nuclear Overhauser n.m.r. spectroscopy. These distances are used as constraints for a restrained Molecular Dynamics (MD) simulation. The two starting structures used for the calculations consist of a beta and gamma turn for model 1 and two gamma turns for model 2. The rms difference in atomic positions of the two conformations is 0.242 nm. They converge during the restrained MD simulation to the same final structure. The positional rms difference of the time averaged (5-14 ps) conformations is 0.011 nm. The hydrogen bond pattern is similar to that of model 1, but in addition we find three more gamma turns. The vicinal NH-C alpha H couplings agree well with those calculated from the time averaged structures.     

Sequence-specific 1H-n.m.r. assignments and peptide backbone conformation in rat epidermal growth factor.

The solution conformation of rat epidermal growth factor (EGF) has been investigated by proton n.m.r. techniques. Two-dimensional proton n.m.r. experiments have allowed sequential resonance assignments to be made for most protons. On the basis of these assignments, two regions of anti-parallel beta-sheet structure have been derived from the n.m.r. data. A beta-sheet segment running from about V19 to V23 (capital letters refer to amino acids in the single-letter notation) is folded onto a beta-sheet segment running from R28 to N32 and joined by a chain reversal from E24 to D27. A second region involves a beta-turn from V34 to Y37, which starts a short beta-sheet up to G39, followed by a chain reversal up to Q43, which leads to folding of the C-terminal beta-sheet segment, i.e. H44-R45, running antiparallel to the short Y37 beta-sheet segment. The N-terminal segment up to G18 exists in a multiple bend conformation and is folded on to the V29-V23/R28-N32 beta-sheet such that Y10, Y13, Y22 and Y29 are proximal to each other. Structural comparison of rat, murine and human EGFs indicates a number of highly conserved structural features common to at least these species of EGF.     

Rapid acquisition of three-dimensional triple-resonance experiments using pulsed field gradient techniques

Summary A rapid method for recording three-dimensional triple-resonance experiments utilising pulsed field gradient techniques is proposed, and applied to the HNCO experiment. In order to optimise the sensitivity of the method, a short phase cycle is used in conjunction with the pulsed field gradients to select the desired coherence transfer pathway. The method is demonstrated for the HU protein.     

Photo-CIDNP study of the interaction between lac repressor headpiece and lac operator DNA

Lac repressor headpiece (HP) and intact lac repressor have been studied using the photo-CIDNP method. At neutral pH histidine 29, tyrosines 7, 12 and 17 and methionine 1 are polarised. His-29 polarizations are weaker and broader in HP59 than in HP51 indicating that the C-terminal octapeptide in HP59 adopts a conformation that allows an interaction with His-29. The photo-CIDNP spectra of intact lac repressor and HP51 are very similar, showing that the same residues are accessible to the photo-excited flavin. An equimolar mixture of HP51 and a 14 base pair lac operator fragment strongly suppresses the photo-CIDNP effect of tyrosines 7 and 17 and abolishes the His-29 polarizations. The results are compared with earlier photo-CIDNP measurements on a complex of headpiece with poly[d(AT)] and with a model derived from a 2D NMR study on a lac headpiece-operator complex.     

Stereospecific assignments of 1H-nmr methyl lines and conformation of valyl residues in the lac repressor headpiece

Two-dimensional ¹H-nmr methods are described to obtain information on the sidechain conformation of valyl residues of the lac repressor headpiece and to assign the resonances of their methyl groups stereospecifically. The spin–spin coupling constants (J_αβ) between C^αand C^β protons are obtained from two-dimensional correlated spectroscopy experiments. Large values for J_αβ(10–12 Hz) corresponding to trans orientations for these protons (g⁺ conformation) are found for all valyl residues in α-helical segments. For these valyl residues, the distance between one methyl group (γ₁)and the valyl amide proton is much shorter than for the other methyl group, so that stereospecific resonance assignments follow from relative intensities of the corresponding cross peaks in a two-dimensional nuclear Overhauser enhancement spectrum. Thus, streospecific assignments could be made for the methyl groups of Val 9, 20, 23, and 38 (of a total of eight valyl residues).     

Determination of protein structures from nuclear magnetic resonance data using a restrained molecular dynamics approach: the lac repressor DNA binding domain

A procedure is described to determine from NMR data the three-dimensional structure of biomolecules. This procedure combines model building with a restrained Molecular Dynamics algorithm, in which distance information from NOEs is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or "headpiece" (amino acids 1-51) of the lac repressor from E. coli, for which no crystal structure is available. The spatial structure of the headpiece is discussed in terms of known physical and biochemical data and of its DNA binding properties.     

Measurement of 15N-1H coupling constants in uniformly 15N-labeled proteins: Application to the photoactive yellow protein

A modified HNHB experiment is presented that allows thedetermination of J(NH) coupling constants directly from the ratio ofcross-peak to diagonal-peak intensities. The experiment was applied to thephotoactive yellow protein (PYP) and yielded the magnitude of 117³J(NH) coupling constants. In addition, 29³J(NH^(i–1)) coupling constantscould be measured, providing information about the backbone angle .These data, in conjunction with the magnitudes of the³J(H^NH) coupling constantsobtained from the HNHA spectrum, effectively discriminate the twopossibilities for the stereospecific assignment of theH resonances in glycine residues. For all eight glycineresidues in PYP that were not subject to conformational averaging and hadnon-degenerate H resonance frequencies, the J-couplingdata, together with limited NOE data, yielded the stereospecific assignmentof the H resonances for these residues. In addition,reliable and precise , dihedral constraints were also derived forthese residues from the J-coupling data.     

Complete 1H, 13C and 15N NMR assignments and secondary structure of the 269-residue serine protease PB92 from Bacillus alcalophilus

Summary The ¹H, ¹³C and ¹⁵N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CH_n carbons, all amide (NH and NH₂) nitrogens and 99.2% of the amide and CH_n protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D two-/three-/four-dimensional - HSQC heteronuclear single-quantum coherence - HMQC heteronuclear multiple-quantum coherence - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - NOE nuclear Overhauser enhancement (connectivity) - NOESY 2D NOE spectroscopy Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667].     

Spatial arrangement of the three alpha helices in the solution conformation of E. coli lac repressor DNA-binding domain

The relative orientations of the 3 helices in the DNA-binding domain ('headpiece') of lac repressor have been determined using distance constraints obtained from 2-dimensional 1H nuclear Overhauser enhancement spectra. The relative orientations of its helices is similar to that of the central 3 helices in the DNA-binding domain of the lambda repressor of the bacteriophage lambda.     

EPR studies of the photodissociation reactions of cytochrome c oxidase-nitric oxide complexes

Three complexes of NO with cytochrome c oxidase are described which are all photodissociable at low temperatures as measured by EPR. The EPR parameters of the cytochrome a²⁺₃-NO complex are the same both in the fully reduced enzyme and in the mixed-valence enzyme. The kinetics of photodissociation of cytochrome a²⁺₃-NO and recombination of NO with cytochrome a²⁺₃ (in the 30–70 K region) revealed no differences in structure between cytochrome a²⁺₃ in the fully reduced and the mixed-valence states. The action spectrum of the photodissociation of cytochrome a²⁺₃-NO as measured by EPR has maxima at 595, 560 and 430 nm, and corresponds to the absorbance spectrum of cytochrome a²⁺₃-NO. Photodissociation of cytochrome a²⁺₃-NO in the mixed-valence enzyme changes the EPR intensity at g 3.03, due to electron transfer from cytochrome a²⁺₃ to cytochrome a³⁺. The extent of electron transfer was found to be temperature dependent. This suggests that a conformational change is coupled to this electron transfer. The complex of NO with oxidized cytochrome c oxidase shows a photodissociation reaction and recombination of NO (in the 20–40 K region) which differ completely from those observed in cytochrome a²⁺₃-NO. The observed recombination occurs at a temperature 15 K lower than that found for the cytochrome a²⁺₃-NO complex. The action spectrum of the oxidized complex shows a novel spectrum with maxima at 640 and below 400 nm; it is assigned to a Cu²⁺_B-NO compound. The triplet species with Δm_s = 2 EPR signals at g 4 and Δm_s = 1 signals at g 2.69 and 1.67, that is observed in partially reduced cytochrome c oxidase treated with azide and NO, can also be photodissociated.