1H, 13C and 15N NMR assignments and secondary structure of the paramagnetic form of rat cytochrome b 5

Summary Modern multidimensional double- and triple-resonance NMR methods have been applied to assign the backbone and side-chain ¹³C resonances for both equilibrium conformers of the paramagnetic form of rat liver microsomal cytochrome b ₅. The assignment of backbone ¹³C resonances was used to confirm previous ¹H and ¹⁵N resonance assignments [Guiles, R.D. et al. (1993) Biochemistry, 32, 8329–8340]. On the basis of short- and medium-range NOEs and backbone ¹³C chemical shifts, the solution secondary structure of rat cytochrome b ₅ has been determined. The striking similarity of backbone ¹³C resonances for both equilibrium forms strongly suggests that the secondary structures of the two isomers are virtually identical. It has been found that the ¹³C chemical shifts of both backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts can be observed, is evaluated through calculations of the magnitude of such shifts.Abbreviations DANTE delays alternating with nutation for tailored excitation - DEAE diethylaminoethyl - DQF-COSY 2D double-quantum-filtered correlation spectroscopy - EDTA ethylenediaminetetraacetic acid - HCCH-TOCSY 3D proton-correlated carbon TOCSY experiment - HMQC 2D heteronuclear multiple-quantum correlation spectroscopy - HNCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons - HNCO 3D triple-resonance experiment correlating amide protons, amide nitrogens and carbonyl carbons - HNCOCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons via carbonyl carbons - HOHAHA 2D homonuclear Hartmann-Hahn spectroscopy - HOHAHA-HMQC 3D HOHAHA relayed HMQC - HSQC 2D heteronuclear single-quantum correlation spectroscopy - IPTG isopropyl thiogalactoside - NOESY 2D nuclear Overhauser enhancement spectroscopy - NOESY-HSQC 3D NOESY relayed HSQC - TOCSY 2D total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP trimethyl silyl propionate     

Gradient-enhanced 3D NOESY-HMQC spectroscopy

Summary A gradient-enhanced 3D NOESY-HMQC experiment is presented and applied to¹⁵N-labeled Mnt repressor (1–76) in¹H₂O. Both coherence selection and¹H₂O suppression are achieved using gradients. A singlescan experiment can be recorded for each time increment, which greatly reduces recording time with respect to conventional methods using phase cycling. This can be of use for kinetic studies and for relatively unstable molecules.     

1H(C) and 1H(N) total NOE correlations in a single 3D NMR experiment. 15N and 13C time-sharing in t1 and t2 dimensions for simultaneous data acquisition

Simultaneous data acquisition in time-sharing (TS) multi-dimensional NMR experiments has been shown an effective means to reduce experimental time, and thus to accelerate structure determination of proteins. This has been accomplished by spin evolution time-sharing of the X and Y heteronuclei, such as ¹⁵N and ¹³C, in one of the time dimensions. In this work, we report a new 3D TS experiment, which allows simultaneous ¹³C and ¹⁵N spin labeling coherence in both t ₁ and t ₂ dimensions to give four NOESY spectra in a single 3D experiment. These spectra represent total NOE correlations between ¹H_N and ¹H_C resonances. This strategy of double time-sharing (2TS) results in an overall four-fold reduction in experimental time compared with its conventional counterpart. This 3D 2TS CN-CN-H HSQC-NOESY-HSQC pulse sequence also demonstrates improvements in water suppression, ¹⁵N spectral resolution and sensitivity, which were developed based on 2D TS CN-H HSQC and 3D TS H-CN-H NOESY-HSQC experiments. Combining the 3D TS and the 3D 2TS NOESY experiments, NOE assignment ambiguities and errors are considerably reduced. These results will be useful for rapid protein structure determination to complement the effort of discerning the functions of diverse genomic proteins.     

HNCO-based measurement of one-bond amide <Superscript>15</Superscript>N-<Superscript>1</Superscript>H couplings with optimized precision

A pair of 3D HNCO-based experiments have been developed with the aim of optimizing the precision of measurement of ¹J_NH couplings. Both pulse sequences record ¹J_NH coupling evolution during the entire constant time interval that ¹⁵N magnetization is dephasing or rephasing with respect to the directly bonded ¹³C′ nucleus, with ¹⁵N¹³C′ multiple quantum coherence maintained during the ¹³C′ evolution period. The first experiment, designed for smaller proteins, produces an apparent doubling of the ¹J_NH coupling without any accompanying increases in line width. The second experiment is a J-scaled TROSY-HNCO experiment in which the ¹J_NH coupling is measured by frequency difference between resonances offset symmetrically about the position of the downfield component of the ¹⁵N doublet (i.e. the TROSY resonance). This experiment delivers significant gains in precision of ¹J_NH coupling measurement compared to existing J-scaled TROSY-HNCO experiments. With the proper choice of acquisition parameters and sufficient sensitivity to acquire a 3D TROSY-HNCO experiment, it is shown that ¹J_NH couplings can be measured with a precision which approaches or exceeds the precision of measurement with which the frequency of the TROSY resonance itself can be determined.     

Comprehensive determination of <Superscript>3</Superscript>J<Subscript>HNHα</Subscript> for unfolded proteins using <Superscript>13</Superscript>C′-resolved spin-echo difference spectroscopy

An experiment is presented to determine ³J_HNHα coupling constants, with significant advantages for applications to unfolded proteins. The determination of coupling constants for the peptide chain using 1D ¹H, or 2D and 3D ¹H-¹⁵N correlation spectroscopy is often hampered by extensive resonance overlap when dealing with flexible, disordered proteins. In the experiment detailed here, the overlap problem is largely circumvented by recording ¹H-¹³C′ correlation spectra, which demonstrate superior resolution for unfolded proteins. J-coupling constants are extracted from the peak intensities in a pair of 2D spin-echo difference experiments, affording rapid acquisition of the coupling data. In an application to the cytoplasmic domain of human neuroligin-3 (hNlg3cyt) data were obtained for 78 residues, compared to 54 coupling constants obtained from a 3D HNHA experiment. The coupling constants suggest that hNlg3cyt is intrinsically disordered, with little propensity for structure.     

1,4-Di(1,2,3,4-tetrazol-2-yl)butane as a precursor of new 2D and 3D coordination polymers of Cu(II)

A series of new coordination polymers of Cu(II) have been prepared in a reaction between copper(II) perchlorate or tetrafluoroborate salt and a novel ligand 1,4-di(1,2,3,4-tetrazol-2-yl)butane (bbtz). The compounds were characterised by an elemental analysis, TG measurements, IR, EPR and UV-Vis spectroscopy. Crystal structures of bbtz and five complexes of Cu(II) were determined by a single crystal X-ray diffraction measurement performed at 100 K. The composition and architecture of the obtained complexes strongly depend on the reaction conditions especially on the kind of solvent. Investigated complexes are composed of polymeric macrocations and non-coordinated anions. In all cases the bbtz molecules act as the bidentate ligand coordinated to metal(II) ions via N4, N4^′ nitrogen atoms from tetrazole rings. The complexes {[Cu(bbtz)₂(MeOH)₂]X₂}_∞ (X=ClO₄⁻, BF₄⁻) crystallise from methanol as 2D coordination polymers. In these compounds central metal ions are coplanar linked by molecules of bbtz and a coordination sphere is completed by axially coordinated solvent molecules. The complexes {[Cu(bbtz)₃]X₂}_∞ (X=ClO₄⁻, BF₄⁻) were synthesised in EtOH/H₂O solvent system and posses a common network topology. In this group of complexes each central atom is linked by ligand molecules to six other in plane arranged central atoms resulting in 2D networks. Reactions between Cu(II) salts and bbtz performed in absolute ethanol resulted in the formation of the next type of product. In {[Cu(bbtz)₃](ClO₄)₂·2EtOH}_∞ neighboured copper(II) ions are linked by ligand molecules in the three directions what leads to the formation of 3D net. A crystal of this complex is composed of two mutually interpenetrated 3D networks.     

The effect of solvent on preparation of CH3NH3PbI3 photodetectors via an antisolvent-free method

During the fabrication of lateral-structured photodetectors based on CH₃NH₃PbI₃ film, antisolvents represented by toluene are usually used to accelerate the crystallization of perovskite. Using antisolvent not only leads to the formation of shrinkage holes at the bottom of the perovskite layer, but the toxicity of antisolvents would also hinder the industrial preparation of perovskite devices. An antisolvent-free method is a possible solution to avoid these problems. Here, we report a lateral-structured photodetector based on an antisolvent-free method. The lateral photodetector exhibited a high responsivity of 1.75 A⋅W⁻¹ and specific detectivity (D*) of 3.54 × 10¹² Jones. In particular, the results indicated that the solvent had an influence on perovskite film morphology, crystallization, and device performance. The prepared CH₃NH₃PbI₃ film presented needle-like crystals and low performance with single precursor solvent N,N-dimethylformamide (DMF). In comparison, appropriate mixing of dimethyl sulfoxide (DMSO) could improve the morphology, crystallization, and performance of the film. In addition, the solvent volume ratio of the precursor had a profound effect on the performance of the as-prepared photodetectors. At a DMSO:DMF volume ratio of 5:5, the as-prepared film had massive perovskite crystals and fewer defects, resulting in optimal device performance, which can be explained by Urbach energy.     

15NH/D-SOLEXSY experiment for accurate measurement of amide solvent exchange rates: application to denatured drkN SH3

Amide solvent exchange rates are regarded as a valuable source of information on structure/dynamics of unfolded (disordered) proteins. Proton-based saturation transfer experiments, normally used to measure solvent exchange, are known to meet some serious difficulties. The problems mainly arise from the need to (1) manipulate water magnetization and (2) discriminate between multiple magnetization transfer pathways that occur within the proton pool. Some of these issues are specific to unfolded proteins. For example, the compensation scheme used to cancel the Overhauser effect in the popular CLEANEX experiment is not designed for use with unfolded proteins. In this report we describe an alternative experimental strategy, where amide ¹⁵N is used as a probe of solvent exchange. The experiment is performed in 50% H₂O–50% D₂O solvent and is based on the (HACACO)NH pulse sequence. The resulting spectral map is fully equivalent to the conventional HSQC. To fulfill its purpose, the experiment monitors the conversion of deuterated species, ¹⁵N^D, into protonated species, ¹⁵N^H, as effected by the solvent exchange. Conceptually, this experiment is similar to EXSY which prompted the name of ¹⁵N^H/D-SOLEXSY (SOLvent EXchange SpectroscopY). Of note, our experimental scheme, which relies on nitrogen rather than proton to monitor solvent exchange, is free of the complications described above. The developed pulse sequence was used to measure solvent exchange rates in the chemically denatured state of the drkN SH3 domain. The results were found to correlate well with the CLEANEX-PM data, r = 0.97, thus providing a measure of validation for both techniques. When the experimentally measured exchange rates are converted into protection factors, most of the values fall in the range 0.5–2, consistent with random-coil behavior. However, elevated values, ca. 5, are obtained for residues R38 and A39, as well as the side-chain indole of W36. This is surprising, given that high protection factors imply hydrogen bonding or hydrophobic burial not expected to occur in a chemically denatured state of a protein. We, therefore, hypothesized that elevated protection factors are an artefact arising from the calculation of the reference (random-coil) exchange rates. To confirm this hypothesis, we prepared samples of several short peptides derived from the sequence of the drkN SH3 domain; these samples were used to directly measure the reference exchange rates. The revised protection factors obtained in this manner proved to be close to 1.0. These results also have implications for the more compact unfolded state of drkN SH3, which appears to be fully permeable to water as well, with no manifestations of hydrophobic burial.     

Synthesis and characterization of stereoregular poly(D-methionyl-L-methionine) and poly(L-methionyl-D-methionyl-L-methionine)

By use of a polycondensation procedure free of racemization, stereoregular polymethionines have been synthesized from C-activated D -methionyl-L -methionine and L -methionyl-D -methionyl-L -methionine. The poly(D -methionyl-L -methionine) and poly(L -methionyl-D -methionyl-L -methionine) so prepared are soluble in chloroform and can be purified through dissolution in this solvent and precipitation by ligroin. Poly(D -Met-L -Met)which is obtained in a 25% yield, is about 5000 in average molecular weight. It has no discernible optical activity when examined between 400 and 600 nm in a trifluoroacetic acid solution. Poly(L -Met-D -Met-L -Met) (40% yield, M. W. = 10,000) is an optically active polymer. [α]₄₃₆²⁴ ≈ + 170° for a chloroformic solution (c = 0.2 CHCl₃).     

C4′/H4′ selective,non-uniformly sampled 4D HC(P)CH experiment for sequential assignments of 13C-labeled RNAs

A through bond, C4′/H4′ selective, “out and stay” type 4D HC(P)CH experiment is introduced which provides sequential connectivity via H4′_(i)–C4′_(i)–C4′_(i?1)–H4′_(i?1) correlations. The ³¹P dimension (used in the conventional 3D HCP experiment) is replaced with evolution of better dispersed C4′ dimension. The experiment fully utilizes ¹³C-labeling of RNA by inclusion of two C4′ evolution periods. An additional evolution of H4′ is included to further enhance peak resolution. Band selective ¹³C inversion pulses are used to achieve selectivity and prevent signal dephasing due to the of C4′–C3′ and C4′–C5′ homonuclear couplings. For reasonable resolution, non-uniform sampling is employed in all indirect dimensions. To reduce sensitivity losses, multiple quantum coherences are preserved during shared-time evolution and coherence transfer delays. In the experiment the intra-nucleotide peaks are suppressed whereas inter-nucleotide peaks are enhanced to reduce the ambiguities. The performance of the experiment is verified on a fully ¹³C, ¹⁵N-labeled 34-nt hairpin RNA comprising typical structure elements.     

Flexibility of the pyranose ring in α- and β-D-glucoses

Conformational energies of α- and β-D -glucopyranoses were computed by varying all the ring bond angles and torsional angles using semiempirical potential functions. Solvent accessibility calculations were also performed to obtain a measure of solvent interaction. The results indicate that the ⁴C₁ (D ) chair is the most favored conformation, both by potential energy and solvent accessibility criteria. The ⁴C₁ (D ) chair conformation is also found to be somewhat flexible, being able to accommodate variations up to 10° in the ring torsional angles without appreciable change in energy. Observed solid-state conformations of these sugars and their derivatives lie in the minimum-energy region, suggesting that the substituents and crystal field forces play a minor role in influencing the pyranose ring conformation. Theory also predicts the variations in the ring torsional angles, i.e., CCCC < CCCO < CCOC, in agreement with the experimental results. The boat and twist-boat conformations are found to be at least 5 kcal mol^?1 higher in energy compared to the ⁴C₁ (D ) chair, suggesting that these forms are unlikely to be present in a polysaccharide chain. The ¹C₄ (D ) chair has energy intermediate between that of the ⁴C₁ (D ) chair and that of the twist-boat conformation. The calculated energy barrier between ⁴C₁ (D ) and ¹C₄ (D ) conformations is high—about 11 kcal mol^?1.     

Compositional and Solvent Engineering in Dion–Jacobson 2D Perovskites Boosts Solar Cell Efficiency and Stability

Hybrid halide 2D perovskites deserve special attention because they exhibit superior environmental stability compared with their 3D analogs. The closer interlayer distance discovered in 2D Dion–Jacobson (DJ) type of halide perovskites relative to 2D Ruddlesden–Popper (RP) perovskites implies better carrier charge transport and superior performance in solar cells. Here, the structure and properties of 2D DJ perovskites employing 3‐(aminomethyl)piperidinium (3AMP²⁺) as the spacing cation and a mixture of methylammonium (MA⁺) and formamidinium (FA⁺) cations in the perovskite cages are presented. Using single‐crystal X‐ray crystallography, it is found that the mixed‐cation (3AMP)(MA_0.75FA_0.25)₃Pb₄I₁₃ perovskite has a narrower bandgap, less distorted inorganic framework, and larger Pb? I? Pb angles than the single‐cation (3AMP)(MA)₃Pb₄I₁₃. Furthermore, the (3AMP)(MA_0.75FA_0.25)₃Pb₄I₁₃ films made by a solvent‐engineering method with a small amount of hydriodic acid have a much better film morphology and crystalline quality and more preferred perpendicular orientation. As a result, the (3AMP)(MA_0.75FA_0.25)₃Pb₄I₁₃‐based solar cells exhibit a champion power conversion efficiency of 12.04% with a high fill factor of 81.04% and a 50% average efficiency improvement compared to the pristine (3AMP)(MA)₃Pb₄I₁₃ cells. Most importantly, the 2D DJ 3AMP‐based perovskite films and devices show better air and light stability than the 2D RP butylammonium‐based perovskites and their 3D analogs.     

A new method for random mutagenesis by error-prone polymerase chain reaction using heavy water

Error-prone polymerase chain reactions (epPCRs) are often used to introduce mutations in random mutagenesis, which has been used as a tool in protein engineering. Here, we developed a new method of epPCR using heavy water as a solvent instead of normal water (H₂O). Rhodopsin cDNA of the Ayu fish (Plecoglossus altivelis) was used as a template and was amplified using five different conditions: (A) 100% H₂O with no Mn²⁺, (B) 100% H₂O/0.6 mM Mn²⁺, (C) 99% D₂O with no Mn²⁺, (D) 99% D₂O/0.6 mM Mn²⁺ and (E) 99% H₂¹⁸O with no Mn²⁺. The 13,960 (for each of the conditions A to D) and 33,504 (for condition E) base pairs were sequenced. A maximum error rate of 1.8 × 10⁻³ errors/bp was detected in condition D, without any particular hot-spot mutations. A high preference for AT → GC transitions was observed in condition D, whereas a high preference for transitions over transversions was observed in condition C. All of the mutations observed in condition E were transversions. When conditions A and C were applied to another template, the honeybee actin gene, the results were comparable to those for Ayu rhodopsin. Based on these results, the use of heavy water, instead of H₂O, as a solvent for epPCR can introduce random mutations without positional bias, template dependency or decreased yield. Our new epPCR method, and possibly combining the use of D₂O and H₂¹⁸O, may be a powerful random mutagenesis technique.     

Triple resonance HNCCCH experiments for correlating exchangeable and nonexchangeable cytidine and uridine base protons in RNA

Summary A set of triple resonance experiments is presented, providing through-bond H₂N/HN to H6 connectivities in uridines and cytidines in ¹³C-/¹⁵N-labeled RNAs. These connectivities provide an important link between the sequential assignment pathways for the exchangeable and nonexchangeable proton resonances in nucleic acids. Both 2D and pseudo-3D HNCCCH experiments were applied to a 30-nucleotide lead-dependent ribozyme, known as the leadzyme. The HN to H6 connectivities for three uridines in the leadzyme were identified from one 2D H(NCCC)H experiment, and the H₂N to H6 connectivities were identified for seven of the eight cytidines from the combination of a 2D H(NCCC)H and a pseudo-3D H(NCC)CH experiment.     

Simultaneous α/β spin-state selection for <Superscript>13</Superscript>C and <Superscript>15</Superscript>N from a time-shared HSQC-IPAP experiment

Two novel HSQC-IPAP approaches are proposed to achieve α/β spin-state editing simultaneously for ¹³C and ¹⁵N in a single NMR experiment. The pulse schemes are based on a time-shared (TS) 2D ¹H,¹³C/¹H,¹⁵N-HSQC correlation experiment that combines concatenated echo elements for simultaneous J(CH) and J(NH) coupling constants evolution, TS evolution of ¹³C and ¹⁵N chemical shifts in the indirect dimension and heteronuclear α/β-spin-state selection by means of the IPAP principle. Heteronuclear α/β-editing for all CH _n (n = 1–3) and NH _n (1–2) multiplicities can be achieved in the detected F2 dimension of a single TS-HSQC-F2-IPAP experiment. On the other hand, an alternative TS-HSQC-F1-IPAP experiment is also proposed to achieve α/β-editing in the indirect F1 dimension. Experimental and simulated data is provided to evaluate these principles in terms of sensitivity and performance simultaneously on backbone and side-chain CH, CH₂, CH₃, NH, and NH₂ spin systems in uniformly ¹³C/¹⁵N-labeled proteins and in small natural-abundance peptides.     

Conformational properties of l-fucose and the tetrasaccharide building block of the sulfated l-fucan from Lytechinus variegatus

Although the 3D structure of carbohydrates is known to contribute to their biological roles, conformational studies of sugars are challenging because their chains are flexible in solution and consequently the number of 3D structural restraints is limited. Here, we investigate the conformational properties of the tetrasaccharide building block of the Lytechinus variegatus sulfated fucan composed of the following structure [l-Fucp4(SO₃⁻)-α(1-3)-l-Fucp2,4(SO₃⁻)-α(1-3)-l-Fucp2(SO₃⁻)-α(1-3)-l-Fucp2(SO₃⁻)] and the composing monosaccharide unit Fucp, primarily by nuclear magnetic resonance (NMR) experiments performed at very low temperatures and using H₂O as the solvent for the sugars rather than using the conventional deuterium oxide. By slowing down the fast chemical exchange rates and forcing the protonation of labile sites, we increased the number of through-space ¹H–¹H distances that could be measured by NMR spectroscopy. Following this strategy, additional conformational details of the tetrasaccharide and l-Fucp in solution were obtained. Computational molecular dynamics was performed to complement and validate the NMR-based measurements. A model of the NMR-restrained 3D structure is offered for the tetrasaccharide.     

Kinetics of oxidation of ferrocene by tris-1,10-phenanthrolinecobalt(III) in t-butyl alcoholwater and acetonewater mixtures

Rate constants and activation parameters (ΔH^≠ and ΔS^≠)are reported for the oxidation of ferrocene by the tris-1,10-phenanthrolinecobalt(III) cation in t-butyl alcoholwater and in acetonewater solvent mixtures. Solvent effects on reactivity trends for these systems, for this same reaction in methanolwater mixtures, and for cobalt(II)-catalysed racemisation of Co(phen)_3³⁺ in t-butyl alcoholwater solvent mixtures are analysed into initial state and transition state contributions. The dependences of solubilities on solvent composition for ferrocene and for [Co(phen)₃](ClO₄)₃ in methanol, t-butyl alcohol, and acetonewater mixtures are also reported; these results are needed in order to establish solvent effects on the initial states of the reactions studied.     

Comparison of <Superscript>13</Superscript>CH<Subscript>3</Subscript>, <Superscript>13</Superscript>CH<Subscript>2</Subscript>D,and <Superscript>13</Superscript>CHD<Subscript>2</Subscript> methyl labeling strategies in proteins

A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins, using ¹³CH₃,¹³CH₂D, and ¹³CHD₂ methyl isotopomers, is presented. For each labeling scheme, ¹H–¹³C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue enzyme, malate synthase G, with ¹³CH₃,¹³CH₂D, and ¹³CHD₂ labeling in Ile δ1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence’s performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence has previously been identified as a transverse relaxation optimized experiment for ¹³CH₃-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (¹³CH₃ HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version of the ¹³CH₃ HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both ¹³C and ¹H spins. The HSQC and HMQC spectra of ¹³CH₂D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, ¹³CH₂D TROSY, that exploits cross-correlation effects to record ¹H–¹³C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of ¹³CHD₂-labeled methyl groups can be recorded with HSQC or HMQC; a new ¹³CHD₂ HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here. When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the ¹³CH₃ labeling strategy is the most beneficial from the perspective of sensitivity (gains ≥2.4 relative to either ¹³CH₂D or ¹³CHD₂ labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented here.     

A solvent effect on the side-chain conformation of phenylalanine derivatives and phenylalanine residuces in dipeptides

Three phenylalanine derivatives, Ac-Phe-NHMe, H-Phe-NHMe, and Ac-Phe-OH, were selected as models of Phe residues situated at the internal, the N-terminal, and the C-terminal positions of peptide chains, respctively. The side-chain conformations of the three compounds were analyzed from the vicnal coupling constants ³J_αβR and ³J_αβS, of their ¹H- nmr spectra measured in various organic sovlent. The two β-protons were unambiguously assined by use of sterospecifically β-monodeuterated phenylalanines. The pro-S β-proton was always situated at lower field than the pro-R one when they were observed separately. The results of a solvent effect on the conformation of the tree compounds demonstrated that the rotamer populations are remarkable sensitive of the three compounds demonstrated that the rotamer populations are remarkably sensitive to solvent polarity and that the tendencies of the solvent effects are quite different from each other. Ac-Phe-OH Showed a trend similar to that of Ac-Phe-OEt reported by early workers. The rotamer populations of other derivatives (Ac-Phe-NMe₂, Ac-Phe-NH₂, Ac-Phe-OBu^t, and Ac-Phe-OBzl) and of Phe residues in some N-acetyl dipeptde esters (Ac-Phe-Gly-OMe, Ac-Phe-Val-OMe, and Ac-Gly-Phe-OMe) were also examined in several sovent, and it was found that substituents of the Phe carboxyl group—amides or esters—determine the tendency of the solvent effect. These results are interesting in the side-chain conformations of Phe residues in peptides and proteins in an environment of low polarity can be disscussed on this experimental basis. Factors responsible for the solvent effect are discussed from (1) a structural comparison of the compunds with various carboxylic substituents, (2) an expriment with cyclohexylalanine derivatives, and (3) the measurement in mixed solvents wiht similar polarity.     

Simultaneous CT-13C and VT-15N chemical shift labelling: Application to 3D NOESY-CH3NH and 3D 13C,15N HSQC-NOESY-CH3NH

Based on the HSQC scheme, we have designed a 2D heterocorrelated experiment which combines constant time (CT) ¹³C and variable time (VT) ¹⁵N chemical shift labelling. Although applicable to all carbons, this mode is particularly suitable for simultaneous recording of methyl-carbon and nitrogen chemical shifts at high digital resolution. The methyl carbon magnetisation is in the transverse plane during the whole CT period (1/J_CC=28.6 ms). The magnetisation originating from NH protons is initially stored in the 2H_zN_z state, then prior to the VT chemical shift labelling period is converted into 2H_zN_y coherence. The VT -¹⁵N mode eliminates the effect of ¹ J _N,CO and ^1,2 J _N,CA coupling constants without the need for band-selective carbon pulses. An optional editing procedure is incorporated which eliminates signals from CH₂ groups, thus removing any potential overlap with the CH₃ signals. The CT-¹³CH₃,VT-¹⁵N HSQC building block is used to construct two 3D experiments: 3D NOESY-CH₃NH and 3D ¹³C,¹⁵N HSQC-NOESY-CH₃NH. Combined use of these experiments yields proton and heteronuclear chemical shifts for moieties experiencing NOEs with CH₃ and NH protons. These NOE interactions are resolved as a consequence of the high digital resolution in the carbon and nitrogen chemical shifts of CH₃ and NH groups, respectively. The techniques are illustrated using a double labelled sample of the CH domain from calponin.