Measurement Of Internuclear Distances Research Articles

2F1600 固体<13>^CNMR距離測定による生体膜結合マストパランXの構造解析

2F1600 固体<13>^CNMR距離測定による生体膜結合マストパランXの構造解析

Retinylidene ligand structure in bovine rhodopsin, metarhodopsin-I, and 10-methylrhodopsin from internuclear distance measurements using 13C-labeling and 1-D rotational resonance MAS NMR.

Rhodopsin is the G-protein coupled photoreceptor that initiates the rod phototransduction cascade in the vertebrate retina. Using specific isotope enrichment and magic angle spinning (MAS) NMR, we examine the spatial structure of the C10-C11=C12-C13-C20 motif in the native retinylidene chromophore, its 10-methyl analogue, and the predischarge photoproduct metarhodopsin-I. For the rhodopsin study 11-Z-[10,20-(13)C(2)]- and 11-Z-[11,20-(13)C(2)]-retinal were synthesized and incorporated into bovine opsin while maintaining a natural lipid environment. The ligand is covalently bound to Lys(296) in the photoreceptor. The C10-C20 and C11-C20 distances were measured using a novel 1-D CP/MAS NMR rotational resonance experimental procedure that was specifically developed for the purpose of these measurements [Verdegem, P. J. E., Helmle, M., Lugtenburg, J., and de Groot, H. J. M. (1997) J. Am. Chem. Soc. 119, 169]. We obtain r(10,20) = 0.304 +/- 0.015 nm and r(11,20) = 0.293 +/- 0.015 nm, which confirms that the retinylidene is 11-Z and shows that the C10-C13 unit is conformationally twisted. The corresponding torsional angle is about 44 degrees as indicated by Car-Parrinello modeling studies. To increase the nonplanarity in the chromophore, 11-Z-[10,20-(13)C(2)]-10-methylretinal and 11-Z-[(10-CH(3)), 13-(13)C(2)]-10-methylretinal were prepared and incorporated in opsin. For the resulting analogue pigment r(10,20) = 0.347 +/- 0.015 nm and r((10)(-)(CH)()3())(,)(13) = 0.314 +/- 0.015 nm were obtained, consistent with a more distorted chromophore. The analogue data are in agreement with the induced fit principle for the interaction of opsin with modified retinal chromophores. Finally, we determined the intraligand distances r(10,20) and r(11,20) also for the photoproduct metarhodopsin-I, which has a relaxed all-E structure. The results (r(10,20) >/= 0.435 nm and r(11,20) = 0.283 +/- 0.015 nm) fully agree with such a relaxed all-E structure, which further validates the 1-D rotational resonance technique for measuring intraligand distances and probing ligand structure. As far as we are aware, these results represent the first highly precise distance determinations in a ligand at the active site of a membrane protein. Overall, the MAS NMR data indicate a tight binding pocket, well defined to bind specifically only one enantiomer out of four possibilities and providing a steric complement to the chromophore in an ultrafast ( approximately 200 fs) isomerization process.

Practical Methods for Solid-State NMR Distance Measurements on Large Biomolecules: Constant-Time Rotational Resonance

Simple modifications of the rotational resonance experiment substantially reduce the total experimental time needed to measure weak homonuclear dipolar couplings, a critical factor for achieving routine internuclear distance measurements in large biomolecular systems. These modifications also address several problems cited in the literature. Here we introduce a constant-time rotational resonance experiment that eliminates the need for control spectra to correct for effects from variable RF heating, particularly critical for accurate long-distance measurements. This reduces the total number of experiments needed by as much as a factor of 2. Other improvements incorporated include achieving selective inversion with a delay rather than a weak pulse (P. R. Costa et al., J. Am. Chem. Soc. 119, 10487–10493, 1997), which we observe results in the elimination of oscillations in peak intensities for short mixing time points. This reduces the total experiment time in two ways. First, there is no longer a need to average different “zero”-time points (S. O. Smith et al., Biochemistry 33, 6334–6341, 1994) to correct for intensity variations. Second, short-mixing-time lineshape differences observed in large membrane-bound proteins only appear with the weak-pulse inversion and not when using the delay inversion. Consistent lineshapes between short and long mixing times permit the use of a single spectrum for subtraction of natural abundance background signals from all labeled-protein time points. Elimination of these effects improves the accuracy and efficiency of rotational resonance internuclear distance measurements.

13C−13C Correlations and Internuclear Distance Measurements with 2D-MELODRAMA

Under magic-angle spinning (MAS), the 2D-MELODRAMA pulse sequence reintroduces homonuclear dipolar couplings, thereby allowing internuclear correlations to be determined. The evolution of the density matrix under the average Hamiltonian established under the 2D-MELODRAMA pulse sequence has been evaluated, and closed-form expressions for the dependence of cross-peak intensity as a function of the dipolar mixing time are presented. In the case of sodium proprionate-2-3-13C2 (>90% 13C) it is shown that analysis of cross-peak intensity vs mixing times allows estimates of dipolar coupling with an accuracy comparable with the 1D-MELODRAMA experiment. The potential for simultaneous multiple internuclear correlations and distance measurements in spin systems for which there is not a large difference in resonance frequencies is illustrated with glucose-13C6 (36% uniformly labeled). It is shown that multiple correlations for each ring carbon can be detected and good estimates of the corresponding internuclear dista...

Rotational Resonance Tickling: Accurate Internuclear Distance Measurement in Solids

Accurate distance measurements between pairs of nuclear spins can provide detailed information about molecular structure in the solid state. The rotational resonance (R2) technique, in particular, has been used to measure internuclear distances in a variety of compounds, often between pairs of 13C nuclei where the outer range is 5−6 Å. Recent studies have revealed that the spin dynamics at rotational resonance are influenced by zero-quantum line shape parameters including T2zq and the dispersion in isotropic chemical shift differences. Errors in the estimation of these parameters are often the limiting factor in determining the accuracy of a distance measurement. Here we present a modification of R2, termed “rotational resonance tickling” (R2T), which uses a ramped rf field to induce fast passage through the dipolar resonance condition, thereby greatly reducing the dependence of the spin dynamics on zero-quantum parameters. Extraction of distance information from the resulting exchange curves is approximately a single-parameter fit, with accuracies in model systems that appear to be on the order of ±0.1 Å or better. An additional feature of the technique is that it does not demand the very high-power 1H decoupling fields typically required in other recoupling experiments to limit signal loss during mixing. We demonstrate the technique in a pair of 13C2-labeled model compounds, tyrosine ethyl ester and glycylglycine hydrochloride, with effective internuclear distances (including intermolecular effects) of 5.05 and 4.3 Å, respectively.

Internuclear Distance Measurements up to 0.44 nm for Retinals in the Solid State with 1-D Rotational Resonance 13C MAS NMR Spectroscopy

Internuclear Distance Measurements up to 0.44 nm for Retinals in the Solid State with 1-D Rotational Resonance ¹³C MAS NMR Spectroscopy

Internuclear distance measurements in solid state nuclear magnetic resonance: Dipolar recoupling via rotor synchronized spin locking

The dipolar coupling in rotating solids, averaged to zero when the rotation is about the magic angle, can be reintroduced in the spin-locking interaction frame by rotor-synchronized 90° phase shifts of an applied spin-locking field. This new technique is an interaction frame analogue of previously proposed experiments. Because the spin-locking fields both truncate and play the role of the chemical shift terms in the Hamiltonian, this technique is insensitive to the chemical shifts as well as chemical shift anisotropies of the coupled spins. Through numerical simulations of the magnetization exchange trajectory (e.g., the mixing time dependence of the cross-peak intensity connecting two coupled spins), the through-space distance between two spin nuclei can be estimated. This technique can also be applied to obtain two-dimensional correlation spectra, or alternatively it can be used as a double-quantum filter. Preliminary experiments demonstrate that the cross peaks in the two-dimensional correlation spectra obtained with this technique have a ‘‘negative–positive–negative’’ pattern to the first, second, etc., coupled neighbor. This spectral feature is useful in the assignment of complex magic angle spinning spectra.

Solid-state NMR study of protein structure. Methods based on the measurement of internuclear distances

Several high-resolutlon solid-state NMR methods have been developed in the last ten years to measure weak homonuclear or heteronuclear dipolar couplings in solid samples. These methods use the magic angle spinning technique and include the rotational resonance and the rf-driven recoupling (RFDR) techniques for the measurement of homonuclear distances and techniques such as REDOR and TEDOR for the measurement of heteronuclear distances. These techniques have been applied to a variety of systems including small peptides, membrane proteins and enzyme-substrate-inhibitor complexes.

Measurement of Internuclear Distances by Switched Angle Spinning NMR

Dipolar switched angle spinning, an NMR technique due to Terao et al. [Terao, T.; Miura H.; Saika, A.J. Chem. Phys. 1986, 85, 3816], has been used to measure [sup 13]C-[sup 13]C distances in the solid state. The experiment involves rotation of the sample at two different angles during different periods of a two-dimensional experiment. An evolution period with off-magic-angle spinning and chemical shift refocusing, followed by detection of the signal under magic angle spinning, yields scaled Pake patterns in [omega][sub 1], correlated with their isotropic shifts in [omega][sub 2] allowing the high-resolution measurement of dipolar couplings. We demonstrate this experiment on samples of doubly [sup 13]C labeled zinc acetate and a 14 amino acid peptide, in which we show that under optimal conditions distances of up to 5 [angstrom] may be measured. 21 refs., 4 figs.

Frequency-selective heteronuclear recoupling in rotating solids

In solid state nuclear magnetic resonance spectroscopy, several methods have recently been introduced for the purpose of restoring dipolar couplings into magic angle spinning experiments. These powerful techniques are useful for the measurement of internuclear distances and, more generally, for filtering and correlation experiments in noncrystalline materials. In the case of heteronuclear spins, the rotational echo double resonance experiment provides an elegant and practical approach to the spectrally nonselective reintroduction of dipolar interactions. In this article, we describe an approach to restore heteronuclear couplings which is spectrally selective—in particular we recouple the observed spins only with those nonobserved spins lying at exact resonance (and at multiples of the spinning frequency) in the triple resonance experiment. We refer to the technique as frequency-selective dipolar recoupling.

Internuclear distance measurement in a reaction intermediate: solid-state carbon-13 NMR rotational resonance determination of the Schiff base configuration in the M photointermediate of bacteriorhodopsin

Rotational resonance (R2) is a technique for selectively recoupling homonuclear dipolar interactions in MAS N M R spectroscopy.l-4 Since the strength of the dipolar interaction is directly related to the internuclear distance, R2 provides an approach for structure determination in polycrystalline or amorphous solids. The method has been used to obtain 13C-13C and 3IP3P distances for a number of proteins in the ground state.5-8 Clearly, the technique is also applicable to structural studies of reaction intermediates if they can be trapped for extended periods. Here we present the first such application. Bacteriorhodopsin (bR), the sole protein in the purple patches of the plasma membrane of Hafobacterium halobium, comprises a single polypeptide chain, folded into seven transmembrane helices, and a retinal chromophore, which is situated in the center of the helix bundle and linked to lysine216 by formation of a Schiff base (SB). During the photocycle of the light-adapted form of the protein, a proton is released on the extracellular side of the membrane and replaced from the intracellular side. The mechanism for this light-driven proton transport involves the photoisomerization of the retinal chromophore from all-trans to 13-cis, a transient drop in the pK of the SB (which leads to deprotonation and reprotonation of the nitrogen in the formation and decay of the M intermediate), and conformational changes that alter the connectivity of the proton transport pathway to prevent proton backflow. Of particular interest is the configuration of the C=N bond of the SB in the M intermediate, as this determines the orientation of the lone pair of the nitrogen at this key step. Previous solid-state N M R studies have used the chemical shifts of the 14-retinal and the c-lysinez16 carbons to

Taxol: quantitative internuclear proton-proton distances in CDCl3 solution from nOe data: 2D nmr ROESY buildup rates at 500 MHz.

Quantitative nmr internuclear proton-proton distance measurements obtained by observation of the initial buildup rates of nOe's in 2D ROESY spectra of taxol [1] in CDCl3 are reported. A comparison to the X-ray crystal structure of taxotere [2] is made, and the results are discussed in terms of previous studies of structure-activity relationships.

Internuclear distance measurement between Deuterium ( I = 1) and a [formula omitted] nucleus in rotating solids

Internuclear distance measurement between Deuterium ( I = 1) and a [formula omitted] nucleus in rotating solids

A strategy for NMR structure determination

Structure determination by two-dimensional NMR is frequently limited by spectral overlap in large macromolecules. The well-resolved peaks in 2D nuclear Overhauser effect spectra are used to determine a limited set of internuclear distances for three-dimensional structure determination. An alternative strategy that includes both resolved and overlapping peaks is to simulate the experimental NOESY spectra as a function of mixing time using a combination of measured and estimated peak volumes. When the experimental and simulated spectra match, the peak volumes can be directly converted to relaxation rates using a matrix approach that accounts for spin diffusion. Correlation times can be measured from the relaxation rates, and the rates can be converted into internuclear distances that can be used for structure determination. By simulation of the spectra at several mixing times, the uncertainties in the distances can be evaluated. This strategy is demonstrated for the eight-base-pair TaqI DNA restriction site d(CTTCGAAG) 2, where a combination of peak overlap and spin diffusion limits the measurement of internuclear distances by conventional methods.

Measurement of internuclear distances in polycrystalline solids. Rotationally enhanced transfer of nuclear spin magnetization

A magic angle spinning (MAS) NMR technique for measurement of the distance between two homonuclear sites separated by as much as 0.5 nm is demonstrated. This is achieved by proton decoupling during the magnetization exchange process, greatly attenuating the influence of abundant nuclear spins, selective isotopic labeling of both sites of interest, so that the system may be approximated as a set of magnetically dilute coupled spin pairs, and MAS with matching of the rotational resonance condition. Tyrosine ethyl ester (TEE), carbon 13 labeled at both the -CH{sub 2}- of the ester moiety and at the 4{prime}-OH aromatic carbon is used as a demonstration of the technique. Calculated data is compared to experimental data.

NMR nutation spectroscopy: A method for determining molecular geometry in amorphous solids

A NMR technique for direct determination of internuclear distances in amorphous solids is described and illustrated with a measurement of the carbon–carbon bond length in acetic acid at −190 °C. The result, 1.488 °A, is sufficiently close to the x-ray value of 1.478 °A to confirm the effectiveness of the overall approach. The pulse sequence used, and the ensuing spectral analysis, ensure a very direct measurement of internuclear distances in amorphour solids.