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De novodetermination of peptide structure withsolid-state magic-angle spinning NMR spectroscopyChad M. Rienstra*†‡, Lisa Tucker-Kellogg§, Christopher P. Jaroniec*†, Morten Hohwy†¶, Bernd Reif*†储,Michael T. McMahon*†, Bruce Tidor*§**††, Toma´s Lozano-Pe´rez§††, and Robert G. Griffin*†,††*Department of Chemistry,†Francis Bitter Magnet Laboratory,§Department of Electrical Engineering and Computer Science, and **Biological EngineeringDivision, Massachusetts Institute of Technology, Cambridge, MA 02139Communicated by John S. Waugh, Massachusetts Institute of Technology, Cambridge, MA, June 10, 2002 (received for review January 25, 2002)The three-dimensional structure of the chemotactic peptide N-formyl-L-Met-L-Leu-L-Phe-OH was determined by using solid-stateNMR (SSNMR). The set of SSNMR data consisted of 1613C–15Ndistances and 18 torsion angle constraints (on 10 angles), recordedfrom uniformly13C,15N- and15N-labeled samples. The peptide’sstructure was calculated by means of simulated annealing and anewly developed protocol that ensures that all of conformationalspace, consistent with the structural constraints, is searched com-pletely. The result is a high-quality structure of a molecule that hasthus far not been amenable to single-crystal diffraction studies.The extensions of the SSNMR techniques and computational meth-ods to larger systems appear promising.Over the last two decades, multidimensional nuclear mag-netic resonance (NMR) methods have been developedwhich permit determinations of globular protein structures insolution (1). To date most structures addressed with thesetechniques involve proteins with molecular weights ⱕ20,000, butthe continued development of new methodology shows promisefor studies of larger systems (2–6). Despite the success of theseapproaches, there remain fundamental limits on the size andphysical state of molecules amenable to study with solution-stateNMR. In contrast, high-resolution solid-state NMR (SSNMR)methods have no inherent molecular weight limit, and have formany years been used to determine details of molecular struc-ture for high molecular weight systems. For example, specificstructural features of intact membrane proteins such as bacte-riorhodopsin (effective molecular weight ⬇85,000) (7, 8) andlarge enzyme complexes such as 5-enolpyruvylshikimate-3-phosphate synthase (46,000) (9) and tryptophan synthase(143,000) (10) have been reported. SSNMR methods have alsobeen used to examine surface-bound peptides (11) and todetermine a low-resolution structure [1.9-Å backbone root-mean-square deviation (rmsd)] of an insoluble peptide fragmentfrom␤-amyloid (12) under experimental conditions inaccessibleto both solution-state NMR and crystallography.To date, essentially all structural NMR studies of solid pep-tides and proteins have relied on site-specific incorporation of apair of spin-1⁄2nuclei, such as13C–13C and13C–15N. This ap-proach has been very successful and will likely continue to beimportant in experiments that address detailed mechanisticquestions in large biomolecular systems. However, recent ad-vances in solid-state NMR methodology, most notably thedevelopment of approaches to perform dipolar recoupling dur-ing magic-angle spinning (MAS) (13, 14), in principle permitmultiple distance and torsion angle measurements on moleculesthat are uniformly13C and15N labeled (15–18). The develop-ment of these approaches considerably simplifies preparation ofsamples for SSNMR experiments and concurrently opens thepossibility of complete structural determinations with solid-stateMAS NMR. In this paper we describe the realization of this goalwith a complete structure determination of the chemotactictripeptide N-formyl-L-Met-L-Leu-L-Phe-OH (f-MLF-OH) (19).The structure of the peptide is based on sets of NMR data thatconstrain 1613C–15N distances and 10 torsion angles derivedfrom a series of MAS NMR experiments performed on uni-formly13C,15N- and15N-labeled samples. Finally, we discussextensions of the solid-state MAS NMR techniques and com-putational methods used here to larger systems.Experimental Proceduresf-MLF-OH samples were synthesized by standard solid-phasemethods and HPLC purification (American Peptide Company,Sunnyvale, CA). One sample, synthesized from U-13C,15N-labeled amino acids (Cambridge Isotope Laboratories, Andover,MA), was used for all resonance assignment experiments and themajority of the three-dimensional (3D) torsion angle experi-ments (1H–15N–13C–1H,1H–13C–13C–1H,15N–13C–13C–15N). Asecond sample was prepared by dilution of the U-13C,15N-labeledf-MLF-OH peptide in natural abundance material in the ratio of1:9 and was used for the frequency-selective rotational-echodouble-resonance (REDOR) experiments. A third sample,synthesized from15N-labeled amino acids, was used for the1H–15N–15N–1H torsion angle experiments. In all cases, micro-crystals of the f-MLF-OH peptides were grown by overnightevaporation from 2-propanol, and ⬇15–20 mg of each polycrys-talline material was packed into a 4-mm zirconia NMR rotor(Varian-Chemagnetics, Fort Collins, CO). Attempts to growsingle crystals suitable for diffraction studies were not successful.The structures of the f-MLF methyl ester (f-MLF-OMe) andother analogs of f-MLF have been determined by diffractionmethods (20), but that of the f-MLF-OH acid form has not.Presumably the acid form does not form large single crystalsbecause of small differences in crystal packing forces, relative tothe methyl ester. We note that the previously published structureof f-MLF-OH includes aD-Phe residue (21), which is not presentin the chemotactically active form (19).MAS NMR experiments were performed on Cambridge In-struments spectrometers operating at 400 and 500 MHz (cour-tesy of D. J. Ruben), together with custom-designed 400- and500-MHz multiple-resonance transmission line probes, or aVarian-Chemagnetics (Fort Collins, CO) 500-MHz triple-resonance probe. All of the probes were equipped with 4-mmMAS spinner modules. The resonance assignment (22) andREDOR experiments (16) were performed at 500 MHz, aswere most of the torsion angle experiments (1H–15N–13C–1H,1H–13C–13C–1H, and15N–13C–13C–15N), with the exception of1H–15N–15N–1H (400 MHz) (23). Typical radiofrequency fieldstrengths were ⬇100–120 kHz on1H during recoupling periods,Abbreviations: SSNMR, solid-state NMR; rmsd, root-mean-square deviation;


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