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De novo determination of peptide structure with solid state magic angle spinning NMR spectroscopy Chad M Rienstra Lisa Tucker Kellogg Christopher P Jaroniec Morten Hohwy Bernd Reif 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 Engineering Division Massachusetts Institute of Technology Cambridge MA 02139 Communicated 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 Nformyl L Met L Leu L Phe OH was determined by using solid state NMR SSNMR The set of SSNMR data consisted of 16 13C 15N distances and 18 torsion angle constraints on 10 angles recorded from uniformly 13C 15N and 15N labeled samples The peptide s structure was calculated by means of simulated annealing and a newly developed protocol that ensures that all of conformational space consistent with the structural constraints is searched completely The result is a high quality structure of a molecule that has thus far not been amenable to single crystal diffraction studies The extensions of the SSNMR techniques and computational methods to larger systems appear promising O ver the last two decades multidimensional nuclear magnetic resonance NMR methods have been developed which permit determinations of globular protein structures in solution 1 To date most structures addressed with these techniques involve proteins with molecular weights 20 000 but the continued development of new methodology shows promise for studies of larger systems 2 6 Despite the success of these approaches there remain fundamental limits on the size and physical state of molecules amenable to study with solution state NMR In contrast high resolution solid state NMR SSNMR methods have no inherent molecular weight limit and have for many years been used to determine details of molecular structure for high molecular weight systems For example specific structural features of intact membrane proteins such as bacteriorhodopsin effective molecular weight 85 000 7 8 and large enzyme complexes such as 5 enolpyruvylshikimate 3phosphate synthase 46 000 9 and tryptophan synthase 143 000 10 have been reported SSNMR methods have also been used to examine surface bound peptides 11 and to determine a low resolution structure 1 9 backbone rootmean square deviation rmsd of an insoluble peptide fragment from amyloid 12 under experimental conditions inaccessible to both solution state NMR and crystallography To date essentially all structural NMR studies of solid peptides and proteins have relied on site specific incorporation of a pair of spin 1 2 nuclei such as 13C 13C and 13C 15N This approach has been very successful and will likely continue to be important in experiments that address detailed mechanistic questions in large biomolecular systems However recent advances in solid state NMR methodology most notably the development of approaches to perform dipolar recoupling during magic angle spinning MAS 13 14 in principle permit multiple distance and torsion angle measurements on molecules that are uniformly 13C and 15N labeled 15 18 The development of these approaches considerably simplifies preparation of samples for SSNMR experiments and concurrently opens the possibility of complete structural determinations with solid state MAS NMR In this paper we describe the realization of this goal with a complete structure determination of the chemotactic tripeptide 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 that constrain 16 13C 15N distances and 10 torsion angles derived 10260 10265 PNAS August 6 2002 vol 99 no 16 from a series of MAS NMR experiments performed on uniformly 13C 15N and 15N labeled samples Finally we discuss extensions of the solid state MAS NMR techniques and computational methods used here to larger systems Experimental Procedures f MLF OH samples were synthesized by standard solid phase methods and HPLC purification American Peptide Company Sunnyvale CA One sample synthesized from U 13C 15Nlabeled amino acids Cambridge Isotope Laboratories Andover MA was used for all resonance assignment experiments and the majority of the three dimensional 3D torsion angle experiments 1H 15N 13C 1H 1H 13C 13C 1H 15N 13C 13C 15N A second sample was prepared by dilution of the U 13C 15N labeled f MLF OH peptide in natural abundance material in the ratio of 1 9 and was used for the frequency selective rotational echo double resonance REDOR experiments A third sample synthesized from 15N labeled amino acids was used for the 1H 15N 15N 1H torsion angle experiments In all cases microcrystals of the f MLF OH peptides were grown by overnight evaporation from 2 propanol and 15 20 mg of each polycrystalline material was packed into a 4 mm zirconia NMR rotor Varian Chemagnetics Fort Collins CO Attempts to grow single crystals suitable for diffraction studies were not successful The structures of the f MLF methyl ester f MLF OMe and other analogs of f MLF have been determined by diffraction methods 20 but that of the f MLF OH acid form has not Presumably the acid form does not form large single crystals because of small differences in crystal packing forces relative to the methyl ester We note that the previously published structure of f MLF OH includes a D Phe residue 21 which is not present in the chemotactically active form 19 MAS NMR experiments were performed on Cambridge Instruments spectrometers operating at 400 and 500 MHz courtesy of D J Ruben together with custom designed 400 and 500 MHz multiple resonance transmission line probes or a Varian Chemagnetics Fort Collins CO 500 MHz tripleresonance probe All of the probes were equipped with 4 mm MAS spinner modules The resonance assignment 22 and REDOR experiments 16 were performed at 500 MHz as were most of the torsion angle experiments 1H 15N 13C 1H 1H 13C 13C 1H and 15N 13C 13C 15N with the exception of 1H 15N 15N 1H 400 MHz 23 Typical radiofrequency field strengths were 100 120 kHz on 1H during recoupling periods Abbreviations SSNMR solid state NMR rmsd root mean square deviation MAS magicangle spinning f MLF OH N formyl L Met L Leu L Phe OH f MLF OMe f MLF OH methyl ester 2D and 3D two and three dimensional REDOR rotational echo double resonance Present address Department of Chemistry

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