DOI: 10.1002/cbic.200600408 Solid-State NMR of Matrix Metalloproteinase 12: An Approach Complementary to Solution NMR StØphane Balayssac, [a] Ivano Bertini,* [a, b] Katja Fälber, [c] Marco Fragai, [a, d] Stefan Jehle, [c, e] Moreno Lelli, [a] Claudio Luchinat, [a, d] Hartmut Oschkinat, [c] and Kwon Joo Yeo [a, b] Solid-state NMR (SS NMR) is a technique that has shown a rapid development in recent years. [1–4] The exciting progress in sample-preparation methods, [5] tailored pulse sequences, [4,6–8] and instrumentation now make the investigation of relatively large proteins possible. In spite of these developments, the number of proteins for which an almost complete solid-state assignment is available is still small. [9] Interestingly, it is general- ly observed that 13 C chemical shifts do not change much on passing from solution to microcrystalline samples (the differen- ces generally being < 1 ppm). [9] This opens the way to a fast liquid-based solid-state assignment, in which the available liquid assignment is transferred to the solid-state spectra, and only a minimal number of solid-state spectra are acquired. The possibility of obtaining a solid-state assignment in a short time is valuable, as it will permit, for instance, the investigation of a protein as part of larger aggregates (oligomerization, protein– protein complexes) without the line broadening due to the in- crease in molecular weight observed with solution NMR. In this work we demonstrate that it is possible to provide a large fraction of the solid-state NMR assignment of a relatively large protein (17 kDa) rapidly by using a pair of experiments (CP MAS proton-driven spin diffusion (PDSD) and J-decoupled PDSD). These spectra can be acquired in a limited amount of time (12–15 h each), and manually assigned in a few days by using the available liquid-state assignment as a guideline. 3D NCACX and NCOCX PDSD spectra fully validate the obtained assignment and further increase the overall fraction of as- signed peaks. However, they require considerably more experi- mental time. For this investigation we selected a microcrystalline sample of the catalytically active domain of the zinc-containing matrix metalloproteinase 12 (Zn-MMP-12, 159 AA, 17.6 kDa), for which one X-ray structure and the solution NMR assignment are avail- able. [10] The crystallographic structure indicates that the catalyt- ically active domain is composed of three a-helices (44 AA, 28% of the total residues) and seven b-strands (27 AA, 17%). The remaining 88 residues do not form regular secondary structure. The protein contains two zinc(II) ions and three cal- cium(II) ions. One of the zinc ions is responsible for the catalyt- ic activity. The SS NMR sample was prepared by crystallizing the pro- tein from 30 % poly(ethylene glycol) (PEG) 8000 according the published procedure. [10] The microcrystalline precipitate began to appear after 12 h, and the crystallization was complete after 1–2 days. Figure 1 shows the 13 C, 13 C CP MAS PDSD spectrum acquired at 16.4 T (700 MHz 1 H Larmor frequency) and a MAS frequency w R /2p = 11.5 kHz, with a mixing time of 15 ms. The assignment of the PDSD spectrum was directly based on the 13 C chemical shifts determined in solution. [10] The extremely good resolution of the spectrum in Figure 1 allowed us to assign up to 75% of the aliphatic 13 C signals by making minor adjustments ACHTUNGTRENNUNG(<1 ppm) to the solution shifts (Table 1), and by checking the consistency with the spin pattern of each residue as far as the aliphatic part of the spectrum was concerned. Further exten- sion of the assignment was prevented by the ambiguities in transferring the solution assignment, mostly in the crowded ACHTUNGTRENNUNGregions of the Ca,Cb correlations and of the leucine Cg,Cd ACHTUNGTRENNUNGcorrelations. Figure 1. Aliphatic region of the 13 C, 13 C CP MAS PDSD of Zn-MMP-12 (16.4 T, 11.5 kHz MAS frequency, 15 ms mixing time). [a] Dr. S. Balayssac, Prof. I. Bertini, Dr. M. Fragai, Dr. M. Lelli, Prof. C. Luchinat, K. J. Yeo Magnetic Resonance Center (CERM), University of Florence Via Luigi Sacconi 6, 50019 Sesto Fiorentino (Italy) Fax: (+ 39)055-457-4271 E-mail: bertini@cerm.unifi.it [b] Prof. I. Bertini, K. J. Yeo Department of Chemistry, University of Florence Via della Lastruccia 3, 50019 Sesto Fiorentino (Italy) [c] Dr. K. Fälber, S. Jehle, Prof. H. Oschkinat Leibnizinstitut für Molekulare Pharmakologie Robert-Rçssle-Strasse 10, 13125 Berlin (Germany) [d] Dr. M. Fragai, Prof. C. Luchinat Department of Agricultural Biotechnology, University of Florence Via Maragliano 75–77, 50144 Florence (Italy) [e] S. Jehle Freie Universität Berlin Takustrasse 3, 14195 Berlin (Germany) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. 486 # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2007, 8, 486 – 489