Paramagnetic shifts in solid-state NMR of proteins to elicit structural information

Abstract

The recent observation of pseudocontact shifts (pcs) in 13 C highresolution solid-state NMR of paramagnetic proteins opens the way to their application as structural restraints. Here, by investigating a microcrystalline sample of cobalt(II)-substituted matrix metalloproteinase 12 [CoMMP-12 (159 AA, 17.5 kDa)], it is shown that a combined strategy of protein labeling and dilution of the paramagnetic species (i.e., 13 C-, 15 N-labeled CoMMP-12 diluted in unlabeled ZnMMP-12, and 13 C-, 15 N-labeled ZnMMP-12 diluted in unlabeled CoMMP-12) allows one to easily separate the pcs contributions originated from the protein internal metal (intramolecular pcs) from those due to the metals in neighboring proteins in the crystal lattice (intermolecular pcs) and that both can be used for structural purposes. It is demonstrated that intramolecular pcs are significant structural restraints helpful in increasing both precision and accuracy of the structure, which is a need in solid-state structural biology nowadays. Furthermore, intermolecular pcs provide unique information on positions and orientations of neighboring protein molecules in the solid phase.S olid-state NMR (SSNMR) on biomolecules is a rapidly growing technique, with an increased interest based on its ability to determine protein structures in the solid phase (1, 2) and to permit the study of noncrystalline biomolecular systems such as membrane proteins (3) and fibrils (4, 5).Limitations in biomolecular structural determination through SSNMR are due to the difficulties in obtaining a large number of restraints to be used for structural purposes (4, 6-9). Most of the structural information is obtained through distance restraints, analogous to nuclear Overhauser effects (NOE) in solution NMR, which in the solid state are obtained through experiments such as proton-driven spin diffusion (PDSD) (1, 6, 10), and CHHC (11), whereas specifically designed sequences can be applied on short peptides (4,(12)(13)(14). The ability to obtain a large number of distance restraints is hampered by the reduced resolution of SSNMR spectra, which increases the amount of ambiguities in the assigned restraints (15), whereas relayed transfer (10, 16) and the effects of the dipolar truncation (17) affect the accuracy of these restraints. These problems have been tackled by working on samples prepared with selective labeling schemes (1, 6) and, more recently, on uniformly labeled proteins with the help of software able to provide automated PDSD/ CHHC assignment and on dealing with a large number of ambiguous restraints (10,15,18). However, even when additional dihedral angle restraints from backbone chemical shiftsthrough Chemical Shift Index (CSI) (19) or TALOS (20) programs-are used, the size of the affordable proteins has been, up to now, limited to small systems (Ͻ100 aa) (10,15,18). In this work, we show how SSNMR paramagnetic restraints such as pseudocontact shifts (pcs) can be used as additional sources of restraints for protein structural determination, even providing information abou...

Magic Angle Spinning NMR Structure Determination of Proteins from Pseudocontact Shifts

“…It should be noted that although only C α PCSs are plotted in this figure, the fittings of the Δχ tensor parameters use all 13 C and 15 N PCSs. 36 in which they observed consistent PCSs in solid and solution state when the paramagnetic metal ions are in an internal, nonsolvent-exposed environment. Their observations reflect the similar local chemical environment around the paramagnetic metal ions in the solid and solution states.…”

“…These advantageous properties of PCS-based approach are expected to accelerate the structure determination of the proteins by solid-state NMR. Indeed, previous studies of PCSs in a metalloprotein 36 have shown great potential of PCSs as a source of long-range restraints for structure determination by solid-state NMR.…”

“…During the past several years, a number of MAS NMR studies of U- 13 C, 15 N enriched proteins containing paramagnetic ions have been reported. 18,32−51 In recent studies of metalloprotein catalytic domain of matrix metalloproteinase 12(CoMMP-12) 36,42 and Co…”

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

“…These effects act up to very long distances, and carry a well-defined dependence on the nuclear position with respect to the paramagnetic center. In solids, measuring pseudocontact shifts is relatively straightforward, and when they occur, they can be used as invaluable structural constraints (20)(21)(22). However, measuring PREs in solids is a prime example where traditional detection methods have difficulty to provide sufficient sensitivity.…”

Solid-state proton NMR of paramagnetic metal complexes: DANTE spin echoes for selective excitation in inhomogeneously broadened lines

“…In recent years, several experimental approaches have been proposed to overcome these intrinsic difficulties both in static solids and under magic-angle spinning (MAS) conditions [2][3][4]. Investigations of systems containing paramagnetic centers have proved to be important for a variety of fields, ranging from the structure determination of metalloproteins [5][6][7][8] to the characterization of catalytic organometallic complexes [9][10][11][12][13][14]. We have recently shown that spin-echo techniques based on combs of rotor-synchronized pulses employing Delays Alternating with Nutation for Tailored Excitation (DANTE) [15][16][17][18] can be used to excite nuclei such as protons that experience severe inhomogeneous broadening due to nearby paramagnetic nuclei [19].…”