Long-Range Structural Restraints in Spin-Labeled Proteins Probed by Solid-State Nuclear Magnetic Resonance Spectroscopy

Nadaud, Philippe S.; Helmus, Jonathan; Höfer, Nicole; Jaroniec, Christopher P.

doi:10.1021/ja072349t

Cited by 110 publications

(154 citation statements)

References 29 publications

Supporting

Mentioning

146

Contrasting

Order By: Relevance

“…While enhanced relaxation caused by a paramagnetic center can be exploited for fast recycling and condensed data collection approaches relying on 13 C detection (23)(24)(25), the addition of paramagnetic dopants is not appropriate for the quantitative measurement of relaxation times as a source of structural and dynamic information. As a result, site-specific PREs in the solid state have only been reported on one small model protein (GB1) by the use of cysteine-containing mutants with paramagnetic tags attached (26,27). …”

mentioning

confidence: 99%

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

Knight

Pell

Bertini

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

179

244

View full text Add to dashboard Cite

We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with 1 H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of 15 N and 13 C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu þ (diamagnetic) or Cu 2þ (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to 1 H-1 H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a Gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.paramagnetism | nuclear relaxation rates | copper | microcrystal S tructure determination of proteins plays a central role in understanding key events in biology. Although the structure of many proteins can be obtained from single-crystal X-ray diffraction, or by solution-state nuclear magnetic resonance (NMR) spectroscopy, there is nevertheless a range of important substrates for which structures cannot be determined today. These include immobile systems lacking long-range order such as protein aggregates, large complexes and membrane-bound systems.Solid-state NMR has the unique potential to study, with atomic resolution, systems of this nature, and spectacular progress has been made in this area over the last decade (1). There are today a small handful of structures obtained by solid-state NMR, from microcrystalline samples to fibrils and membrane-associated systems (2). Additionally, solid-state NMR is uniquely sensitive to site-specific protein dynamics over a broad range of timescales, and a number of demonstration studies have recently appeared for model proteins (3).The use of perdeuterated proteins has very recently opened the way to highly sensitive proton-detected solid-state experiments (4). Despite early proof-of-principle papers (5), this approach only became popular with the realization that amide sites must be only partially reprotonated (typically 10-30% back exchange) (6-8) to yield well-resolved 1 H spectra. This represented a significant compromise in sensitivity to gain resolution, and effectively made the determination of internuclear distances impractical, with few exceptions (9-11). We have recently shown how this problem can be completely overcome by using 100% reprotonation of exchangeable sites, without loss of resolution, if perdeuteration is combined wit...

show abstract

mentioning

confidence: 99%

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

Knight

Pell

Bertini

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

179

244

View full text Add to dashboard Cite

show abstract

“…Spin labels allow one to obtain long-range distance constraints, [10] particularly in large proteins. [11] Herein, we demonstrate DNP-MAS in a solvent-free amorphous powder of the decapeptide Fmoc-Gly-Ala-Arg(Pbf)-GlyAsp(OtBu)-d-Phe-Lys(Z)-Arg(Pbf)-Gly-OH) (DP), of which a fraction f 16 % was labeled by covalent attachment of the biradical TOTAPOL (DP*).…”

mentioning

confidence: 99%

Fractional Spin‐Labeling of Polymers for Enhancing NMR Sensitivity by Solvent‐Free Dynamic Nuclear Polarization

et al. 2011

View full text Add to dashboard Cite

Dynamic nuclear polarization (DNP) [1] combined with magic angle spinning (MAS) [2] can-under favorable conditions-enhance the nuclear spin polarization, that is, the difference between the populations of the Zeeman levels j ai and j bi of a spin I = 1 = 2 by up to two orders of magnitude (e DNP 10 2 ) with respect to the Boltzmann distribution at thermal equilibrium at ca. 100 K, while accelerating relaxation and hence reducing recovery delays by more than an order of magnitude (k = R DNP / R 1 = T 1 /t DNP > 10), thus providing a means of shortening measurement times by up to five orders of magnitude. Where suitable solvents can be found, typical enhancements for 13

show abstract

“…Pioneering works have shown that paramagnetic proteins are also affordable by SSNMR by using either perdeuterated substrates (35) or selective labeling (36). More recently, uniformly labeled paramagnetic proteins have also been studied (37)(38)(39), taking advantage of the absence of paramagnetic relaxation mechanisms related to the molecular tumbling (as Curie relaxation terms) (40). The prospective availability of fast and ultrafast MAS probes should allow one to reduce the limits posed by the presence of metals inducing large shift anisotropies (33,40,41).…”

mentioning

confidence: 99%

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

Balayssac

Bertini

Bhaumik

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

141

View full text Add to dashboard Cite

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...

show abstract

Long-Range Structural Restraints in Spin-Labeled Proteins Probed by Solid-State Nuclear Magnetic Resonance Spectroscopy

Cited by 110 publications

References 29 publications

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

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

Fractional Spin‐Labeling of Polymers for Enhancing NMR Sensitivity by Solvent‐Free Dynamic Nuclear Polarization

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

Contact Info

Product

Resources

About