Protein Side-Chain Dynamics Observed by Solution- and Solid-State NMR:  Comparative Analysis of Methyl <sup>2</sup>H Relaxation Data

Reif, Bernd; Xue, Yi; Agarwal, Vipin; Pavlova, Maria S.; Hologne, Maggy; Diehl, Anne; Ryabov, Yaroslav; Skrynnikov, Nikolai R.

doi:10.1021/ja062808a

Cited by 59 publications

(71 citation statements)

References 31 publications

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“…As observed for increasing weight percentage of NaF, salt activation is accompanied by a large ⌬⌬S † and a negligible ⌬⌬H † , providing further evidence that the activation effect is primarily entropic/dynamic in nature. (29)(30)(31), were precluded by the high molecular weight of subtilisin and the large homonuclear dipolar couplings associated with solid enzyme samples. However, prior evidence that bulk relaxation measurements in the solid state are a valid measure of enzyme dynamics has been provided previously where side-chain dynamics (measured via T 1D ) of ␣-spectrin SH3 were found to be roughly constant regardless of their position in the amino acid sequence (29).…”

Section: Resultsmentioning

confidence: 99%

Biocatalyst activity in nonaqueous environments correlates with centisecond-range protein motions

Eppler

Hudson

Chase

et al. 2008

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

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Recent studies exploring the relationship between enzymatic catalysis and protein dynamics in the aqueous phase have yielded evidence that dynamics and enzyme activity are strongly correlated. Given that protein dynamics are significantly attenuated in organic solvents and that proteins exhibit a wide range of motions depending on the specific solvent environment, the nonaqueous milieu provides a unique opportunity to examine the role of protein dynamics in enzyme activity. Variable-temperature kinetic measurements, X-band electron spin resonance spectroscopy, 1 H NMR relaxation, and 19 F NMR spectroscopy experiments were performed on subtilisin Carlsberg colyophilized with several inorganic salts and suspended in organic solvents. The results indicate that salt activation induces a greater degree of transition-state flexibility, reflected by a more positive ⌬⌬S † , for the more active biocatalyst preparations in organic solvents. In contrast, ⌬⌬H † was negligible regardless of salt type or salt content. Electron spin resonance spectroscopy and 1 H NMR relaxation measurements, including spin-lattice relaxation, spin-lattice relaxation in the rotating frame, and longitudinal magnetization exchange, revealed that the enzyme's turnover number (k cat) was strongly correlated with protein motions in the centisecond time regime, weakly correlated with protein motions in the millisecond regime, and uncorrelated with protein motions on the piconanosecond timescale. In addition, 19 F chemical shift measurements and hyperfine tensor measurements of biocatalyst formulations inhibited with 4-fluorobenzenesulfonyl fluoride and 4-ethoxyfluorophosphinyl-oxy-TEMPO, respectively, suggest that enzyme activation was only weakly affected by changes in active-site polarity.enzyme activation ͉ enzyme dynamics ͉ NMR spectroscopy ͉ organic solvents ͉ subtilisin Carlsberg T he direct coupling of protein dynamics to enzymatic catalysis is strongly supported by recent studies of enzymes in aqueous solution (1-3). These studies have revealed insights into the connection between enzyme activity and protein dynamics [e.g., conformation exchange in CypA (4), Aquifex Adk lid movement (5), Met-20 loop dynamics in DHFR (6), loop motions leading to conformational change in RNase A (7), and modulation of surfaceglycosylated chymotrypsin structural dynamics and activity (8)], yet the relatively small range of enzyme activities accessible under aqueous conditions, without the use of protein modifications or alteration of the solvent, hinders thorough investigation of the central hypothesis that enzyme catalysis is intimately coupled to protein dynamics. One strategy for broadening the range of activities over which enzyme catalysis and protein dynamics can be studied concurrently is to employ nonaqueous or organic solvents. For example, a correlation between molecular dynamics, as evidenced by a reduced rate of amide H/D exchange, and biocatalyst activity has been observed for PEGylated subtilisin Carlsberg (SC) in 1,4-dioxane (9).Nonaqueous biocata...

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Section: Resultsmentioning

confidence: 99%

Biocatalyst activity in nonaqueous environments correlates with centisecond-range protein motions

Eppler

Hudson

Chase

et al. 2008

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

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“…2 H NMR has long been used to determine the degree to which amino acid side chain dynamics are sensitive to structural environment, for example, the perturbation of the motion of leucine side chains in collagen fibrils (30), the dynamic impact of polylysine adsorption onto silica and hydroxyapatite surfaces (31), and the dynamics of hydrophobic side chains in membrane-associated proteins (32)(33)(34). Additionally, 13 C-2 H NMR correlation techniques have been used by Reif and coworkers to identify the dynamics of deuterated valine and leucine side chains in uniformly 13 C-labeled micro-crystalline proteins (35,36).…”

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confidence: 99%

Sum frequency generation and solid-state NMR study of the structure, orientation, and dynamics of polystyrene-adsorbed peptides

Weidner

Breen

et al. 2010

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

134

221

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The power of combining sum frequency generation (SFG) vibrational spectroscopy and solid-state nuclear magnetic resonance (ssNMR) spectroscopy to quantify, with site specificity and atomic resolution, the orientation and dynamics of side chains in synthetic model peptides adsorbed onto polystyrene (PS) surfaces is demonstrated in this study. Although isotopic labeling has long been used in ssNMR studies to site-specifically probe the structure and dynamics of biomolecules, the potential of SFG to probe side chain orientation in isotopically labeled surface-adsorbed peptides and proteins remains largely unexplored. The 14 amino acid leucine-lysine peptide studied in this work is known to form an α-helical secondary structure at liquid-solid interfaces. Selective, individual deuteration of the isopropyl group in each leucine residue was used to probe the orientation and dynamics of each individual leucine side chain of LKα14 adsorbed onto PS. The selective isotopic labeling methods allowed SFG analysis to determine the orientations of individual side chains in adsorbed peptides. Side chain dynamics were obtained by fitting the deuterium ssNMR line shape to specific motional models. Through the combined use of SFG and ssNMR, the dynamic trends observed for individual side chains by ssNMR have been correlated with side chain orientation relative to the PS surface as determined by SFG. This combination provides a more complete and quantitative picture of the structure, orientation, and dynamics of these surface-adsorbed peptides than could be obtained if either technique were used separately.protein | surface | isotope labels | solid-liquid interface

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“…We and others could show previously, that deuteration and back-substitution of exchangeable protons allows for sensitive detection of protons [29][30][31], determination of long range 1 H, 1 H distances [32][33][34], and permits to localize mobile water molecules in the protein structure [35]. In addition, the 2 H spin can be used to probe side chain dynamics [36][37][38][39]. In contrast to the experimental scheme that was suggested by Ramamoorthy et al [40], use of high power decoupling is redundant using the deuteration approach.…”

Section: Introductionmentioning

confidence: 96%

Sensitivity enhancement using paramagnetic relaxation in MAS solid-state NMR of perdeuterated proteins

Linser

Chevelkov

Diehl

et al. 2007

Journal of Magnetic Resonance

Self Cite

127

109

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Previously, Ishii et al., could show that chelated paramagnetic ions can be employed to significantly decrease the recycle delay of a MAS solid-state NMR experiment [N.P. Wickramasinghe, M. Kotecha, A. Samoson, J. Past, Y. Ishii, Sensitivity enhancement in C-13 solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing H-1 T-1 relaxation, J. Magn. Reson. 184 (2007) 350-356]. Application of the method is limited to very robust samples, for which sample stability is not compromised by RF induced heating. In addition, probe integrity might be perturbed in standard MAS PRE experiments due to the use of very short duty cycles. We show that these deleterious effects can be avoided if perdeuterated proteins are employed that have been re-crystallized from D 2 O:H 2 O = 9:1 containing buffer solutions. The experiments are demonstrated using the SH3 domain of chicken a-spectrin as a model system. The labeling scheme allows to record proton detected 1 H, 15 N correlation spectra with very high resolution in the absence of heteronuclear dipolar decoupling. Cu-edta as a doping reagent yields a reduction of the recycle delay by up to a factor of 15. In particular, we find that the 1 H T 1 for the bulk H N magnetization is reduced from 4.4 s to 0.3 s if the Cu-edta concentration is increased from 0 mM to 250 mM. Possible perturbations like chemical shift changes or line broadening due to the paramagnetic chelate complex are minimal. No degradation of our samples was observed in the course of the experiments.

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Protein Side-Chain Dynamics Observed by Solution- and Solid-State NMR: Comparative Analysis of Methyl ²H Relaxation Data

Cited by 59 publications

References 31 publications

Biocatalyst activity in nonaqueous environments correlates with centisecond-range protein motions

Biocatalyst activity in nonaqueous environments correlates with centisecond-range protein motions

Sum frequency generation and solid-state NMR study of the structure, orientation, and dynamics of polystyrene-adsorbed peptides

Sensitivity enhancement using paramagnetic relaxation in MAS solid-state NMR of perdeuterated proteins

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Protein Side-Chain Dynamics Observed by Solution- and Solid-State NMR: Comparative Analysis of Methyl 2H Relaxation Data

Cited by 59 publications

References 31 publications

Biocatalyst activity in nonaqueous environments correlates with centisecond-range protein motions

Biocatalyst activity in nonaqueous environments correlates with centisecond-range protein motions

Sum frequency generation and solid-state NMR study of the structure, orientation, and dynamics of polystyrene-adsorbed peptides

Sensitivity enhancement using paramagnetic relaxation in MAS solid-state NMR of perdeuterated proteins

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Protein Side-Chain Dynamics Observed by Solution- and Solid-State NMR: Comparative Analysis of Methyl ²H Relaxation Data