Chemical-Shift Anisotropy Measurements of Amide and Carbonyl Resonances in a Microcrystalline Protein with Slow Magic-Angle Spinning NMR Spectroscopy

Wylie, Benjamin J.; Sperling, Lindsay J.; Frericks, Heather L.; Shah, Gautam J.; Franks, W. Trent; Rienstra, Chad M.

doi:10.1021/ja0701199

Cited by 79 publications

(118 citation statements)

References 24 publications

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“…Data collected at neutral pH and low potassium indicate that E71 and D80 retain the same protonation states at high and low potassium. As a control we also measured the tensor of the backbone carbonyl of the α-helical residue A32 and compared it to previously measured α-helical tensors (24). The agreement is good.…”

Section: E71a Mutantmentioning

confidence: 89%

Protonation state of E71 in KcsA and its role for channel collapse and inactivation

Bhate

McDermott

2012

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

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The prototypical prokaryotic potassium channel KcsA alters its pore depending on the ambient potassium; at high potassium, it exists in a conductive form, and at low potassium, it collapses into a nonconductive structure with reduced ion occupancy. We present solidstate NMR studies of KcsA in which we test the hypothesis that an important channel-inactivation process, known as C-type inactivation, proceeds via a state similar to this collapsed state. We test this using an inactivation-resistant mutant E71A, and show that E71A is unable to collapse its pore at both low potassium and low pH, suggesting that the collapsed state is structurally similar to the inactivated state. We also show that E71A has a disordered selectivity filter. Using site-specific K þ titrations, we detect a local change at E71 that is coupled to channel collapse at low K þ . To gain more insight into this change, we site specifically measure the chemical shift tensors of the side-chain carboxyls of E71 and its hydrogen bond partner D80, and use the tensors to assign protonation states to E71 and D80 at high K þ and neutral pH. Our measurements show that E71 is protonated at pH 7.5 and must have an unusually perturbed pK a (>7.5) suggesting that the change at E71 is a structural rearrangement rather than a protonation event. The results offer new mechanistic insights into why the widely used mutant KcsA-E71A does not inactivate and establish the ambient K þ level as a means to populate the inactivated state of KcsA in a controlled way.membrane proteins | ion channel | chemical-shift anisotropy

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Section: E71a Mutantmentioning

confidence: 89%

Protonation state of E71 in KcsA and its role for channel collapse and inactivation

Bhate

McDermott

2012

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

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“…Backbone CA shifts report primarily on backbone conformation, [19][20][21][22] whereas 15 N chemical shifts depend upon not only conformation but are especially sensitive to electrostatics and hydrogen bonding. 23,24 Carbonyl chemical shifts also show a dependence on hydrogen bonding, as well as backbone conformation. 19,25,26 However, the C', CA and backbone 15 N chemical shifts are not the most prominent reporters on the subtle changes among crystal formulations including intermolecular crystal contacts, protonation state of the ionizable sidechains, aromatic ring interactions and salt bridge geometry.…”

Section: Introductionmentioning

confidence: 99%

Crystal Polymorphism of Protein GB1 Examined by Solid-State NMR Spectroscopy and X-ray Diffraction

et al. 2007

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The study of micro-or nanocrystalline proteins by magic-angle spinning (MAS) solid-state NMR (SSNMR) gives atomic-resolution insight into structure in cases when single crystals cannot be obtained for diffraction studies. Subtle differences in the local chemical environment around the protein, including the characteristics of the co-solvent and the buffer, determine whether a protein will form single crystals. The impact of these small changes in formulation is also evident in the SSNMR spectra, but leads only to correspondingly subtle changes in the spectra. Here we demonstrate that several formulations of GB1 microcrystals yield very high-quality SSNMR spectra, although only a subset of conditions enable growth of single crystals. We have characterized these polymorphs by X-ray powder diffraction and assigned the SSNMR spectra. Assignments of the 13 C and 15 N SSNMR chemical shifts confirm that the backbone structure is conserved, indicative of a common protein fold, but sidechain chemical shifts are changed on the surface of the protein, in a manner dependent upon crystal packing and electrostatic interactions with salt in the mother liquor. Our results demonstrate the ability of SSNMR to reveal minor structural differences among crystal polymorphs. This ability has potential practical utility for studying formulation chemistry of industrial and therapeutic proteins, as well as for deriving fundamental insights into the phenomenon of single crystal growth.

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“…Nevertheless, this should not present a fundamental problem when simulating PDSD in proteins, since in that case the 13 C CSA parameters are accessible through ab initio calculations, 82 or can be estimated from the isotropic chemical shift values. 83 The exchange of the protons in the nearby -NH 3 and -CH 3 groups should also be taken into account in the simulations. Freezing this exchange in the serine, for example, leads to noticeable overestimation of the constants, with the largest relative errors observed for the CO-CA pair (2% mean and 9% standard deviation).…”

Section: B Factors Affecting the Accuracymentioning

confidence: 99%

Proton-driven spin diffusion in rotating solids via reversible and irreversible quantum dynamics

Veshtort

Griffin

2011

The Journal of Chemical Physics

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Proton-driven spin diffusion (PDSD) experiments in rotating solids have received a great deal of attention as a potential source of distance constraints in large biomolecules. However, the quantitative relationship between the molecular structure and observed spin diffusion has remained obscure due to the lack of an accurate theoretical description of the spin dynamics in these experiments. We start with presenting a detailed relaxation theory of PDSD in rotating solids that provides such a description. The theory applies to both conventional and radio-frequency-assisted PDSD experiments and extends to the non-Markovian regime to include such phenomena as rotational resonance (R 2 ). The basic kinetic equation of the theory in the non-Markovian regime has the form of a memory function equation, with the role of the memory function played by the correlation function. The key assumption used in the derivation of this equation expresses the intuitive notion of the irreversible dissipation of coherences in macroscopic systems. Accurate expressions for the correlation functions and for the spin diffusion constants are given. The theory predicts that the spin diffusion constants governing the multi-site PDSD can be approximated by the constants observed in the two-site diffusion. Direct numerical simulations of PDSD dynamics via reversible Liouville-von Neumann equation are presented to support and compliment the theory. Remarkably, an exponential decay of the difference magnetization can be observed in such simulations in systems consisting of only 12 spins. This is a unique example of a real physical system whose typically macroscopic and apparently irreversible behavior can be traced via reversible microscopic dynamics. An accurate value for the spin diffusion constant can be usually obtained through direct simulations of PDSD in systems consisting of two 13 C nuclei and about ten 1 H nuclei from their nearest environment. Spin diffusion constants computed by this method are in excellent agreement with the spin diffusion constants obtained through equations given by the relaxation theory of PDSD. The constants resulting from these two approaches were also in excellent agreement with the results of 2D rotary resonance recoupling proton-driven spin diffusion (R 3 -PDSD) experiments performed in three model compounds, where magnetization exchange occurred over distances up to 4.9 Å. With the methodology presented, highly accurate internuclear distances can be extracted from such data. Relayed transfer of magnetization between distant nuclei appears to be the main (and apparently resolvable) source of uncertainty in such measurements. The non-Markovian kinetic equation was applied to the analysis of the R 2 spin dynamics. The conventional semi-phenomenological treatment of relxation in R 2 has been shown to be equivalent to the assumption of the Lorentzian spectral density function in the relaxatoin theory of PDSD. As this assumption is a poor approximation in real physical systems, the conventional R 2 treatment is li...

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Chemical-Shift Anisotropy Measurements of Amide and Carbonyl Resonances in a Microcrystalline Protein with Slow Magic-Angle Spinning NMR Spectroscopy

Cited by 79 publications

References 24 publications

Protonation state of E71 in KcsA and its role for channel collapse and inactivation

Protonation state of E71 in KcsA and its role for channel collapse and inactivation

Crystal Polymorphism of Protein GB1 Examined by Solid-State NMR Spectroscopy and X-ray Diffraction

Proton-driven spin diffusion in rotating solids via reversible and irreversible quantum dynamics

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