Quantitative Interpretation of Solvent Paramagnetic Relaxation for Probing Protein–Cosolute Interactions

Szabó, Attila; Clore, G. Marius

doi:10.1021/jacs.0c00747

Cited by 25 publications

(52 citation statements)

References 81 publications

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“…Tetramethyl nitroxide compounds such as PROXYL or TEMPOL are known to preferentially interact with hydrophobic patches on protein surface (23,26,27). Because the hydrophobic moiety of PROXYL is identical for the two PROXYL derivatives, it is likely that the hydrophobic contribution in each U term is approximately cancelled in ΔU ENS .…”

Section: Resultsmentioning

confidence: 99%

De novo determination of near-surface electrostatic potentials by NMR

Pletka

Pettitt

et al. 2021

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

118

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Electrostatic potentials computed from three-dimensional structures of biomolecules by solving the Poisson–Boltzmann equation are widely used in molecular biophysics, structural biology, and medicinal chemistry. Despite the approximate nature of the Poisson–Boltzmann theory, validation of the computed electrostatic potentials around biological macromolecules is rare and methodologically limited. Here, we present a unique and powerful NMR method that allows for straightforward and extensive comparison with electrostatic models for biomolecules and their complexes. This method utilizes paramagnetic relaxation enhancement arising from analogous cationic and anionic cosolutes whose spatial distributions around biological macromolecules reflect electrostatic potentials. We demonstrate that this NMR method enables de novo determination of near-surface electrostatic potentials for individual protein residues without using any structural information. We applied the method to ubiquitin and the Antp homeodomain–DNA complex. The experimental data agreed well with predictions from the Poisson–Boltzmann theory. Thus, our experimental results clearly support the validity of the theory for these systems. However, our experimental study also illuminates certain weaknesses of the Poisson–Boltzmann theory. For example, we found that the theory predicts stronger dependence of near-surface electrostatic potentials on ionic strength than observed in the experiments. Our data also suggest that conformational flexibility or structural uncertainties may cause large errors in theoretical predictions of electrostatic potentials, particularly for highly charged systems. This NMR-based method permits extensive assessment of near-surface electrostatic potentials for various regions around biological macromolecules and thereby may facilitate improvement of the computational approaches for electrostatic potentials.

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Section: Resultsmentioning

confidence: 99%

De novo determination of near-surface electrostatic potentials by NMR

Pletka

Pettitt

et al. 2021

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

118

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“…For sPREs this expression is approximate, with the quantitative relationship between Eq. 2 and solvent PRE being somewhat more complicated ( Okuno et al, 2020 ), and yet this formulation has been employed with some success. In this vein, our residue-based water-edited SSNMR-derived surface area data are fit to values computed from molecular structure using Eq.…”

Section: Resultsmentioning

confidence: 99%

Water Accessibility Refinement of the Extended Structure of KirBac1.1 in the Closed State

Amani

Schwieters

Borcik

et al. 2021

Front. Mol. Biosci.

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NMR structures of membrane proteins are often hampered by poor chemical shift dispersion and internal dynamics which limit resolved distance restraints. However, the ordering and topology of these systems can be defined with site-specific water or lipid proximity. Membrane protein water accessibility surface area is often investigated as a topological function via solid-state NMR. Here we leverage water-edited solid-state NMR measurements in simulated annealing calculations to refine a membrane protein structure. This is demonstrated on the inward rectifier K+ channel KirBac1.1 found in Burkholderia pseudomallei. KirBac1.1 is homologous to human Kir channels, sharing a nearly identical fold. Like many existing Kir channel crystal structures, the 1p7b crystal structure is incomplete, missing 85 out of 333 residues, including the N-terminus and C-terminus. We measure solid-state NMR water proximity information and use this for refinement of KirBac1.1 using the Xplor-NIH structure determination program. Along with predicted dihedral angles and sparse intra- and inter-subunit distances, we refined the residues 1–300 to atomic resolution. All structural quality metrics indicate these restraints are a powerful way forward to solve high quality structures of membrane proteins using NMR.

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“…This compound is a conjugate of OAc − and a paramagnetic group, 2,2,5,5-tetramethyl-pyrrolidinooxy (PROXYL). Recently, for ubiquitin, Okuno et al used carboxy-PROXYL and its neutral analog, carbamoyl-PROXYL to observe weak protein–cosolute interactions through PRE measurements ( 35 ). This type of PRE, which is conventionally referred to as solvent PRE, occurs through dipole–dipole interactions between the unpaired electrons of the cosolutes and nuclei of a protein ( 36 ).…”

Section: Resultsmentioning

confidence: 99%

“…In general, protein 1 H nuclei near the molecular surface exhibit large PRE arising from paramagnetic cosolutes. If the paramagnetic cosolutes are distributed uniformly around the protein, the PRE profile can be predicted from the protein structure using an empirical approach proposed by Pintacuda and Otting ( 37 ) and by Hernández et al ( 38 ) However, for nitroxide compounds involving a tetramethyl moiety (e.g., TEMPOL, PROXYL, and their derivatives), the spatial distribution around a protein is known to be nonuniform and biased due to hydrophobic interactions with apolar patches on protein surfaces ( 35 , 37 ). A similar bias in the spatial distribution of PROXYL derivatives may also occur on DNA surfaces ( 39 ).…”

Section: Resultsmentioning

confidence: 99%

Quantifying and visualizing weak interactions between anions and proteins

Pletka

Iwahara

2020

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

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The molecular properties of proteins are influenced by various ions present in the same solution. While site-specific strong interactions between multivalent metal ions and proteins are well characterized, the behavior of other ions that are only weakly interacting with proteins remains elusive. In the current study, using NMR spectroscopy, we have investigated anion–protein interactions for three proteins that are similar in size but differ in overall charge. Using a unique NMR-based approach, we quantified anions accumulated around the proteins. The determined numbers of anions that are electrostatically attracted to the charged proteins were notably smaller than the overall charge valences and were consistent with predictions from the Poisson–Boltzmann theory. This NMR-based approach also allowed us to measure ionic diffusion and characterize the anions interacting with the positively charged proteins. Our data show that these anions rapidly diffuse while bound to the proteins. Using the same experimental approach, we observed the release of the anions from the protein surface upon the formation of the Antp homeodomain–DNA complex. Using paramagnetic relaxation enhancement (PRE), we visualized the spatial distribution of anions around the free proteins and the Antp homeodomain–DNA complex. The obtained PRE data revealed the localization of anions in the vicinity of the highly positively charged regions of the free Antp homeodomain and provided further evidence of the release of anions from the protein surface upon the protein–DNA association. This study sheds light on the dynamic behavior of anions that electrostatically interact with proteins.

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Quantitative Interpretation of Solvent Paramagnetic Relaxation for Probing Protein–Cosolute Interactions

Cited by 25 publications

References 81 publications

De novo determination of near-surface electrostatic potentials by NMR

De novo determination of near-surface electrostatic potentials by NMR

Water Accessibility Refinement of the Extended Structure of KirBac1.1 in the Closed State

Quantifying and visualizing weak interactions between anions and proteins

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