Broadband Dynamics of Ubiquitin by Anionic and Cationic Nanoparticle Assisted NMR Spin Relaxation

Wardenfelt, Stacey; Xiang, Xinyao; Xie, Mouzhe; Yu, Lei; Bruschweiler‐Li, Lei; Brüschweiler, Rafael

doi:10.1002/anie.202007205

Cited by 15 publications

(31 citation statements)

References 30 publications

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“…This agreement indicates that on the ps to low-μs time-scale range probed here, the methyl group dynamics in ubiquitin is largely confined to the ps to low-ns regime. It mirrors our previous observation for the ubiquitin backbone dynamics, which also lack ns−μs motions according to 15 N-Δ R 2 NASR measurements . The average of S axis 2 (ΔR + ) and S axis 2 (ΔΓ) values, termed S axis 2 (NASR), were then mapped onto the ubiquitin crystal structure in Figure D.…”

Section: Resultssupporting

confidence: 78%

“…Levasil CS40–120 (previously known as Bindzil 2040) colloidal anionic silica nanoparticles (SNPs) with average size of 20 nm diameters were obtained from Nouryon (previously AkzoNobel) and were characterized previously. , Our previously reported TEM data shows a nearly symmetric distribution of the radius of gyration with ⟨ R g ⟩ = 19.5 ± 5.3 nm . SNPs were dialyzed into corresponding buffers (20 mM sodium phosphate at pH 7.0 for ubiquitin and 50 mM sodium phosphate at pH 7.0 for Im7) then directly mixed with proteins to prepare the SNP-containing NMR samples.…”

Section: Materials and Methodsmentioning

confidence: 99%

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Observation of Sub-Microsecond Protein Methyl-Side Chain Dynamics by Nanoparticle-Assisted NMR Spin Relaxation

Xiang

Hansen

et al. 2021

J. Am. Chem. Soc.

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Amino-acid side-chain properties in proteins are key determinants of protein function. NMR spin relaxation of side chains is an important source of information about local protein dynamics and flexibility. However, traditional solution NMR relaxation methods are most sensitive to sub-nanosecond dynamics lacking information on slower ns−μs time-scale motions. Nanoparticle-assisted NMR spin relaxation (NASR) of methyl-side chains is introduced here as a window into these ns−μs dynamics. NASR utilizes the transient and nonspecific interactions between folded proteins and slowly tumbling spherical nanoparticles (NPs), whereby the increase of the relaxation rates reflects motions on time scales from ps all the way to the overall tumbling correlation time of the NPs ranging from hundreds of ns to μs. The observed motional amplitude of each methyl group can then be expressed by a model-free NASR S 2 order parameter. The method is demonstrated for 2H-relaxation of CH2D methyl moieties and cross-correlated relaxation of CH3 groups for proteins Im7 and ubiquitin in the presence of anionic silica-nanoparticles. Both types of relaxation experiments, dominated by either quadrupolar or dipolar interactions, yield highly consistent results. Im7 shows additional dynamics on the intermediate time scales taking place in a functionally important loop, whereas ubiquitin visits the majority of its conformational substates on the sub-ns time scale. These experimental observations are in good agreement with 4–10 μs all-atom molecular dynamics trajectories. NASR probes side-chain dynamics on a much wider range of motional time scales than previously possible, thereby providing new insights into the interplay between protein structure, dynamics, and molecular interactions that govern protein function.

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Section: Resultssupporting

confidence: 78%

Section: Materials and Methodsmentioning

confidence: 99%

Observation of Sub-Microsecond Protein Methyl-Side Chain Dynamics by Nanoparticle-Assisted NMR Spin Relaxation

Xiang

Hansen

et al. 2021

J. Am. Chem. Soc.

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“…By contrast, the addition of SNPs (to 0.84 μM final SNP concentration) generates a differential line-broadening effect, from the weakest for GLY to the strongest for TMG, which rather nicely follows the degree of N -methylation (Figure A). Importantly, line broadening and the absence of changes in resonance positions observed here, which is indicative of fast exchange dynamics, are consistent with previous findings for SNP interactions with free amino acids, intrinsically disordered polypeptides, , and globular proteins. , …”

supporting

confidence: 92%

Degree of N-Methylation of Nucleosides and Metabolites Controls Binding Affinity to Pristine Silica Surfaces

Xie

Brüschweiler

2020

J. Phys. Chem. Lett.

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Biological molecules interact with silica (SiO2) surfaces with binding affinities that greatly vary depending on their physical–chemical properties. However, the quantitative characterization of biological compounds adsorbed on silica surfaces, especially of compounds involved in fast, reversible interactions, has been challenging, and the driving forces are not well understood. Here, we show how carbon-13 NMR spin relaxation provides quantitative atomic-detail information about the transient molecular binding to pristine silica surfaces, represented by colloidally dispersed silica nanoparticles (SNPs). Based on the quantitative analysis of almost two dozen biological molecules, we find that the addition of N-methyl motifs systematically increases molecular binding affinities to silica in a nearly quantitatively predictable manner. Among the studied compounds are methylated nucleosides, which are common in epigenetic signaling in nucleic acids. The quantitative understanding of N-methylation may open up new ways to detect and separate methylated nucleic acids or even regulate their cellular functions.

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“…26,27 In addition, when proteins are intrinsically disordered or contain long, flexible loops, the motions of flexible residues can lead to different broadening behaviors for each residue. 19,28,29 However, for many circumstances, the situation becomes much simpler: when proteins are globular and lack long loops, and when the kinetics of desorption are slow on the NMR time scale, 30 all signals from the adsorbed proteins are uniformly broadened beyond detection. The intensity of the observed NMR signal therefore is proportional to the concentration of free proteins, which can be used to quantify the bound proteins.…”

Section: Introductionmentioning

confidence: 99%

“…In the study of protein–NP association, the line broadening effect that occurs upon binding is particularly important in studies of protein corona . Multiple outcomes are possible: residues that are weakly coupled to the NP surface can exhibit lifetime line broadening, becoming broader in the presence of NPs. , In addition, when proteins are intrinsically disordered or contain long, flexible loops, the motions of flexible residues can lead to different broadening behaviors for each residue. ,, However, for many circumstances, the situation becomes much simpler: when proteins are globular and lack long loops, and when the kinetics of desorption are slow on the NMR time scale, all signals from the adsorbed proteins are uniformly broadened beyond detection. The intensity of the observed NMR signal therefore is proportional to the concentration of free proteins, which can be used to quantify the bound proteins.…”

Section: Introductionmentioning

confidence: 99%

Quantitative Measurement of Multiprotein Nanoparticle Interactions Using NMR Spectroscopy

Alom

Fitzkee

2021

Anal. Chem.

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An effective intensity-based reference is a cornerstone for quantitative nuclear magnetic resonance (NMR) studies, as the molecular concentration is encoded in its signal. In theory, NMR is well suited for the measurement of competitive protein adsorption onto nanoparticle (NP) surfaces, but current referencing systems are not optimized for multidimensional experiments. Presented herein is a simple and novel referencing system using 15 N tryptophan (Trp) as an external reference for 1 H− 15 N 2D NMR experiments. The referencing system is validated by the determination of the binding capacity of a single protein onto gold NPs. Then, the Trp reference is applied to protein mixtures, and signals from each protein are accurately quantified. All results are consistent with previous studies, but with substantially higher precision, indicating that the Trp reference can accurately calibrate the residue peak intensities and reduce systematic errors. Finally, the proposed Trp reference is used to kinetically monitor in situ and in real time the competitive adsorption of different proteins. As a challenging test case, we successfully apply our approach to a mixture of protein variants differing by only a single residue. Our results show that the binding of one protein will affect the binding of the other, leading to an altered NP corona composition. This work therefore highlights the importance of studying protein−NP interactions in protein mixtures in situ, and the referencing system developed here enables the quantification of binding kinetics and thermodynamics of multiple proteins using various 1 H− 15 N 2D NMR techniques.

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Broadband Dynamics of Ubiquitin by Anionic and Cationic Nanoparticle Assisted NMR Spin Relaxation

Cited by 15 publications

References 30 publications

Observation of Sub-Microsecond Protein Methyl-Side Chain Dynamics by Nanoparticle-Assisted NMR Spin Relaxation

Observation of Sub-Microsecond Protein Methyl-Side Chain Dynamics by Nanoparticle-Assisted NMR Spin Relaxation

Degree of N-Methylation of Nucleosides and Metabolites Controls Binding Affinity to Pristine Silica Surfaces

Quantitative Measurement of Multiprotein Nanoparticle Interactions Using NMR Spectroscopy

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