scholarly journals A suite of 19F based relaxation dispersion experiments to assess biomolecular motions

2020 ◽  
Vol 74 (12) ◽  
pp. 753-766
Author(s):  
Jan H. Overbeck ◽  
Werner Kremer ◽  
Remco Sprangers
Keyword(s):  
Structural Information ◽  
Chemical Exchange ◽  
Chemical Shifts ◽  
Nmr Relaxation ◽  
Relaxation Dispersion ◽  
Cold Shock Protein ◽  
X Ray Crystallography ◽  
Kinetic And Thermodynamic Parameters ◽  
Low Costs ◽  
Exchange Parameters

Abstract Proteins and nucleic acids are highly dynamic bio-molecules that can populate a variety of conformational states. NMR relaxation dispersion (RD) methods are uniquely suited to quantify the associated kinetic and thermodynamic parameters. Here, we present a consistent suite of 19F-based CPMG, on-resonance R1ρ and off-resonance R1ρ RD experiments. We validate these experiments by studying the unfolding transition of a 7.5 kDa cold shock protein. Furthermore we show that the 19F RD experiments are applicable to very large molecular machines by quantifying dynamics in the 360 kDa half-proteasome. Our approach significantly extends the timescale of chemical exchange that can be studied with 19F RD, adds robustness to the extraction of exchange parameters and can determine the absolute chemical shifts of excited states. Importantly, due to the simplicity of 19F NMR spectra, it is possible to record complete datasets within hours on samples that are of very low costs. This makes the presented experiments ideally suited to complement static structural information from cryo-EM and X-ray crystallography with insights into functionally relevant motions. Graphic abstract

scholarly journals Probing the excited-state chemical shifts and exchange parameters by nitrogen-decoupled amide proton chemical exchange saturation transfer (HNdec-CEST)

2017 ◽  
Vol 53 (61) ◽  
pp. 8541-8544 ◽  
Author(s):  
Qinglin Wu ◽  
Benjamin A. Fenton ◽  
Jessica L. Wojtaszek ◽  
Pei Zhou
Keyword(s):  
Excited State ◽  
Chemical Exchange ◽  
Chemical Shifts ◽  
Chemical Exchange Saturation Transfer ◽  
Amide Proton ◽  
Saturation Transfer ◽  
State Information ◽  
State Chemical ◽  
Exchange Parameters

The HNdec-CEST experiment enables robust extraction of excited-state information of macromolecules.

Mutational investigation of protein folding transition states by Φ-value analysis and beyond: lessons from SH3 domain foldingThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (2) ◽  
pp. 231-238 ◽  
Author(s):  
Arash Zarrine-Afsar ◽  
Sung Lun Lin ◽  
Philipp Neudecker
Keyword(s):  
Protein Folding ◽  
Transition State ◽  
Structural Information ◽  
Peer Review Process ◽  
Transition States ◽  
Nmr Relaxation ◽  
Relaxation Dispersion ◽  
Folding Kinetics ◽  
Value Analysis ◽  
Kinetic Effects

Understanding how proteins adopt their unique native structures requires a complete structural characterization of the rate-limiting transition state(s) along the folding pathway. By definition, transition states are not significantly populated and are only accessible via folding kinetics studies. In this respect, interpreting the kinetic effects of amino acid substitutions (especially to Ala) via Φ-value analysis is the most common method to probe the structure of these transient, yet important states. A critical review of the key assumptions required for rigorous interpretation of Φ values reveals that a multiple substitution strategy in which a position of interest is mutated to a variety of amino acids, and not exclusively to Ala, provides the best means to characterize folding transition states. This approach has proven useful in revealing non-native interactions and (or) conformations in folding transition states. Moreover, by simultaneously examining the folding kinetics of multiple substitutions made at a single surface-exposed position using the Brønsted analysis the backbone conformation in a folding transition state can be investigated. For folding equilibria with exchange rates on the order of milliseconds, the kinetic parameters for Φ-value analysis can be obtained from NMR relaxation dispersion experiments, under fully native conditions, along with a wealth of high-resolution structural information about the states in exchange (native, denatured, and intermediate states that populate the pathway). This additional structural information, which is not readily obtained through stopped-flow based methods, can significantly facilitate the interpretation of Φ values because it often reports on the validity of the assumptions required for a rigorous interpretation of Φ values.

scholarly journals Chemical shift prediction of RNA imino groups: application toward characterizing RNA excited states

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yanjiao Wang ◽  
Ge Han ◽  
Xiuying Jiang ◽  
Tairan Yuwen ◽  
Yi Xue
Keyword(s):  
Chemical Shift ◽  
Base Pair ◽  
Chemical Shifts ◽  
Nmr Relaxation ◽  
Semiempirical Method ◽  
Relaxation Dispersion ◽  
Lookup Table ◽  
Imino Group ◽  
Chemical Shift Prediction ◽  
Central Base

AbstractNH groups in proteins or nucleic acids are the most challenging target for chemical shift prediction. Here we show that the RNA base pair triplet motif dictates imino chemical shifts in its central base pair. A lookup table is established that links each type of base pair triplet to experimental chemical shifts of the central base pair, and can be used to predict imino chemical shifts of RNAs to remarkable accuracy. Strikingly, the semiempirical method can well interpret the variations of chemical shifts for different base pair triplets, and is even applicable to non-canonical motifs. This finding opens an avenue for predicting chemical shifts of more complicated RNA motifs. Furthermore, we combine the imino chemical shift prediction with NMR relaxation dispersion experiments targeting both 15N and 1HN of the imino group, and verify a previously characterized excited state of P5abc subdomain including an earlier speculated non-native G•G mismatch.

scholarly journals Rapid measurement of Watson-Crick to Hoogsteen exchange in unlabeled DNA duplexes using high-power SELOPE imino <sup>1</sup>H CEST

2021 ◽  
Author(s):  
Bei Liu ◽  
Atul Rangadurai ◽  
Honglue Shi ◽  
Hashim Al-Hashimi
Keyword(s):  
High Power ◽  
Dna Sequences ◽  
Dynamic Equilibrium ◽  
Chemical Exchange ◽  
Nmr Relaxation ◽  
Relaxation Dispersion ◽  
Duplex Dna ◽  
Dna Duplexes ◽  
Base Pairs ◽  
1H Cest

Abstract. In duplex DNA, Watson-Crick A-T and G-C base pairs (bps) exist in dynamic equilibrium with an alternative Hoogsteen conformation, which is low in abundance and short-lived. Measuring how the Hoogsteen dynamics varies across different DNA sequences, structural contexts and physiological conditions is key for understanding the role of these non-canonical bps in DNA recognition and repair. However, such studies are hampered by the need to prepare 13C or 15N isotopically enriched DNA samples for NMR relaxation dispersion (RD) experiments. Here, using SELective Optimized Proton Experiments (SELOPE) 1H CEST experiments employing high-power radiofrequency fields (B1 > 250 Hz) targeting imino protons, we demonstrate accurate and robust characterization of Waston-Crick to Hoogsteen exchange, without the need for isotopic enrichment of the DNA. For 13 residues in three DNA duplexes under different temperature and pH conditions, the exchange parameters deduced from high-power imino 1H CEST were in very good agreement with counterparts measured using off-resonance 13C/15N spin relaxation in the rotating frame (R1ρ). It is shown that 1H-1H NOE effects which typically introduce artifacts in 1H based measurements of chemical exchange can be effectively suppressed by selective excitation, provided that the relaxation delay is short (≤ 100 ms). The 1H CEST experiment can be performed with ~10X higher throughput and ~100X lower cost relative to 13C/15N R1ρ, and enabled Hoogsteen chemical exchange measurements undetectable by R1ρ. The results reveal an increased propensity to form Hoogsteen bps near terminal ends and a diminished propensity within A-tract motifs. The 1H CEST experiment opens the door to more comprehensively characterizing Hoogsteen breathing in duplex DNA.

Identification of a Collapsed Intermediate with Non-native Long-range Interactions on the Folding Pathway of a Pair of Fyn SH3 Domain Mutants by NMR Relaxation Dispersion Spectroscopy

2006 ◽  
Vol 363 (5) ◽  
pp. 958-976 ◽  
Author(s):  
Philipp Neudecker ◽  
Arash Zarrine-Afsar ◽  
Wing-Yiu Choy ◽  
D. Ranjith Muhandiram ◽  
Alan R. Davidson ◽  
...  
Keyword(s):  
Long Range ◽  
Nmr Relaxation ◽  
Sh3 Domain ◽  
Relaxation Dispersion ◽  
Folding Pathway ◽  
Long Range Interactions

Examining the structure of the mature amyloid fibril

2002 ◽  
Vol 30 (4) ◽  
pp. 521-525 ◽  
Author(s):  
O. S. Makin ◽  
L. C. Serpell
Keyword(s):  
Self Assembly ◽  
Rational Design ◽  
Amyloid Fibrils ◽  
Structural Information ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Fibre Diffraction ◽  
Complementary Techniques ◽  
Mature Fibril

The pathogenesis of the group of diseases known collectively as the amyloidoses is characterized by the deposition of insoluble amyloid fibrils. These are straight, unbranching structures about 70–120 å (1 å = 0.1 nm) in diameter and of indeterminate length formed by the self-assembly of a diverse group of normally soluble proteins. Knowledge of the structure of these fibrils is necessary for the understanding of their abnormal assembly and deposition, possibly leading to the rational design of therapeutic agents for their prevention or disaggregation. Structural elucidation is impeded by fibril insolubility and inability to crystallize, thus preventing the use of X-ray crystallography and solution NMR. CD, Fourier-transform infrared spectroscopy and light scattering have been used in the study of the mechanism of fibril formation. This review concentrates on the structural information about the final, mature fibril and in particular the complementary techniques of cryo-electron microscopy, solid-state NMR and X-ray fibre diffraction.

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