Derivation of structural restraints using a thiol‐reactive chelator

Dvoretsky, Alex; Gaponenko, Vadim; Rosevear, Paul R.

doi:10.1016/s0014-5793(02)03297-0

Cited by 71 publications

(63 citation statements)

References 28 publications

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“…However, in many RNA systems, paramagnetic metals lead to alternative, often inactive or incorrectly folded, structures. Thus, a more general approach will be to develop paramagnetic tags [66] that can be connected to an RNA, thereby leading to a very different alignment tensor for the molecule. As ultrahigh magnetic fields and easily incorporated nonperturbing paramagnetic tags become more available, these techniques will become valuable tools for the alignment of RNAs.…”

Section: Methods For Aligning Rna For Measurement Of Rdcsmentioning

confidence: 99%

NMR Methods for Studying the Structure and Dynamics of RNA

et al. 2005

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Proper functioning of RNAs requires the formation of complex three-dimensional structures combined with the ability to rapidly interconvert between multiple functional states. This review covers recent advances in isotope-labeling strategies and NMR experimental approaches that have promise for facilitating solution structure determinations and dynamics studies of biologically active RNAs. Improved methods for the production of isotopically labeled RNAs combined with new multidimensional heteronuclear NMR experiments make it possible to dramatically reduce spectral crowding and simplify resonance assignments for RNAs. Several novel applications of experiments that directly detect hydrogen-bonding interactions are discussed. These studies demonstrate how NMR spectroscopy can be used to distinguish between possible secondary structures and identify mechanisms of ligand binding in RNAs. A variety of recently developed methods for measuring base and sugar residual dipolar couplings are described. NMR residual dipolar coupling techniques provide valuable data for determining the long-range structure and orientation of helical regions in RNAs. A number of studies are also presented where residual dipolar coupling constraints are used to determine the global structure and dynamics of RNAs. NMR relaxation data can be used to probe the dynamics of macromolecules in solution. The power dependence of transverse rotating-frame relaxation rates was used here to study dynamics in the minimal hammerhead ribozyme. Improved methods for isotopically labeling RNAs combined with new types of structural data obtained from a growing repertoire of NMR experiments are facilitating structural and dynamic studies of larger RNAs.

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Section: Methods For Aligning Rna For Measurement Of Rdcsmentioning

confidence: 99%

NMR Methods for Studying the Structure and Dynamics of RNA

et al. 2005

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“…In the case of EDTA-conjugation to proteins, disulfide bond linkage to a Cys side-chain is commonly used (9,(41)(42)(43). As the reaction is reversible, the conversion rate cannot be 100% and there is always a small percentage of unconverted diamagnetic species.…”

Section: Considerations Of Sample Stability For 1 H-γ 2 Measurementsmentioning

confidence: 99%

Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules

Iwahara

Tang

Clore

2007

Journal of Magnetic Resonance

245

385

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The use of 1 H transverse paramagnetic relaxation enhancement (PRE) has seen a resurgence in recent years as method for providing long-range distance information for structural studies and as a probe of large amplitude motions and lowly populated transient intermediates in macromolecular association. In this paper we discuss various practical aspects pertaining to accurate measurement of PRE 1 H transverse relaxation rates (Γ 2 ). We first show that accurate Γ 2 rates can be obtained from a two time-point measurement without requiring any fitting procedures or complicated error estimations, and no additional accuracy is achieved from multiple time-point measurements recorded in the same experiment time. Optimal setting of the two time-points that minimize experimental errors is also discussed. Next we show that the simplistic single time-point measurement that has been commonly used in the literature, can substantially underestimate the true value of Γ 2 , unless a relatively long repetition delay is employed. We then examine the field dependence of Γ 2 , and show that Γ 2 exhibits only a very weak field dependence at high magnetic fields typically employed in macromolecular studies. The theoretical basis for this observation is discussed. Finally, we investigate the impact of contamination of the paramagnetic sample by trace amounts (≤5%) of the corresponding diamagnetic species on the accuracy of Γ 2 measurements. Errors in Γ 2 introduced by such diamagnetic contamination are potentially sizeable, but can be significantly reduced by using a relatively short time interval for the two time-point Γ 2 measurement.

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“…For these situations, we used an alternative method for introducing metal quenchers: using small cysteinereactive metal binding groups to coordinate the metals (23). Synthetic metal binding molecules such as nitrilotriacetic acid (NTA), EDTA, and iminodiacetic acid (IDA), can bind transition metal ions such as Ni 2ϩ , Cu 2ϩ , and Co 2ϩ , with affinities in the picomolar range, higher than the affinity of most native sites in proteins.…”

Section: Labeling Peptides With Cysteine-reactive Metal-binding Molecmentioning

confidence: 99%

Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

Taraska

Puljung

Zagotta

2009

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

119

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The structure and dynamics of proteins underlies the workings of virtually every biological process. Existing biophysical methods are inadequate to measure protein structure at atomic resolution, on a rapid time scale, with limited amounts of protein, and in the context of a cell or membrane. FRET can measure distances between two probes, but depends on the orientation of the probes and typically works only over long distances comparable with the size of many proteins. Also, common probes used for FRET can be large and have long, flexible attachment linkers that position dyes far from the protein backbone. Here, we improve and extend a fluorescence method called transition metal ion FRET that uses energy transfer to transition metal ions as a reporter of short-range distances in proteins with little orientation dependence. This method uses a very small cysteine-reactive dye monobromobimane, with virtually no linker, and various transition metal ions bound close to the peptide backbone as the acceptor. We show that, unlike larger fluorophores and longer linkers, this donor-acceptor pair accurately reports shortrange distances and changes in backbone distances. We further extend the method by using cysteine-reactive metal chelators, which allow the technique to be used in protein regions of unknown secondary structure or when native metal ion binding sites are present. This improved method overcomes several of the key limitations of classical FRET for intramolecular distance measurements.fluorescence ͉ peptides ͉ FRET F luorescence resonance energy transfer (FRET) occurs when excitation energy is transferred from a donor fluorophore to an acceptor dye through weak dipole-dipole resonance interactions (1, 2). FRET is extremely sensitive to the distance between the two dyes, with the transfer rate inversely proportional to the sixth power of the distance between them. This steep distance dependence has suggested that FRET could be used as a spectroscopic ruler on the molecular scale (3, 4). In addition to distance, five other factors influence the rate of energy transfer: (i) the overlap of the donor's and acceptor's excitation and absorbance spectra, respectively; (ii) the refractive index of the solvent; (iii) the relative orientation of the dyes; (iv) the quantum yield of the donor; and (v) the extinction coefficient of the acceptor.Although FRET is sensitive to the distance between probes, several factors have prevented the widespread use of FRET to easily map backbone distances within proteins. First, most commonly used FRET pairs have R 0 values (distance at which FRET efficiency is 50%) between Ϸ30 and 60 Å and are only able to measure distances between Ϸ20 and 80 Å. Thus, to accurately measure distances, the probes must be separated by large distances relative to the size of many proteins. Also, the large size of many dyes and their long attachment linkers position fluorophores far from the backbone of the protein. These flexible linkers also increase the conformational space sampled by the dye (4, 5). Becau...

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Derivation of structural restraints using a thiol‐reactive chelator

Cited by 71 publications

References 28 publications

NMR Methods for Studying the Structure and Dynamics of RNA

NMR Methods for Studying the Structure and Dynamics of RNA

Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules

Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

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