Structural Characterization of Proteins with an Attached ATCUN Motif by Paramagnetic Relaxation Enhancement NMR Spectroscopy

Donaldson, Logan W.; Skrynnikov, Nikolai R.; Choy, Wing‐Yiu; Muhandiram, D. R.; Sarkar, Bibudhendra; Forman-Kay, Julie D.; Kay, Lewis E.

doi:10.1021/ja011241p

Cited by 167 publications

(194 citation statements)

References 40 publications

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“…In unfolded proteins, R red 2 rates show considerable variability along the peptide chain; accurate measurement of R red 2 requires a full-scale relaxation experiment. 103 As an alternative to Eq. (19), we propose a simpler expression:…”

Section: Resultsmentioning

confidence: 99%

“…Initially, we measured the PRE rates using the sequence by Donaldson et al, 103 as well as the variant of this sequence published by Iwahara et al 104 Both experiments are 1 H N spin-echo relaxation experiments that record the magnetization decay profiles. The difference is that in the former case the effect of 3 J HN,Ha coupling is eliminated by means of the 1 H N -selective pulse, whereas in the latter case it is eliminated via point-by-point division of the two decay curves (from paramagnetic and diamagnetic samples).…”

Section: Resultsmentioning

confidence: 99%

“…The HSQC sequence used to quantify V ox and V red was fashioned from the experiment of Kay and coworkers. 103 to eliminate the effect of 3 J HN,Ha coupling during the INEPT periods. The spectra were acquired using a long recycling delay, 5 s; shorter recovery delays lead to an unsatisfactory situation when certain signals recover fully in paramagnetic samples, but only partially in the reduced sample.…”

Section: Resultsmentioning

confidence: 99%

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Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

et al. 2009

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Site-directed spin labeling in combination with paramagnetic relaxation enhancement (PRE) measurements is one of the most promising techniques for studying unfolded proteins. Since the pioneering work of Gillespie and Shortle (J Mol Biol 1997;268:158), PRE data from unfolded proteins have been interpreted using the theory that was originally developed for rotational spin relaxation. At the same time, it can be readily recognized that the relative motion of the paramagnetic tag attached to the peptide chain and the reporter spin such as 1 H N is best described as a translation. With this notion in mind, we developed a number of models for the PRE effect in unfolded proteins: (i) mutual diffusion of the two tethered spheres, (ii) mutual diffusion of the two tethered spheres subject to a harmonic potential, (iii) mutual diffusion of the two tethered spheres subject to a simulated mean-force potential (Smoluchowski equation); (iv) explicit-atom molecular dynamics simulation. The new models were used to predict the dependences of the PRE rates on the 1 H N residue number and static magnetic field strength; the results are appreciably different from the Gillespie-Shortle model. At the same time, the Gillespie-Shortle approach is expected to be generally adequate if the goal is to reconstruct the distance distributions between 1 H N spins and the paramagnetic center (provided that the characteristic correlation time is known with a reasonable accuracy). The theory has been tested by measuring the PRE rates in three spin-labeled mutants of the drkN SH3 domain in 2M guanidinium chloride. Two modifications introduced into the measurement scheme-using a reference compound to calibrate the signals from the two samples (oxidized and reduced) and using peak volumes instead of intensities to determine the PRE rates-lead to a substantial improvement in the quality of data. The PRE data from the denatured drkN SH3 are mostly consistent with the model of moderately expanded random-coil protein, although part of the data point toward a more compact structure (local hydrophobic cluster). At the same time, the radius of gyration reported by Choy et al. (J Mol Biol 2002;316:101) suggests that the protein is highly expanded. This seemingly contradictory evidence can be reconciled if one assumes that denatured drkN SH3 forms a conformational ensemble that is dominated by extended conformations, yet also contains compact (collapsed) species. Such behavior is apparently more complex than predicted by the model of a random-coil protein in good solvent/poor solvent.Additional Supporting Information may be found in the online version of this article.Abbreviations: drkN SH3, N-terminal SH3 domain of the Drosophila adapter protein drk; EDTA, ethylenediaminetetraacetic acid; ESR, electron spin resonance; FRET, fluorescence resonance energy transfer; MTSL, methanethiosulfonate spin label; NOE, nuclear Overhauser effect; NAG, N-acetyl-glycine.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

et al. 2009

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“…Fig. 1 shows the pulse sequence we have been using for 1 H-Γ 2 measurements (19,23,24,25,27), which is essentially the same as that proposed by Kay and co-workers (8 A two-time-point measurement provides a simple means of obtaining Γ 2 rates and their corresponding errors without making use of any fitting procedures. In this approach, 1 H-Γ 2 rates are determined from two time points (T = 0 and ΔT) for transverse relaxation using Eqs.…”

Section: Experiments For 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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“…9-11, is the normalized form of the potential applied in ref. 56. The normalization used here ensures that restraints with identical relative uncertainty are weighted equally in the structure calculation.…”

Section: Methodsmentioning

confidence: 99%

Determination of the geometric structure of the metal site in a blue copper protein by paramagnetic NMR

Hansen

Led

2006

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

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The biological function of metalloproteins is closely tied to the geometric and electronic structures of the metal sites. Here, we show that the geometric structure of the metal site of a metalloprotein in solution can be determined from experimentally measured electron-nuclear spin-spin interactions obtained by NMR. Thus, the geometric metal site structure of plastocyanin from Anabaena variabilis was determined by including the paramagnetic relaxation enhancement of protons close to the copper site as restraints in a conventional NMR structure determination, together with the distribution of the unpaired electron onto the ligand atoms. Also, the interproton distances (nuclear Overhauser enhancements) and dihedral angles (scalar nuclear spin-spin couplings) normally used in NMR structure determinations were included as restraints. The structure calculations were carried out with the program X-PLOR and a module that takes into account the specific characteristics of the paramagnetic restraints. A well defined metal site structure was obtained with the structural characteristics of the blue copper site, including a distorted tetrahedral geometry, a short Cu-Cys S ␥ bond, and a long Cu-Met S ␦ bond. Overall, the agreement of the obtained metal site structure of Anabaena variabilis plastocyanin with those of other plastocyanins obtained by x-ray crystallography confirms the reliability of the approach.metal site structure ͉ metalloproteins ͉ paramagnetic nuclear relaxation T he biological function of many metalloproteins stems from the geometric and electronic structures of the metal sites imposed by the protein environment. In the blue copper proteins, such as plastocyanins, amicyanins, and azurins, the geometry of the copper site is unusual as compared with smallmolecule copper complexes (1-15). In particular, the metal sites of blue copper proteins are characterized by a short coppersulfur bond. This unusual geometry is believed to be the main reason for the strong covalency of the metal site (10,16,17) and, thus, responsible for the rapid and long-range electron transfer reactivity (18-22) that characterizes the blue copper proteins. Detailed knowledge of the geometric and electronic metal site structures of the blue copper proteins is, therefore, imperative for understanding the function of the proteins at the molecular level.So far, the geometric structure of the metal site in blue copper proteins (12-14) has been determined primarily by x-ray crystallography and extended x-ray absorption fine structure (3,23,24), whereas the electronic structure of the blue copper site has been determined theoretically from quantum chemical calculations (5, 25-27) and experimentally by x-ray absorption spectroscopy (16, 17), and more recently by nuclear paramagnetic relaxation (28-31). These structures have formed the basis for a detailed understanding of the biological function of the proteins by elucidating the interplay between the electronic and geometric structure of the metal site (8,15,(32)(33)(34). However, the geomet...

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Structural Characterization of Proteins with an Attached ATCUN Motif by Paramagnetic Relaxation Enhancement NMR Spectroscopy

Cited by 167 publications

References 40 publications

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

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

Determination of the geometric structure of the metal site in a blue copper protein by paramagnetic NMR

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