Characterization of long-range structure in the denatured state of staphylococcal nuclease. I. paramagnetic relaxation enhancement by nitroxide spin labels

Gillespie, Joel R.; Shortle, David

doi:10.1006/jmbi.1997.0954

Cited by 305 publications

(321 citation statements)

References 29 publications

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“…Rather than directly measuring relaxation rates from a series of twodimensional experiments, which is tedious and time-consuming, relaxation rates were indirectly extracted from ratios of cross-peak heights between oxidized and reduced spectra (I ox / I red ) according to equations given under Materials and Methods. This procedure is nearly identical to the heteronuclear work done on unfolded and 15 N-labeled staphyloccocal nuclease, which showed that the intensity ratio procedure gave similar results to directly measured relaxation rates (16). For this work, two-dimensional peak fitting with the program nlimNS was used to expedite analysis, rather than fitting of one-dimensional slices.…”

Section: Resultsmentioning

confidence: 89%

Utilization of Site-Directed Spin Labeling and High-Resolution Heteronuclear Nuclear Magnetic Resonance for Global Fold Determination of Large Proteins with Limited Nuclear Overhauser Effect Data

2000

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To test whether distances derived from paramagnetic broadening of 15 N heteronuclear single quantum coherence (HSQC) resonances could be used to determine the global fold of a large, perdeuterated protein, we used site-directed spin-labeling of 5 amino acids on the surface of 15 N-labeled eukaryotic translation initiation factor 4E (eIF4E). eIF4E is a 25 kDa translation initiation protein, whose solution structure was previously solved in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) micelle of total molecular mass ∼45-50 kDa. Distance-dependent line broadening consistent with the three-dimensional structure of eIF4E was observed for all spin-label substitutions. The paramagnetic broadening effects (PBEs) were converted into distances for modeling by a simple method comparing peak heights in 15 N-HSQC spectra before and after reduction of the nitroxide spin label with ascorbic acid. The PBEs, in combination with HN-HN nuclear Overhauser effects (NOEs) and chemical shift index (CSI) angle restraints, correctly determined the global fold of eIF4E with a backbone precision of 2.3 Å (1.7 Å for secondary structure elements). The global fold was not correctly determined with the HN-HN NOEs and CSI angles alone. The combination of PBEs with simulated restraints from another nuclear magnetic resonance (NMR) method for global fold determination of large proteins (methylprotonated, highly deuterated samples) improved the quality of calculated structures. In addition, the combination of the two methods simulated from a crystal structure of an all R-helical protein (40 kDa farnesyl diphoshphate synthase) correctly determined the global fold where neither method individually was successful. These results show the potential feasibility of obtaining medium-resolution structures for proteins in the 40-100 kDa range via NMR.There has been an increase in research aimed at calculating global folds of proteins with the limited NOE 1 data obtained from deuterated samples (1). The motivation is the molecular weight size gap between what can be sequentially assigned and for what a sufficient number of NOEs can be determined for structure calculations. Backbone resonances in macromolecular systems as large as 65 kDa have been assigned with deuterated samples (2) and recent advances with transverse relaxation-optimized spectroscopy (TROSY) experiments promise to extend the theoretical limit for backbone assignment to beyond 100 kDa (3, 4). However, the high levels of deuteration required to increase the relaxation times of the aliphatic 13 C-carbon resonances remove most of the side-chain protons for NOE analysis. The most simple methodology for global fold determination is to use the remaining NOEs between nitrogen-attached protons (HN) that can be obtained from 3D or 4D 15 N-NOESY-HSQC experiments of a deuterated protein (5). This limited subset of NOEs results in very poor quality structures that in many cases cannot correctly determine the global fold of the protein. Use of HN NOEs is particul...

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Section: Resultsmentioning

confidence: 89%

Utilization of Site-Directed Spin Labeling and High-Resolution Heteronuclear Nuclear Magnetic Resonance for Global Fold Determination of Large Proteins with Limited Nuclear Overhauser Effect Data

2000

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“…In this approach, 1 H-Γ 2 rates are determined from two time points (T = 0 and ΔT) for transverse relaxation using Eqs. 1 and 2 as follows: [5] where I dia and I para are the peak intensities for the diamagnetic and paramagnetic states, respectively. It should be noted that effects of homonuclear 3 J HNHα -modulation during T are cancelled out by using identical times and taking ratios for the two states.…”

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

244

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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“…[40][41][42] PRE measurements can readily detect interactions between residues up to $25 Å from the spin label. We measured PRE in aS at pH 3.0 using a number of single cysteine mutants (S9C, G31C, E61C, A85C, E110C, and E130C) conjugated to MTSL (see Fig.…”

Section: Paramagnetic Relaxation Enhancementmentioning

confidence: 99%

Charge neutralization and collapse of the C‐terminal tail of alpha‐synuclein at low pH

2009

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Alpha-synuclein (aS) is the primary component of Lewy bodies, the pathological hallmark of Parkinson's Disease. Aggregation of aS is thought to proceed from a primarily disordered state with nascent secondary structure through intermediate conformations to oligomeric forms and finally to mature amyloid fibrils. Low pH conditions lead to conformational changes associated with increased aS fibril formation. Here we characterize these structural and dynamic changes using solution state NMR measurements of secondary chemical shifts, relaxation parameters, residual dipolar couplings, and paramagnetic relaxation enhancement. We find that the neutralization of negatively charged side-chains eliminates electrostatic repulsion in the C-terminal tail of aS and leads to a collapse of this region at low pH. Hydrophobic contacts between the compact C-terminal tail and the NAC (non-amyloid-b component) region are maintained and may lead to the formation of a globular domain. Transient long-range contacts between the C-terminus of the protein and regions N-terminal to the NAC region are also preserved. Thus, the release of long-range contacts does not play a role in the increased aggregation of aS at low pH, which we instead attribute to the increased hydrophobicity of the protein.

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Characterization of long-range structure in the denatured state of staphylococcal nuclease. I. paramagnetic relaxation enhancement by nitroxide spin labels

Cited by 305 publications

References 29 publications

Utilization of Site-Directed Spin Labeling and High-Resolution Heteronuclear Nuclear Magnetic Resonance for Global Fold Determination of Large Proteins with Limited Nuclear Overhauser Effect Data

Utilization of Site-Directed Spin Labeling and High-Resolution Heteronuclear Nuclear Magnetic Resonance for Global Fold Determination of Large Proteins with Limited Nuclear Overhauser Effect Data

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

Charge neutralization and collapse of the C‐terminal tail of alpha‐synuclein at low pH

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