Assessment of protein alignment using 1H–1H residual dipolar coupling measurements

Wang, Junfeng; Valafar, Homayoun; Prestegard, James H.

doi:10.1016/j.jmr.2004.03.012

Cited by 5 publications

(4 citation statements)

References 23 publications

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“…There has been considerable interest in the development of membrane mimetics and other media that can spontaneously align in the presence of an external magnetic field. − Studies have reported the magnetic alignment of bicelles and macro–nanodiscs with the bilayer normal perpendicular to the magnetic field axis. ,,, The alignment direction of nanodiscs can be changed by varying the magnetic susceptibility with the addition of paramagnetic metal ions (such as lanthanide ions including Yb 3+ ) as reported previously. ,− The addition of paramagnetic lanthanide ions has been shown to 90°-flip bicelles or macro–nanodiscs to result in the parallel orientation of the lipid bilayer normal with the magnetic field axis. − Although magnetically aligned media are commonly used to measure RDCs, this study demonstrates the feasibility of measuring RDCs using two different media that have orthogonal alignment directions. We used a negatively charged SMA-EA polymer nanodisc as an alignment medium to measure 1 H– 15 N RDCs from the ∼21 kDa flavin mononucleotide binding domain (FBD) of cytochrome-P450 reductase (CPR).…”

Section: Introductionmentioning

confidence: 83%

Measurement of Residual Dipolar Couplings Using Magnetically Aligned and Flipped Nanodiscs

Ravula

Ramamoorthy

2021

Langmuir

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Recent developments in lipid nanodisc technology have successfully overcome the major challenges in the structural and functional studies of membrane proteins and drug delivery. Among the different types of nanodiscs, the use of synthetic amphiphilic polymers created new directions including the applications of solution and solid-state NMR spectroscopy. The ability to magnetically align large-size (>20 nm diameter) polymer nanodiscs and flip them using paramagnetic lanthanide ions has enabled high-resolution studies on membrane proteins using solid-state NMR techniques. The use of polymer-based macro–nanodiscs (>20 nm diameter) as an alignment medium to measure residual dipolar couplings (RDCs) and residual quadrupole couplings by NMR experiments has also been demonstrated. In this study, we demonstrate the use of magnetically aligned and 90°-flipped polymer nanodiscs as alignment media for structural studies on proteins by solution NMR spectroscopy. These macro–nanodiscs, composed of negatively charged SMA-EA polymers and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids, were used to measure residual 1H–15N dipolar couplings (RDCs) from the water-soluble ∼21 kDa uniformly 15N-labeled flavin mononucleotide binding domain (FBD) of cytochrome-P450 reductase. The experimentally measured 1H–15N RDC values are compared with the values calculated from the crystal structures of cytochrome-P450 reductase that lacks the transmembrane domain. The N–H RDCs measured using aligned and 90°-flipped nanodiscs show a modulation by the function (3 cos2 θ – 1), where θ is the angle between the N–H bond vector and the applied magnetic field direction. This successful demonstration of the use of two orthogonally oriented alignment media should enable structural studies on a variety of systems including small molecules, DNA, and RNA.

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Section: Introductionmentioning

confidence: 83%

Measurement of Residual Dipolar Couplings Using Magnetically Aligned and Flipped Nanodiscs

Ravula

Ramamoorthy

2021

Langmuir

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“…A fast method for assessing directly the degree of protein alignment from proton-proton residual dipolar couplings has been proposed and demonstrated for the case of unlabeled Cp-rubredoxin for which RDCs between aromatic protons were measured from proton homonuclear J-resolved experiments and the alignment assessed without the use of extensive resonance assignment. 178 It has been demonstrated that exclusive 13 C-only spectroscopy can be routinely used to measure an extensive set of the side-chain residual 13 C- 13 C dipolar couplings in uniformly deuterated and 13 C-labeled proteins. 179 An excellent correlation between measured RDCs and the 3D structure of the protein, human ubiquitin in the presence of bacteriophage Pf1 was observed indicating a potential use of the 13 C- 13 C RDCs in the structure determination of perdeuterated proteins.…”

Section: New Experimentalmentioning

confidence: 99%

Oriented molecules

Khetrapal¹,

Ramanathan²

1998

Nuclear Magnetic Resonance

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“…Elimination of the S zz term is the reason for the appearance of the z 2 term in the first two columns of the A matrix in Equation 4. Other modifications of the system of equations shown in Equation 4, with their corresponding adaptations of SVD, have also been introduced in order to accommodate conformational rotation of side chain methyl and phenyl groups [62,63]. …”

Section: Introductionmentioning

confidence: 99%

Advances in the REDCAT software package

2013

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BackgroundResidual Dipolar Couplings (RDCs) have emerged in the past two decades as an informative source of experimental restraints for the study of structure and dynamics of biological macromolecules and complexes. The REDCAT software package was previously introduced for the analysis of molecular structures using RDC data. Here we report additional features that have been included in this software package in order to expand the scope of its analyses. We first discuss the features that enhance REDCATs user-friendly nature, such as the integration of a number of analyses into one single operation and enabling convenient examination of a structural ensemble in order to identify the most suitable structure. We then describe the new features which expand the scope of RDC analyses, performing exercises that utilize both synthetic and experimental data to illustrate and evaluate different features with regard to structure refinement and structure validation.ResultsWe establish the seamless interaction that takes place between REDCAT, VMD, and Xplor-NIH in demonstrations that utilize our newly developed REDCAT-VMD and XplorGUI interfaces. These modules enable visualization of RDC analysis results on the molecular structure displayed in VMD and refinement of structures with Xplor-NIH, respectively. We also highlight REDCAT’s Error-Analysis feature in reporting the localized fitness of a structure to RDC data, which provides a more effective means of recognizing local structural anomalies. This allows for structurally sound regions of a molecule to be identified, and for any refinement efforts to be focused solely on locally distorted regions.ConclusionsThe newly engineered REDCAT software package, which is available for download via the WWW from http://ifestos.cse.sc.edu, has been developed in the Object Oriented C++ environment. Our most recent enhancements to REDCAT serve to provide a more complete RDC analysis suite, while also accommodating a more user-friendly experience, and will be of great interest to the community of researchers and developers since it hides the complications of software development.

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Assessment of protein alignment using 1H–1H residual dipolar coupling measurements

Cited by 5 publications

References 23 publications

Measurement of Residual Dipolar Couplings Using Magnetically Aligned and Flipped Nanodiscs

Measurement of Residual Dipolar Couplings Using Magnetically Aligned and Flipped Nanodiscs

Oriented molecules

Advances in the REDCAT software package

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