19F-Modified Proteins and 19F-Containing Ligands as Tools in Solution NMR Studies of Protein Interactions

Sharaf, Naima G.; Gronenborn, Angela M.

doi:10.1016/bs.mie.2015.05.014

Cited by 65 publications

(68 citation statements)

References 62 publications

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“…33,35 Briefly, competent E. coli BL21 ai cells (Invitrogen) were co-transformed with the vector encoding the TAG containing constructs (above), and the pDule2 RS vector encoding the orthogonal amber tRNA/tRNA synthetase pair. Several transformants were screened for expression.…”

Section: Methodsmentioning

confidence: 99%

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Conformational Plasticity of the NNRTI-Binding Pocket in HIV-1 Reverse Transcriptase: A Fluorine Nuclear Magnetic Resonance Study

2016

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HIV-1 reverse transcriptase (RT) is a major drug target in the treatment of HIV-1 infection. RT inhibitors currently in use include non-nucleoside, allosteric RT inhibitors (NNRTIs), which bind to a hydrophobic pocket, distinct from enzyme's active site. We investigated RT-NNRTI interactions by solution 19F NMR, using singly 19F labeled RT proteins. Comparison of 19F chemical shifts of fluorinated RT and drug-resistant variants revealed that the fluorine resonance is a sensitive probe for identifying mutation-induced changes in the enzyme. Our data show that in the unliganded enzyme, the NNRTI-binding pocket is highly plastic and not locked into a single conformation. Upon inhibitor binding, the binding pocket rigidifies. In the inhibitor-bound state, the 19F signal of RT is similar to that of drug-resistant mutant enzymes, distinct from what is observe for the free state. Our results demonstrate the power of 19F NMR spectroscopy to characterize conformational properties using selectively 19F labeled protein.

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Section: Methodsmentioning

confidence: 99%

“…In addition the 19 F shielding is dominated by a large paramagnetic term, which makes it exquisitely sensitive to its local environment (the 19 F chemical shift range is ~100 fold larger than that of 1 H). 31–33 …”

Section: Introductionmentioning

confidence: 99%

Conformational Plasticity of the NNRTI-Binding Pocket in HIV-1 Reverse Transcriptase: A Fluorine Nuclear Magnetic Resonance Study

2016

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“…It can therefore serve as a highly sensitive probe of protein and peptide structure, folding and aggregation, dynamics and ligand binding (reviewed in (Chen et al . 2013; Sharaf & Gronenborn, 2015). Specific 19 F labels can be installed by peptide synthesis, conjugation or fluorination of cysteine and lysine residues, or by supplementing the culture media with fluorinated amino acids (which results in amino acid specific labeling).…”

Section: Protein Engineering Approaches For Tackling Outstanding Cmentioning

confidence: 99%

“…Specific 19 F labels can be installed by peptide synthesis, conjugation or fluorination of cysteine and lysine residues, or by supplementing the culture media with fluorinated amino acids (which results in amino acid specific labeling). Increasingly, amber suppression is becoming the site-specific incorporation method of choice ((Marsh & Suzuki, 2014; Sharaf & Gronenborn, 2015). There are several orthogonal tRNA/synthetase systems and fluorinated UAAs available, each imparting different spectroscopic signatures with respect to chemical shift anisotropy and relaxation.…”

Section: Protein Engineering Approaches For Tackling Outstanding Cmentioning

confidence: 99%

A molecular engineering toolbox for the structural biologist

Debelouchina

Muir

2017

Quart. Rev. Biophys.

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Exciting new technological developments have pushed the boundaries of structural biology, and have enabled studies of biological macromolecules and assemblies that would have been unthinkable not long ago. Yet, the enhanced capabilities of structural biologists to pry into the complex molecular world have also placed new demands on the abilities of protein engineers to reproduce this complexity into the test tube. With this challenge in mind, we review the contents of the modern molecular engineering toolbox that allow the manipulation of proteins in a site-specific and chemically well-defined fashion. Thus, we cover concepts related to the modification of cysteines and other natural amino acids, native chemical ligation, intein and sortase-based approaches, amber suppression, as well as chemical and enzymatic bio-conjugation strategies. We also describe how these tools can be used to aid methodology development in X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy and in the studies of dynamic interactions. It is our hope that this monograph will inspire structural biologists and protein engineers alike to apply these tools to novel systems, and to enhance and broaden their scope to meet the outstanding challenges in understanding the molecular basis of cellular processes and disease.

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“…NMR spectroscopy and cryo-EM appear to be particularly complementary because they both do not require crystallization [20]. 19 F-labeling of proteins appears poised for a renaissance [109]. Solid state NMR with ultra-fast magic angle spinning [94] and signal enhancement by DNP [106] are expected to continue their rapid development.…”

Section: Our Crystal Ball To the Futurementioning

confidence: 99%

Biomolecular NMR: Past and future

Markley

Westler

2017

Archives of Biochemistry and Biophysics

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The editors of this special volume suggested this topic, presumably because of the perspective lent by our combined >90-year association with biomolecular NMR. What follows is our personal experience with the evolution of the field, which we hope will illustrate the trajectory of change over the years. As for the future, one can confidently predict that it will involve unexpected advances. Our narrative is colored by our experience in using the NMR Facility for Biomedical Studies at Carnegie-Mellon University (Pittsburgh) and in developing similar facilities at Purdue (1977–1984) and the University of Wisconsin-Madison (1984-). We have enjoyed developing NMR technology and making it available to collaborators and users of these facilities. Our group’s association with the Biological Magnetic Resonance data Bank (BMRB) and with the Worldwide Protein Data Bank (wwPDB) has also been rewarding. Of course, many groups contributed to the early growth and development of biomolecular NfMR, and our brief personal account certainly omits many important milestones.

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19F-Modified Proteins and 19F-Containing Ligands as Tools in Solution NMR Studies of Protein Interactions

Cited by 65 publications

References 62 publications

Conformational Plasticity of the NNRTI-Binding Pocket in HIV-1 Reverse Transcriptase: A Fluorine Nuclear Magnetic Resonance Study

Conformational Plasticity of the NNRTI-Binding Pocket in HIV-1 Reverse Transcriptase: A Fluorine Nuclear Magnetic Resonance Study

A molecular engineering toolbox for the structural biologist

Biomolecular NMR: Past and future

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