Cyano Groups as Probes of Protein Microenvironments and Dynamics

Zimmermann, Jörg; Thielges, Megan C.; Seo, Young Jun; Dawson, Philip E.; Romesberg, Floyd E.

doi:10.1002/ange.201101016

Cited by 12 publications

(3 citation statements)

References 49 publications

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“…-N 3 [27,[49][50][51], or -C≡N [28,52] groups, many of which have been summarized in recent reviews [53,54]. Of course, with such labels one gives up one of the beauties of IR spectroscopy, i.e., one has to chemically modify the protein, but many of these IR labels are small perturbations in comparison to for example fluorescence labels.…”

Section: Advancing the Selectivitymentioning

confidence: 99%

Fast infrared spectroscopy of protein dynamics: advancing sensitivity and selectivity

Koziol

Johnson

Stucki-Buchli

et al. 2015

Current Opinion in Structural Biology

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2D-IR spectroscopy has matured to a powerful technique to study the structure and dynamics of peptides, but its extension to larger proteins is still in its infancy, the major limitations being sensitivity and selectivity. Site-selective information requires measuring single vibrational probes at sub-millimolar concentrations where most proteins are still stable, which is a severe challenge for conventional (FT)IR spectroscopy. Besides its ultrafast time-resolution, a so far largely underappreciated potential of 2D-IR spectroscopy lies in its sensitivity gain. The present paper sets the goals and outlines strategies how to use that sensitivity gain together with properly designed vibrational labels to make IR spectroscopy a versatile tool to study a wide class of proteins. Abstract Abstract: 2D-IR spectroscopy has matured to a powerful technique to study the structure

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Section: Advancing the Selectivitymentioning

confidence: 99%

Fast infrared spectroscopy of protein dynamics: advancing sensitivity and selectivity

Koziol

Johnson

Stucki-Buchli

et al. 2015

Current Opinion in Structural Biology

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“…The non‐canonical amino acid (ncAA) 4‐cyano‐L‐phenylalanine (pCNF) is a vibrational reporter which has been incorporated into a number of peptides including the MLCK peptide, the villin headpiece subdomain, the human islet amyloid polypeptide, and the amyloid peptide (A β 16‐22), by standard solid‐phase peptide synthesis. Genetic incorporation through the amber codon suppression method has also been used to incorporate pCNF into several protein systems including myoglobin, the N‐terminal domain of the L9 protein (NTL9), cytochrome c , superfolder green fluorescent protein (sfGFP), the N‐terminal Src homology 3 domain of the murine adaptor protein Crk‐II (nSH3), plastocyanin protein (Pc), and the heme nitric oxide and/or oxygen (H‐NOX) protein from Caldanaerobacter subterraneus ( Cs H‐NOX) . Finally, nitrile vibrational reporters have also been incorporated post‐translationally though the conversion of a cysteine to a thiocyanate (SCN) or by using enzyme inhibitors containing nitriles.…”

Section: Introductionmentioning

confidence: 99%

Paired Spectroscopic and Crystallographic Studies of Proteases

et al. 2019

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The active sites of subtilisin and trypsin have been studied by paired IR spectroscopic and X‐ray crystallographic studies. The active site serines of the proteases were reacted with 4‐cyanobenzenesulfonyl fluoride (CBSF), an inhibitor that contains a nitrile vibrational reporter. The nitrile stretch vibration of the water‐soluble inhibitor model, potassium 4‐cyanobenzenesulfonate (KCBSO), and the inhibitor were calibrated by IR solvent studies in H2O/DMSO and the frequency‐temperature line‐slope (FTLS) method in H2O and THF. The inhibitor complexes were examined by FTLS and the slopes of the best fit lines for subtilisin‐CBS and trypsin‐CBS in aqueous buffer were both measured to be −3.5x10−2 cm−1/°C. These slopes were intermediate in value between that of KCBSO in aqueous buffer and CBSF in THF, which suggests that the active‐site nitriles in both proteases are mostly solvated. The X‐ray crystal structures of the subtilisin‐CBS and trypsin‐CBS complexes were solved at 1.27 and 1.32 Å, respectively. The inhibitor was modelled in two conformations in subtilisin‐CBS and in one conformation in the trypsin‐CBS. The crystallographic data support the FTLS data that the active‐site nitrile groups are mostly solvated and participate in hydrogen bonds with water molecules. The combination of IR spectroscopy utilizing vibrational reporters paired with X‐ray crystallography provides a powerful approach to studying protein structure.

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“…Therefore, the past decade has seen a continued effort to develop extrinsic IR probes that can circumvent this limitation. 1,5 For instance, to improve the structural and spatial resolution of IR measurements, non-natural amino acids consisting of a unique vibrational probe, such as a ACN, ASCN, or AN 3 moiety, have been utilized in various studies, including those concerning protein folding, 6 amyloid formation, 7 the structure and function of membrane proteins, 8,9 electrostatic field of enzymes, [10][11][12][13][14] and drug binding. 15,16 Recently, Pazos et al 17 have shown that the ester carbonyl stretching vibration is a useful and convenient site-specific IR probe of protein electrostatics because (1) its frequency is linearly correlated with the local electrostatic field, even when the carbonyl group is engaged in hydrogen-bonding interactions; (2) its frequency is located in the spectral range of 1700-1800 cm 21 , wherein no intrinsic protein vibrations show up at neutral pH; and (3) it has a relatively high extension coefficient.…”

Section: Introductionmentioning

confidence: 99%

Simple method to introduce an ester infrared probe into proteins

Ahmed

Gai

2017

Protein Science

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The ester carbonyl stretching vibration has recently been shown to be a sensitive and convenient infrared (IR) probe of protein electrostatics due to the linear dependence of its frequency on local electric field. While an ester moiety can be easily incorporated into peptides via solid-phase synthesis, currently there is no method available to site-specifically incorporate it into a large protein. Herein, we show that it is possible to use a cysteine alkylation reaction to achieve this goal and demonstrate the feasibility of this simple method by successfully incorporating a methyl ester group (CH COOCH ) into a model peptide (YGGCGG), two amyloid-forming peptides derived from the insulin B chain and Aβ, and bovine serum albumin (BSA). IR results obtained with those peptide and protein systems further confirm the utility of this vibrational probe in monitoring, for example, the structural integrity of amyloid fibrils and ligand binding-induced changes in protein local hydration status.

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Cyano Groups as Probes of Protein Microenvironments and Dynamics

Cited by 12 publications

References 49 publications

Fast infrared spectroscopy of protein dynamics: advancing sensitivity and selectivity

Fast infrared spectroscopy of protein dynamics: advancing sensitivity and selectivity

Paired Spectroscopic and Crystallographic Studies of Proteases

Simple method to introduce an ester infrared probe into proteins

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