Decomposition of Vibrational Shifts of Nitriles into Electrostatic and Hydrogen-Bonding Effects

Fafarman, Aaron T.; Sigala, Paul A.; Herschlag, Daniel; Boxer, Steven G.

doi:10.1021/ja104573b

Cited by 146 publications

(304 citation statements)

References 33 publications

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“…6A. As elucidated previously (20,37) and discussed in more detail in SI Text, changes in the IR frequency for each nitrile probe across the series of bound phenols report on changes in the local electrostatic field experienced by each probe due to repositioning of charge within the hydrogen bond network.…”

Section: Measuring the Electrostatic Field Changes From Proton Transfersmentioning

confidence: 69%

“…KSI mutants were expressed and purified from Escherichia coli using published methods (8). Nitrile labeling and uniform 13 C-Tyr incorporation were performed as previously described (20,29,37). 19 F and 13 C NMR spectra were acquired at 20°C on 500-and 600-MHz (proton frequency) Varian UNITY INOVA NMR spectrometers using previously published methods (8,9,37).…”

Section: Methodsmentioning

confidence: 99%

“…Nitrile labeling and uniform 13 C-Tyr incorporation were performed as previously described (20,29,37). 19 F and 13 C NMR spectra were acquired at 20°C on 500-and 600-MHz (proton frequency) Varian UNITY INOVA NMR spectrometers using previously published methods (8,9,37). IR spectra were acquired at room temperature and at 80 K as previously reported (20,29,37).…”

Section: Methodsmentioning

confidence: 99%

“…19 F and 13 C NMR spectra were acquired at 20°C on 500-and 600-MHz (proton frequency) Varian UNITY INOVA NMR spectrometers using previously published methods (8,9,37). IR spectra were acquired at room temperature and at 80 K as previously reported (20,29,37). Cocrystals of pKSI D40N-3-F-4-NO 2 -phenolate and D40N/M116C-CN-equilenin were obtained at 20°C using hanging drop vapor diffusion in accordance with previously published methods (8,20).…”

Section: Methodsmentioning

confidence: 99%

“…In prior work, we site-specifically incorporated nitrile groups into pKSI by cyanylating a unique Cys introduced by mutation at position M116, M105, or F86 within a Cys-free C69S/C81S/C97S background (20,29,37). These positions were selected on the basis of their proximity (3-11 Å) to the key catalytic groups (Fig.…”

Section: Quantitative Modeling Of Ionization States Within the Ksi Hymentioning

confidence: 99%

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Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

Sigala¹,

Fafarman²,

Schwans³

et al. 2013

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

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Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated sitespecific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol-Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations.computational modeling | enzyme catalysis | protein electrostatics | protein semisynthesis | active site environment H ydrogen bond networks are ubiquitous structural features within proteins, and they play key roles linking secondary and tertiary structural elements and spanning protein-protein interfaces. Such networks are especially common within enzyme active sites, where they position protein and substrate groups for catalysis, stabilize charge rearrangements during chemical transformations, and mediate proton transfers (1). Despite the prevalence and critical structural and functional roles of hydrogen bond networks, incisive dissection of their physical properties within the idiosyncratic interior of folded proteins remains difficult.Hydrogen-bonded protons are not observed in the vast majority of protein X-ray structures due to the low X-ray scattering power of hydrogen atoms (2). Thus, the presence of hydrogen bond networks is typically inferred from the proximity and orientation of hydrogen bond donor and acceptor groups within refined protein structural models. The inherent inability of most X-ray diffraction studies to monitor proton positions imposes additional challenges for dissecting the physical features that influence the equilibrium protonation states of specific residues along a hydrogen-bonded proton transfer network. Furthermore, it remains extremely challenging t...

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Section: Measuring the Electrostatic Field Changes From Proton Transfersmentioning

confidence: 69%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Quantitative Modeling Of Ionization States Within the Ksi Hymentioning

confidence: 99%

See 3 more Smart Citations

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

Sigala¹,

Fafarman²,

Schwans³

et al. 2013

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

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Cyano Groups as Probes of Protein Microenvironments and Dynamics

et al. 2011

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Sondentest: Die Cyangruppe reagiert auf ihre Umgebung, absorbiert in einem einzigartigen Bereich von Protein‐IR‐Spektren und kann an Aminosäuren gebunden werden. Mit stationären und zeitaufgelösten Methoden wurde sie in mehreren Cytochrom‐c‐Varianten (siehe Struktur der Hämtasche) als nützliche ortsspezifische Sonde für Proteinmikroumgebungen und ‐dynamiken belegt; doch sie kann ihre Umgebung auch stören und den gefalteten Proteinzustand destabilisieren.

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Combinatorial Catalysis Employing a Visible Enzymatic Beacon in Real Time: Synthetically Versatile (Pseudo)Halometalation/Carbocyclizations

et al. 2011

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Combinatorial approaches to catalysis have made an impact in targeted transformation development, including silvermediated carbene insertion, [1] scandium/pybox-based (pybox = bis(oxazolinyl)pyridine) asymmetric cyclopropanation, [2] and rhodium/iridium-based asymmetric hydrogenation.[3] Useful design elements have emerged from these studies, for example, the value of ligand self-assembly, [4] or of the inclusion of peptide-like structural elements [5][6][7] in building ligand arrays. Efficient screening methods are of paramount importance for such efforts. Methods based on fluorescence, [8] REMPI, [9] MS, [10] NMR, [11] and IR thermography [12] have appeared. A chromophore may be installed into the substrate [13] or product [14] of the reaction under study. Alternatively, one can exploit chromophores inherent in proteins [15] or enzyme-associated reactions, [16] and use these sensors to report back on product formation and composition.Our group has developed an in situ enzymatic screening (ISES) approach whereby an organometallic reaction under study is coupled to an enzymatic reporting reaction in real time.[17] This screening method led to the discovery of the first asymmetric allylic amination with nickel(0) [18] and to the identification of novel salen [salen = N,N'-bis(salicylidene)-ethylenediamine)] ligands with promise for asymmetric synthesis.

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Decomposition of Vibrational Shifts of Nitriles into Electrostatic and Hydrogen-Bonding Effects

Cited by 146 publications

References 33 publications

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

Cyano Groups as Probes of Protein Microenvironments and Dynamics

Combinatorial Catalysis Employing a Visible Enzymatic Beacon in Real Time: Synthetically Versatile (Pseudo)Halometalation/Carbocyclizations

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