Cyanylated Cysteine: A Covalently Attached Vibrational Probe of Protein−Lipid Contacts

McMahon, Heather A.; Alfieri, Katherine N.; Clark, Katherine; Londergan, Casey H.

doi:10.1021/jz1000177

Cited by 62 publications

(100 citation statements)

References 29 publications

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“…20−23,30 Thus, the GmACP3 probe vibration should exhibit a pronounced redshift compared to the cyanylated ACT ACP probe according to prior reports.…”

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confidence: 56%

Probing the Phosphopantetheine Arm Conformations of Acyl Carrier Proteins Using Vibrational Spectroscopy

2014

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Acyl carrier proteins (ACPs) are universal and highly conserved domains central to both fatty acid and polyketide biosynthesis. These proteins tether reactive acyl intermediates with a swinging 4′-phosphopantetheine (Ppant) arm and interact with a suite of catalytic partners during chain transport and elongation while stabilizing the growing chain throughout the biosynthetic pathway. The flexible nature of the Ppant arm and the transient nature of ACP–enzyme interactions impose a major obstacle to obtaining structural information relevant to understanding polyketide and fatty acid biosynthesis. To overcome this challenge, we installed a thiocyanate vibrational spectroscopic probe on the terminal thiol of the ACP Ppant arm. This site-specific probe successfully reported on the local environment of the Ppant arm of two ACPs previously characterized by solution NMR, and was used to determine the solution exposure of the Ppant arm of an ACP from 6-deoxyerythronolide B synthase (DEBS). Given the sensitivity of the probe’s CN stretching band to conformational distributions resolved on the picosecond time scale, this work lays a foundation for observing the dynamic action-related structural changes of ACPs using vibrational spectroscopy.

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“…20−23,30 Thus, the GmACP3 probe vibration should exhibit a pronounced redshift compared to the cyanylated ACT ACP probe according to prior reports.…”

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confidence: 56%

Probing the Phosphopantetheine Arm Conformations of Acyl Carrier Proteins Using Vibrational Spectroscopy

2014

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“…(17) Because Ala10 was characterized as a solvent-exposed residue by EPR, A10C* was previously chosen as a control for the examination of cyanylated cysteine’s sensitivity to membrane burial versus solvent-exposure. (19) If C* is sensitive to membrane burial and the tenth residue position is solvent-exposed, then there should be no change in the CN stretching band of A10C* in a lipid versus aqueous environment.…”

Section: Resultsmentioning

confidence: 99%

“…(19–26) This band, which is moderately strong and can be observed clearly in aqueous samples of 25 μm thickness at approximately 1 mM concentration, is sensitive to its local environment in two ways. The frequency depends on water exposure (via a blue shift due to weak hydrogen bonds donated by water) and the local electrostatic environment (via a red shift with increasing solvent polarity).…”

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confidence: 99%

“…Two peptides derived from natural sequences, of which CM15 is one, were used recently in contact with DPC micelles to demonstrate that the CN stretching band of C* is also a sensitive probe of the membrane exposure of labeled side chains. (19) C* is projected to be a widely useful probe of membrane-protein interactions, since it can be incorporated into sequences of arbitrary size via site-directed mutagenesis to cysteine followed by chemical ligation of the CN group at the free thiol. Here C* is used to map the membrane-bound structure of CM15, a short peptide, but its usefulness is not limited to such short sequences.…”

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confidence: 99%

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Using Infrared Spectroscopy of Cyanylated Cysteine To Map the Membrane Binding Structure and Orientation of the Hybrid Antimicrobial Peptide CM15

2011

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The synthetic antimicrobial peptide CM15, a hybrid of N-terminal sequences from cecropin and melittin peptides, has been shown to be extremely potent. Its mechanism of action has been speculated to involve pore formation based on prior site-directed spin labeling studies. This study examines four single-site β-thiocyanatoalanine variants of CM15 in which the artificial amino acid side chain acts as a vibrational reporter of its local environment through the frequency and lineshape of the unique CN stretching band in the infrared spectrum. Circular dichroism experiments indicate that the placements of the artificial side chain have only small perturbative effects on the membrane-bound secondary structure of the CM15 peptide. All variant peptides were placed in buffer solution, in contact with dodecylphosphatidylcholine micelles, and in contact with vesicles formed from E. coli polar lipid extract. At each site, the CN stretching band reports a different behavior. Time-dependent attenuated total reflectance infrared spectra were also collected for each variant as it was allowed to remodel the E. coli lipid vesicles. These experiments agree with the previously proposed formation of toroidal pores, in which each peptide finds itself in an increasingly homogeneous and curved local environment without apparent peptide-peptide interactions. This work also demonstrates the excellent sensitivity of the SCN stretching vibration to small changes in peptide-lipid interfacial structure. Keywordsantimicrobial peptides; cyanylated cysteine; nitrile vibrations; membrane-active peptides; sitedirected labeling; toroidal pores Characterizing the structure, structural distribution, and membrane binding geometry of peripheral membrane proteins (PMPs) is a challenge that requires new experimental approaches. Recent advances in crystallography using surfactants(1) and solid state NMR of oriented samples(2) have led to new insights into the structure and function of a number of such proteins and peptides. However, a hallmark of these studies is that the experimental approach used depends to a large extent on the lipid and protein system of interest without a single, unified approach that is applicable to proteins of any size in arbitrary lipid systems. EPR spectroscopy of site-directed spin labels has this sought-after flexibility when applied to PMPs,(3) with documented limitations due to the size and chemical nature of the most typical spin label(4) and the need for external solution-or lipid-phase paramagnetic species to address directly the extent of membrane burial.(3) Antimicrobial peptides (AMPs) are a subset of membrane-active species generating much recent interest due to their possible use as antibiotic agents in an era of global overuse of antibiotic drugs.(5) The relative simplicity * to whom correspondence may be addressed; telephone 610-896-1217, fax 610-896-4963, clonderg@haverford.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2012 December 27. NIH-PA Author Manusc...

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“…Recently, cyanylated cysteine, in which the cysteine thiol has been converted into a thiocyanate, has been the subject of several studies. [37][38][39][40] These studies illustrate that the thiocyanate is very sensitive to its local environment, making it an excellent probe of the local dynamics. Furthermore, because it is prepared by modifying cysteine in the protein, the synthetic methods to incorporate the probe site selectively are straightforward.…”

Section: Introductionmentioning

confidence: 84%

Two-dimensional infrared study of 3-azidopyridine as a potential spectroscopic reporter of protonation state

Nydegger

Dutta

Cheatum

2010

The Journal of Chemical Physics

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The lack of general spectroscopic probes that can be used in a range of systems to probe kinetics and dynamics is a major obstacle to the widespread application of two-dimensional infrared (2D IR) spectroscopy. We have studied 3-azidopyridine to characterize its potential as a probe of the protonation state of the pyridine ring. We find that the azido-stretching vibration is split by accidental Fermi resonance interactions with one or more overtones and combination states. Using 2D IR spectroscopy, we determine the state structure of the resulting eigenstates for complexes of 3-azidopyridine with formic acid and trifluoroacetic acid in which the pyridine ring is unprotonated and protonated, respectively. Based on the measurements, we develop a two-oscillator depurturbation model to determine the energies and couplings of the zeroth-order azido-stretching state and the perturbing dark state that couples to it. Based on these results, we conclude that the azido-stretching vibration is, in fact, sensitive to the protonation state of the pyridine shifting up in frequency by 8 cm(-1) in the complex with trifluoroacetic acid relative to the formic acid complex. These results suggest that, although 3-azidopyridine is not suitable as a spectroscopic probe, the approach of employing an organic azide as a remote probe of protonation state holds significant promise.

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Cyanylated Cysteine: A Covalently Attached Vibrational Probe of Protein−Lipid Contacts

Cited by 62 publications

References 29 publications

Probing the Phosphopantetheine Arm Conformations of Acyl Carrier Proteins Using Vibrational Spectroscopy

Probing the Phosphopantetheine Arm Conformations of Acyl Carrier Proteins Using Vibrational Spectroscopy

Using Infrared Spectroscopy of Cyanylated Cysteine To Map the Membrane Binding Structure and Orientation of the Hybrid Antimicrobial Peptide CM15

Two-dimensional infrared study of 3-azidopyridine as a potential spectroscopic reporter of protonation state

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