Site-Specific Conversion of Cysteine Thiols into Thiocyanate Creates an IR Probe for Electric Fields in Proteins

Abstract

In this paper we develop a straightforward and general method to introduce the thiocyanate nitrile stretch as a site-specific electric field probe for proteins. Electrostatics affect nearly all aspects of protein function, but general, site-specific probes for these fields are not yet available. The nitrile stretch is a particularly attractive probe because the frequency is in a relatively uncluttered region of the IR spectrum ( -2240 cm −1 ), is typically quite intense (ε∼ 50−1000 M −1 cm −1 ), and is sensitive to electric fields, that is, it has a relatively large Stark tuning rate [∼ 0.4 − 1.1 cm −1 /(MV/cm)] 1 . In some cases it is possible to deliver the nitrile probe on a substrate or inhibitor 2 , and it may prove possible to introduce nitrilecontaining amino acids such as 4-CN-Phe site-specifically into proteins by semi-synthesis or nonsense suppression 3 , but both methods often yield smaller quantities of modified protein than typically used for biophysical studies and are not readily compatible with diverse expression systems or multi-subunit protein assemblies. Since the introduction of cysteine residues is used widely as a site-specific labeling strategy (e.g. for spin 4 or fluorescent labels), we exploit a well-known chemical modification of cysteine residues to form thiocyanates as a general method for introducing a very small IR-based probe of protein electric fields.The strategy outlined in Scheme 1 for converting cysteine thiols into thiocyanates 5,6 is routinely employed as the first step in the selective cleavage of peptide bonds at cysteine residues 7 . Briefly, the protein in buffer at pH 7 was reacted with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB or Ellman's reagent 8 ) to form the mixed protein-thionitrobenzoic acid disulfide (PS-TNB), followed by displacement by cyanide (CN − ), to form the protein-thiocyanate (PS-CN) 9 . The electronic absorption of 2-nitro-5-thiobenzoate (TNB) anion byproduct is conveniently monitored at 412 nm (ε 412 =13600 M −1 cm −1 ) 8 to follow the course of reaction. We observed that the PS-CN products in the examples that follow are stable when stored at 4°C over 4 days at pH 7 10 , consistent with previous reports 11 .We have chosen three very different systems to demonstrate the versatility and scope of this method: modification of S-peptide bound to the ribonuclease S-protein (RNase S) 12 , human aldose reductase (hALR2), which has multiple cysteine residues and for which a number of CN-containing inhibitors related to diabetes control are available 2,13 , and the bacterial photosynthetic reaction center (RC), which is a multi-subunit, integral membrane protein containing many prosthetic groups.The sensitivity of a vibrational frequency to an electric field is calibrated by vibrational Stark effect (VSE) spectroscopy 14-16 . Thiocyanate was introduced into RNase S and the VSE spectrum recorded (Figure 1). RNase S is a non-covalent complex between residues 1−20 (S- peptide) and 21−124 (S-protein) of bovine ribonuclease A 12 and is an extensively studied vehic...

Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase

“…All the four mutant enzymes (C69S/C81S/C97S/D40N)/M116C, (C69S/C81S/C97S/D40N)/F86C, (C69S/C81S/C97S/D40N)/I17C, and (C69S/C81S/ C97S/D40N)/M105C were expressed and purified according to a previously published protocol (43). The purified enzymes were labeled with K 13 C 15 N according to a previously published protocol (58). Isotopically labeled − 13 C 15 N was used to shift the spectator probe frequencies as far as possible from the nitrile on C183 (see Fig.…”

Triggered infrared spectroscopy for investigating metalloprotein chemistry

“…Since the frequency of the vibrational band of the probe is sensitive to local environment, IR spectra of the modified protein reported on ligand binding to the haem. Chemically modified cysteine residues converted to thiocyanate in a method reported by Fafarman et al (2006) offer similar possibilities. In conjunction with two-dimensional IR methods, introduction of probes by chemical or genetic modification may allow resolution of structural factors and interactions between proteins, paralleling spin probes introduced into proteins for distance detection in electron paramagnetic resonance methods.…”

“…Application of IR spectroscopy to proteins has often targeted the amide I and II bands (1700-1600 and 1580-1480 cm −1 ) of the polypeptide backbone as probes for gross structural change (Siebert & Hildebrandt 2008). The amide I band arises predominantly from (Schultz et al 2006) and (ii) cysteine converted to a thiocyanate by Fafarman et al (2006).…”

“…The frequencies for the vibrational bands of these groups coordinated to metal centres generally occur in regions of the IR spectrum that are distinguishable from the amide bands and thus can be identified readily (figure 2a). Other interesting opportunities arise from localized probes that give rise to IR-active vibrations, such as the non-natural amino acid cyanophenylalanine introduced into a protein (Schultz et al 2006) or cysteine converted to a thiocyanate (Fafarman et al 2006; figure 1d). These could be exploited to examine hydrogen-bonding interactions between the protein and IR probe, the electrostatic character of sites in a protein or possibly to confirm proximity between residues or follow protein-protein interactions or folding.…”

Dynamics of the folded and unfolded villin headpiece (HP35) measured with ultrafast 2D IR vibrational echo spectroscopy

“…For time-dependent studies, nitrile can produce reasonable signals, and it is sensitive to the electric field (23). CN is very useful because it can be inserted into many biological molecules via molecular engineering techniques (24,25). These techniques include the in vivo genetic incorporation of unnatural amino acids developed by Schultz et al, which allows the introduction of CN probes virtually anywhere in a protein (26).…”