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...

We present studies on a series of photosynthetic reaction center (RC) mutants created in the background of the Rhodobacter capsulatus D(LL) mutant, in which the D helix of the M subunit has been substituted with that from the L subunit. Previous work on the D(LL) mutant in chromatophore preparations showed that RCs assembled without the bacteriopheophytin H(L) electron acceptor and performed no charge separation following light absorption. We have successfully isolated poly-His-tagged D(LL) RCs by using the detergent Deriphat 160-C and shown that the RCs are devoid of H(L). The excited state of the primary electron donor, P*, is found to have a lifetime of 180 +/- 20 ps and to decay exclusively (>95%) via internal conversion to the ground state, with no evidence for formation of any charge-separated intermediates. By additional mutation in the D(LL) background of two residues that affect the P/P+ oxidation potential and one that facilitates M-side electron transfer, we achieve an unprecedented 70% yield of P+ H(M)-, more than doubling the highest yield of this state achieved previously. This result underscores the importance of the relative free energies of P* and the charge-separated states in governing the rates and yields of electron transfer in bacterial RCs and provides a basis for systematically investigating M-side electron transfer without any competition from the native L-side pathway.

No abstract

-lauryl-N,N-dimethylamine-N-oxide (LDAO) is used to suspend the RCs, the excited state of the primary electron donor (P*) decays to the ground state with an average lifetime at 77 K of 330 or 170 ps, respectively; however, in both cases the time constant obtained from single-exponential fits varies markedly as a function of the probe wavelength. These findings on the D LL RC are most easily explained in terms of a heterogeneous population of RCs. Similarly, the complex results for D LL -FY L F M in Deriphatglycerol glass at 77 K are most simply explained using a model that involves (minimally) two distinct populations of RCs with very different photochemistry. Within this framework, in 50% of the D LL -FY L F M RCs in Deriphat-glycerol glass at 77 K, P* deactivates to the ground state with a time constant of ∼400 ps, similar to the deactivation of P* in the D LL mutant at 77 K. In the other 50% of D LL -FY L F M RCs, P* has a 35 ps lifetime and decays via electron transfer to the M branch, giving P + H M -in high yield (g80%). This result indicates that P* f P + H M -is roughly a factor of 2 faster at 77 K than at 295 K. In alternative homogeneous models the rate of this M-side electron-transfer process is the same or up to 2-fold slower at low temperature. A 2-fold increase in rate with a reduction in temperature is the same behavior found for the overall L-side process P* f P + H L -in wild-type RCs. Our results suggest that, as for electron transfer on the L side, the M-side electron-transfer reaction P* f P + H M -is an activationless process.

The 13 C 2 and d 2 isotopomers of 1,1,2,2-tetrafluoroethane (TFEA) have been synthesized. Raman spectra of these new species have been recorded, and infrared spectra of all three isotopomers, including some regions with high-resolution at -100°C, have also been recorded. Guided by recently published calculations of frequencies and infrared intensities and the new spectra, we have revised the previous assignments of fundamentals for the two rotamers of the normal species of TFEA. Assignments of the fundamentals for both rotamers of the 13 C 2 and d 2 isotopomers are proposed. The anti rotamer is the more abundant species in the gas phase and, to a lesser extent, in the liquid phase and the only species in the crystal phase. Thus, the assignments of the anti rotamer of all three isotopic species are complete and supported by isotope product rules, but the assignments for the gauche rotamers are incomplete. Estimates of the missing frequencies for the gauche rotamer of the normal species are supplied.