Many peptidases are thought to require non-active site interaction surfaces, or exosites, to recognize and cleave physiological substrates with high specificity and catalytic efficiency. However, the existence and function of protease exosites remain obscure owing to a lack of effective methods to identify and characterize exosite-interacting substrates. To address this need, we modified the cellular libraries of peptide substrates (CLiPS) methodology to enable the discovery of exosite-interacting peptide ligands. Invariant cleavage motifs recognized by the active sites of thrombin and caspase-7 were displayed on the outer surface of bacteria adjacent to a candidate exosite-interacting peptide. Exosite peptide libraries were then screened for ligands that accelerate cleavage of the active site recognition motif using two-color flow cytometry. Exosite CLiPS (eCLiPS) identified exosite-binding peptides for thrombin that were highly similar to a critical exosite interaction motif in the thrombin substrate, protease-activated receptor 1. Protease activity probes incorporating exosite-binding peptides were cleaved ten-fold faster than substrates without exosite ligands, increasing their sensitivity to thrombin activity in vitro. For comparison, screening with caspase-7 yielded peptides that modestly enhanced (two-fold) substrate cleavage rates. The eCLiPS method provides a new tool to profile the ligand specificity of protease exosites and to develop improved substrates.

Caspases are a powerful class of cysteine proteases. Introduction of activated caspases in healthy or cancerous cells results in induction of apoptotic cell death. In this study, we have designed and characterized a version of caspase‐7 that can be inactivated under oxidizing extracellular conditions and then reactivated under reducing intracellular conditions. This version of caspase‐7 is allosterically inactivated when two of the substrate‐binding loops are locked together via an engineered disulfide. When this disulfide is reduced, the protein regains its full function. The inactive loop‐locked version of caspase‐7 can be readily observed by immunoblotting and mass spectrometry. The reduced and reactivated form of the enzyme observed crystallographically is the first caspase‐7 structure in which the substrate‐binding groove is properly ordered even in the absence of an active‐site ligand. In the reactivated structure, the catalytic‐dyad cysteine–histidine are positioned 3.5 Å apart in an orientation that is capable of supporting catalysis. This redox‐controlled version of caspase‐7 is particularly well suited for targeted cell death in concert with redox‐triggered delivery vehicles.

The infrared spectra of ethylmethylgermane (CH(3)CH(2)GeH(2)CH(3)) have been studied as a vapour in the 4000-400 and 500-100 cm(-1) regions and as an amorphous solid at 78 K. Raman spectra of the sample as a liquid were recorded at 293 K and polarization data were obtained. Additional Raman spectra were recorded at various temperatures between 293 and 143 K, and small intensity changes of certain bands with temperature were detected. The sample was also investigated as an amorphous solid on a cold finger of copper or a Cslwindow at 78 K. No crystallization was ever obtained in the Raman or infrared cryostats in spite of extensive annealing.The compound exists a priori in two conformers, anti and gauche, and the variable temperature Raman spectra suggest an equilibrium in which the gauche conformer has 0.4 kJ mol(-1) lower enthalpy than anti in the liquid.B3LYP and MP2 calculations with various basis sets and the CBS-QB3 and G2 models were employed, yielding an average conformational enthalpy difference Delta H(anti-gauche) = 0.24 kJ mol(-1). Infrared and Raman intensities, polarization ratios and vibrational frequencies for the gauche and anti conformers were calculated. Instead of scaling the calculated wavenumbers in the harmonic approximation, performed in our previous publications, calculations from B3LYP/cc-pVTZ were derived in the anharmonic approximation. In most cases these values gave a good agreement with the experimental results for 34 observed modes of the gauche and 10 modes of the anti conformer. (C) 2010 Elsevier B.V. All rights reserved.

The active sites of caspases are composed of four mobile loops. A loop (L2) from one half of the dimer interacts with a loop (L2') from the other half of the dimer to bind substrate. In an inactive form, the two L2' loops form a cross-dimer hydrogen-bond network over the dimer interface. Although the L2' loop has been implicated as playing a central role in the formation of the active-site loop bundle, its precise role in catalysis has not been shown. A detailed understanding of the active and inactive conformations is essential to control the caspase function. We have interrogated the contributions of the residues in the L2' loop to catalytic function and enzyme stability. In wild-type and all mutants, active-site binding results in substantial stabilization of the complex. One mutation, P214A, is significantly destabilized in the ligand-free conformation, but is as stable as wild type when bound to substrate, indicating that caspase-7 rests in different conformations in the absence and presence of substrate. Residues K212 and I213 in the L2' loop are shown to be essential for substrate-binding and thus proper catalytic function of the caspase. In the crystal structure of I213A, the void created by side-chain deletion is compensated for by rearrangement of tyrosine 211 to fill the void, suggesting that the requirements of substrate-binding are sufficiently strong to induce the active conformation. Thus, although the L2' loop makes no direct contacts with substrate, it is essential for buttressing the substrate-binding groove and is central to native catalytic efficiency.

The infrared spectra of ethoxytrichlorosilane (trichlorosilyl ethyl ether, SiCl 3 OCH 2 CH 3 ) have been recorded in vapour, liquid and amorphous solid state at 78 K in the 4000-50 cm - 1 range, and in argon matrices at ca. 5 K. Raman spectra of the liquid were recorded at room temperature and at various temperatures between 158 and 296 K. In spite of many attempts, it was not possible to crystallize this molecule in the range 60-200 K. In the variable temperature Raman spectra, certain bands changed in intensity and were interpreted in terms of conformational equilibrium. Employing two band pairs, the conformational enthalpy difference ΔH(gauche-anti) in the liquid was calculated to be 2.0 kJ.mol - 1 . With the statistical weight of 1:2 for the anti/gauche ratio, the two conformers should therefore have approximately equal abundance at room temperature. Some intensity variations were observed in the IR spectra when the argon matrix was annealed, but these could not be related to conformer identity. Ab initio and DFT calculations were carried out using a variety of basis sets. Only in the MP2(FC) calculations a distinct minimum in the potential curve appeared for the gauche conformer, whereas only a small deflection around 120° from anti was apparent in the other calculations. An enthalpy difference of AH (gauche-anti) = 2.0 kJ.mol - 1 was obtained in the MP2/aug-cc-pVTZ calculations, in excellent agreement with the observed value.

The infrared spectra (3200-30 cm(-1)) of ethyl bromogermane, CH3CH2GeH2Br, in the gaseous phase and of amorphous and annealed solid samples as well as the Raman spectrum (3200-100 cm(-1)) of the liquid have been recorded. Variable-temperature (-105 to -150 degreesC) studies of the infrared spectra of a sample dissolved in liquid krypton have been carried out. From these data, the enthalpy difference between the gauche and trans conformers has been determined to be 158 +/- 16 cm(-1) (1.89 +/- 0.20 kJ/mol), with the gauche conformer being the more stable form. This result is consistent with the prediction from ab initio calculations at both the Hartree-Fock level and with full electron correlation by the perturbation method to second order. It is estimated that at ambient temperature 81 +/- 2% of the sample is in the gauche form. A relatively complete vibrational assignment is proposed for both the gauche and trans conformers based on infrared band contours, relative intensities, depolarization values, and group frequencies which is supported by normal coordinate calculations utilizing the force constants from the ab initio MP2/6-31G(d) calculations. The optimized geometries and conformational stabilities have also been obtained from ab initio calculations utilizing several different basis sets with full electron correlation by the perturbation method up to MP2/6-311+G(2d,2p). Additionally, the potential functions governing the conformation interchange have been estimated from ab initio MP2/6-31G(d) and density functional theory calculations by the B3LYP method, with barriers to conformational interchange of 560, 924, and 852 cm(-1) from MP2/6-31G(d) and 418, 771, and 606 cm(-1) from B3LYP/6-31G(d) calculations for the trans to gauche, gauche to gauche, and gauche to trans conformers, respectively. The results are discussed and compared to some corresponding quantities for several related molecules.