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 0 ) from the other half of the dimer to bind substrate. In an inactive form, the two L2 0 loops form a cross-dimer hydrogen-bond network over the dimer interface. Although the L2 0 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 0 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 0 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 0 loop makes no direct contacts with substrate, it is essential for buttressing the substrate-binding groove and is central to native catalytic efficiency.

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 10-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 (twofold) 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.

were obtained in the vapour, liquid, and crystalline solid phases in the range 4000-50 cm −1 . Additional spectra in argon matrices at 5 K were recorded before and after annealing to temperatures 20-34 K. Raman spectra of the compounds as liquids were recorded at various temperatures between 296 and 183 K and spectra of the amorphous and crystalline solids were obtained.The spectra revealed the existence of two conformers (anti and gauche) in the fluid phases and in the matrices. When the two vapours were shock-frozen on a cold finger at 78 K, they turned partly crystalline immediately. After subsequent annealing to 140-150 K, ca 7-9 Raman bands of both molecules present in the liquids vanished in the crystal. Similar variations in intensity were observed in the corresponding infrared spectra before and after annealing. The spectra revealed the existence of one conformer (anti) in the crystal.From Raman intensity variations of three independent pairs of anti and gauche bands between 298 and 173 K for the parent compound, and 298 and 183 K for the deuterated analogue, the values 1 conf H o .gauche−anti/ = 4.1 ± 0.3 kJ mol −1 for the parent compound and the same value for the deuterated species were obtained in the liquid state. Annealing experiments in the matrices show that the gauche bands vanish after annealing, demonstrating that the anti conformer also has the lower energy here and that the barrier to gauche → anti inter-conversion is around 5-6 kJ mol −1 . The spectra of both conformers have been interpreted in detail.Ab initio and DFT calculations at the HF/6-311G**, B3LYP/6-311 G** and MP2/6-311 G** levels gave optimized geometries, infrared and Raman intensities and vibrational wavenumbers for the anti and gauche conformers. The conformational enthalpy difference derived from the calculations was between 6.0 and 4.1 kJ mol −1 with anti being the low energy conformer.

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 °C) 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.Key words: conformational stability, Raman spectra, infrared spectra; ab initio calculations, ethyl bromogermane.