Synthesis and evaluation of the first cis-cyclobutane-containing receptor for lipid A.

The first example of a designed receptor containing a cis-1,3-disubstituted cyclobutane ring has been synthesized. This molecule binds diphosphoryl lipid A (a conserved portion of the Gram-(-) bacterial cell membrane, and the causative agent of septic shock) with an affinity comparable to previously described ter-cycloalkane based lipid A-binding compounds.

A designed receptor for pH-switchable ion binding in water.

Molecules with conditional (switchable) properties are of considerable fundamental interest and are potentially useful for a broad range of applications, including chemical sensing. We have prepared a novel receptor, derived from the peracylation of cyclohexane 1,3,5-trimethanol with tyrosine, that suggests that the phenol-amine hydrogen bond may be an effective structural tool in the preparation of molecules with pH-switchable conformations. This conclusion is based on several key observations. First, the proton longitudinal relaxation rates for this molecule change in a pH-dependent fashion, while those for closely related structures do not. Second, NOESY spectra for the receptor change markedly depending on pH, with the spectrum acquired at pH 9.5 displaying NOEs consistent with the calculated "closed" conformation. Third, this molecule serves as a receptor for anions and cations in aqueous solution at high pH, but not at low pH, as demonstrated by UV-vis titrations and isothermal titration calorimetry (ITC).

The active site cysteine of ubiquitin-conjugating enzymes has a significantly elevated pKa: functional implications.

Ubiquitin-conjugating enzymes (E2s or Ubcs) are essential components in the ubiquitination apparatus. These enzymes accept ubiquitin from an E1 enzyme and then, usually with the aid of an E3 enzyme, donate the ubiquitin to the target protein. The function of E2 relies critically on the chemistry of its active site cysteine residue since this residue must form a thioester bond with the carboxyl terminus of ubiquitin. Despite the plethora of structural information that is available, there has been a notable dearth of information regarding the chemical basis of E2 function. Toward filling this large void in our understanding of E2 function, we have examined the pK(a) of the active site cysteine using a combination of experimental and theoretical approaches. We find, remarkably, that the pK(a) of the active site cysteine residue is elevated by approximately 2 pH units above that of a free cysteine. We have identified residues that contribute to the increase in this pK(a). On the basis of experimental values obtained with three different E2 proteins, we believe this to be a general and important characteristic of E2 protein chemistry. Sequence comparison suggests that the electrostatic environment is maintained not through strict residue conservation but through different combinations of residues near the active site. We propose that the elevated pK(a) is a regulatory mechanism that prevents the highly exposed cysteine residue in free E2 from reacting promiscuously with electron deficient chemical moieties in the cell.

Direct determination of thiol pKa by isothermal titration microcalorimetry.

Knowledge of the acid dissociation constant (pKa) of a molecule is a critical step toward understanding its structure and reactivity. Current methods for pKa measurement, including electrochemical, spectroscopic, and spectrophotometric titrations, have proven to be useful but also have significant limitations. To overcome these limitations, we report the use of isothermal titration calorimetry (ITC) as a new method for pKa determination. We demonstrate by the measurement of the pKa values for free cysteine, glutathione, and a cysteine residue in a protein that this method is rapid and accurate.