It has long been appreciated that both temperature and density play roles in determining the extremely super-Arrhenius, low-temperature behavior of the viscosity and long α-relaxation times that characterize fragile supercooled liquids. But what has not been generally appreciated, and what we believe we have established (by focusing on a model-free analysis in terms of temperature and density, rather than upon temperature and pressure) is that over the range of densities and temperatures spanned by the experiments carried out at 1 atm pressure, temperature is the dominant control variable. This information is essential input to the formulation of a theory or model of the long-time dynamics of low-temperature fragile liquids, and it suggests a focus on activated dynamics rather than on free volume. This work indicates that, except possibly at very high densities (very high pressures), the glass transition is not a result of congestion due to a lack of free volume.

The fragile glass-forming liquid triphenyl phosphite (TPP) melts at 298 K, has a glass transition at about 186 K, and can undergo a first order transition to a metastable, solid, apparently amorphous phase (denoted the glacial phase) at about 230 K. Though apparently amorphous (on the basis of preliminary X-ray data), we have shown that the glacial phase is well described as a plastic crystal composed of nanocrystallites; it is thus not a second liquid or glass nor a poor ordinary molecular crystal. This picture of the glacial phase with its close connection to the supercooled liquid from which it forms is placed in the context of the frustrationlimited-domain theory of supercooled liquids, and the glacial phase is associated with the defect-ordered phase predicted by that theory. In examining the applicability of this picture, we carried out extensive experimental studies, many of them by means of NMR at both ambient and high pressures, on the glacial phase and on the dynamics of the transformations to and from this phase.

The mechanism of herpesviral protease activation upon dimerization was studied using two independent spectroscopic assays augmented by directed mutagenesis. Spectroscopic changes, attributable to dimer interface conformational plasticity, were observed upon dimerization of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr). KSHV Pr's dissociation constant of 585 +/- 135 nM at 37 degrees C was measured by a concentration-dependent, 100-fold increase in specific activity to a value of 0.275 +/- 0.023 microM product min(-1) (microM enzyme)(-1). A 4 nm blue-shifted fluorescence emission spectrum and a 25% increase in ellipticity at 222 nm were detected by circular dichroism upon dimer association. This suggested enhanced hydrophobic packing within the dimer interface and/or core, as well as altered secondary structures. To better understand the structure-activity relationship between the monomer and the dimer, KSHV Pr molecules were engineered to remain monomeric via substitution of two separate residues within the dimer interface, L196 and M197. These mutants were proteolytically inactive while exhibiting the spectroscopic signature and thermal stability of wild type, dissociated monomers (T(M) = 75 degrees C). KSHV Pr conformational changes were found to be relevant in vivo, as the autoproteolytic inactivation of KSHV Pr at its dimer disruption site [Pray et al. (1999) J. Mol. Biol. 289, 197-203] was detected in viral particles from KSHV-infected cells. This characterization of structural plasticity suggests that the structure of the KSHV Pr monomer is stable and significantly different from its structure in the dimer. This structural uniqueness should be considered in the development of compounds targeting the dimer interface of KSHV Pr monomers.

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