Glass transition in thaumatin crystals revealed through temperature-dependent radiation-sensitivity measurements

Abstract

The temperature-dependence of radiation damage to thaumatin crystals between T = 300 and 100 K is reported. The amount of damage for a given dose decreases sharply as the temperature decreases from 300 to 220 K and then decreases more gradually on further cooling below the protein-solvent glass transition. Two regimes of temperature-activated behavior were observed. At temperatures above $200 K the activation energy of 18.0 kJ mol À1 indicates that radiation damage is dominated by diffusive motions in the protein and solvent. At temperatures below $200 K the activation energy is only 1.00 kJ mol À1 , which is of the order of the thermal energy. Similar activation energies describe the temperaturedependence of radiation damage to a variety of solvent-free small-molecule organic crystals over the temperature range T = 300-80 K. It is suggested that radiation damage in this regime is vibrationally assisted and that the freezing-out of amino-acid scale vibrations contributes to the very weak temperature-dependence of radiation damage below $80 K. Analysis using the radiation-damage model of Blake and Phillips [Blake & Phillips (1962), Biological Effects of Ionizing Radiation at the Molecular Level, indicates that large-scale conformational and molecular motions are frozen out below T = 200 K but become increasingly prevalent and make an increasing contribution to damage at higher temperatures. Possible alternative mechanisms for radiation damage involving the formation of hydrogen-gas bubbles are discussed and discounted. These results have implications for mechanistic studies of proteins and for studies of the protein glass transition. They also suggest that data collection at T ' 220 K may provide a viable alternative for structure determination when cooling-induced disorder at T = 100 is excessive.

Spatial distribution of radiation damage to crystalline proteins at 25–300 K

“…As has been previously discussed, global sensitivity estimates exhibit significant ($50%) sample-to-sample variability (Meents et al, 2007(Meents et al, , 2010Warkentin & Thorne, 2010;Warkentin et al, 2012). The B-factor-derived dose estimates given above should thus be considered accurate to within 50%.…”

“…The number of data sets and the dose per data set were chosen so that B factors could still be determined from the final set, so that each set exhibited a significant increment in damage (a scaling B-factor increase of $0.5 Å 2 ) and so that the total number of sets (4-20) was sufficient to allow averaging over set-to-set scatter to obtain a reliable measure of increases in atomic B factors as a function of dose. The sensitivities of the thaumatin crystals were known from our previous work and they informed our choice of dose per data set at each temperature (Warkentin & Thorne, 2010). At T = 25, 100 and 180 K crystals were matched to the beam size (100 mm) and no attenuation was used.…”

“…At T = 25, 100 and 180 K crystals were matched to the beam size (100 mm) and no attenuation was used. Because of the rapid increase in global sensitivity of protein crystals above T ' 200 K (Warkentin & Thorne, 2010), at higher temperatures large crystals ($500 mm) were used to spread absorption and scattering over a larger volume along the beam path (with fixed beam diameter) and the beam was attenuated by a factor of 4. (Even so, only four data sets were obtained from thaumatin at 240 K.) For urease, which has an $500 000 Å 3 unit cell, only three sets were obtained at T = 300 K owing to the large dose required per complete set.…”

“…Protein crystals are approximately 10-130 times more sensitive to global damage at room temperature than at cryogenic temperatures ($100 K; Blake & Phillips, 1962;Teng & Moffat, 2002;Kmetko et al, 2006Kmetko et al, , 2011Southworth-Davies et al, 2007;Barker et al, 2009;Warkentin & Thorne, 2010). Solvent and atomic radical diffusion and diffusive motions of protein side chains and larger structural elements are suppressed below the proteinsolvent glass-transition temperature near T ' 200 K and become negligible by $100 K (Rodgers, 1994;Garman & Schneider, 1997;Weik, Kryger et al, 2001;Garman, 2003;Weik et al, 2004Weik et al, , 2005Warkentin & Thorne, 2009, 2010.…”

Active-site protein dynamics and solvent accessibility in nativeAchromobacter cycloclastescopper nitrite reductase

“…Maintaining crystal order and diffraction quality in the range between this transition temperature and RT is challenging, but recently experimental advances have opened this regime for study. In this work, we have obtained a crystal structure of wild-type AcNiR at the standard cryogenic temperature of 100 K and a series of structures from one crystal (MSOX; Horrell et al, 2016) at 240 K, a temperature that allows anharmonic motion while still extending the resolution and crystal lifetime beyond those achievable at RT (Warkentin & Thorne, 2010). In addition, we have examined the active-site protein dynamics and solvent accessibility using all-atom molecular dynamics and DFT calculations based on the crystal structures.…”