Modulation of Active Site Picosecond Dynamics in Mutant Forms of a Thermophilic Alcohol Dehydrogenase (HT-ADH)

Abstract

Previous kinetic and solvent-exchange data have suggested that an Arrhenius break observed at 30oC is attributed to a shift in the equilibrium populations of inactive and active microstates accompanied with a change in protein flexibility [1,2]. We employed time-correlated single photon counting to investigate the statistical nature of the fluorescence dynamics as a function of temperature in ht-ADH. Two single-tryptophan variants were used as reporter probes; Trp87 is fortuitously located at the active site and Trp167 is a surface tryptophan >25A from the catalytic center. The temperature dependence of the time-resolved Stokes shifts reveal that the active site Trp contains highly temperature-dependent relaxation dynamics while the surface Trp is virtually temperature-independent. Though no break is observed that correlates to catalysis, such results are discussed in the context of slower, more global dynamics as a major contributor to catalytic behavior. Using both single-Trp variants, Ala was then introduced to Tyr25 at the dimer interface and Val260 within the cofactor binding site. Such alterations were previously shown to either eliminate or exacerbate the magnitude of the temperature break, respectively [3,4]. The fluorescence dynamics reveal that the temperature-dependent dynamics at Trp87 are drastically altered relative to its wild-type counterpart, while Trp167 shows minor differences. Such results are discussed in the context of an altered conformational landscape that further corroborates an equilibration process occurring more slowly than the ps-timescale.[1] Kohen, A., et al (1999) Nature, 399, 496-499.[2] Liang, Z. X., et al (2004) PNAS, 101, 9556-9561.[3] Nagel, Z. D., et al (2011) PNAS, 108, 10520-10525.[4] Nagel, Z. D., et al (2013) JBC, 288, 14807-14097.