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. 2014 Oct 22;136(42):14821-33.
doi: 10.1021/ja506667k. Epub 2014 Oct 14.

Picosecond-resolved fluorescence studies of substrate and cofactor-binding domain mutants in a thermophilic alcohol dehydrogenase uncover an extended network of communication

Corey W Meadows  1 , Jonathan E Tsang, Judith P Klinman
Affiliations

Picosecond-resolved fluorescence studies of substrate and cofactor-binding domain mutants in a thermophilic alcohol dehydrogenase uncover an extended network of communication

Corey W Meadows et al. J Am Chem Soc. .

Abstract

Time-resolved fluorescence dynamics are investigated in two mutants of a thermophilic alcohol dehydrogenase (ht-ADH): Y25A (at the dimer interface) and V260A (at the cofactor-binding domain). These residues, ca. 32 Å apart, are shown to exhibit opposing low-temperature effects on the hydride tunneling step. Using single-tryptophan constructs at the active site (Trp87) and a remote, surface-exposed site (Trp167), time-dependent Stokes shifts and collisional quenching data allow an analysis of intra-protein dynamical communication. A double mutant, Y25A:V260A, was also inserted into each single-Trp construct and analyzed accordingly. None of the mutations affect fluorescence lifetimes, Stokes shift relaxation rates, and quenching data for the surface-exposed Trp167 to an appreciable extent. By contrast, fluorescent probes of the active-site tryptophan 87 reveal distinctive forms of dynamical communication. Stokes shifts show that the distal Y25A increases active-site flexibility, V260A introduces a temperature-dependent equilibration process not previously reported by such measurements, and the double mutant (Y25A:V260A) eliminates the temperature-dependent transition sensed by the active-site tryptophan in the presence of V260A. Collisional quenching data at Trp87 further show a structural change in the active-site environment/solvation for V260A. In the aggregate, the temperature dependencies of the fluorescence data are distinct from the breaks in behavior previously reported for catalysis and hydrogen/deuterium exchange, attributed to time scales for the interconversion of protein conformational substates that are slower and more global than the local motions monitored within. An extended network of dynamical communication between the protein dimer surface and substrate- and cofactor-binding domains emerges from the flourescent data.

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Figures

Figure 1
Figure 1
Structures of ht-ADH (PDB: 1RJW). Top panel: The structure is rotated to show the network of β-sheets (blue) connecting Tyr25 (red) at the dimer interface to the substrate-binding site Trp87 (red). In the crystal structure, Trp87 lies within van der Waals contact of the substrate analogue trifluorethanol (black) which coordinates to the catalytic Zn2+ (gray). Middle panel: Close-up of the cofactor-binding site and residues Leu176 (yellow) and Val260 (orange). Both residues lie within van der Waals of NAD+ (blue) in the docked structure. Image reproduced with permission from ref (37). Bottom panel: Monomeric structure of ht-ADH showing the relative positioning of both residues of interest, Tyr25 (magenta) and Val260 (blue), and the tryptophans used as probes (red).
Figure 2
Figure 2
Fluorescence transients of W87in:Y25A (left), W87in:V260A (middle), and W87in:Y25A:V260A (right) at each mutant’s steady-state peak emission wavelength at 30 °C. Each decay panel contains the instrument response function (black), the raw fluorescence counts (green) and the fit (red). The residuals associated with the number of exponentials used to fit the data are shown under each decay panel. The reduced χ2 values were 12.9, 1.4, and 1.1 for one, two, and three exponentials used to fit W87in:Y25A, respectively. For W87in:V260A, the values were 9.8 and 1.0 for one and two exponentials; for W87in:Y25A:V260A, these respective values were 9.7 and 1.0.
Figure 3
Figure 3
Fluorescence transients of W167in:Y25A (left), W167in:V260A (middle), and W167in:Y25A:V260A (right) at each mutant’s respective peak emission wavelength at 30 °C. The color scheme is the same as that used in Figure 2. The data for all mutants could be fit to two exponentials. The reduced χ2 values were found to be 1.7 and 1.1 for W167in:Y25A, 1.5 and 1.2 for W167in:V260A, and 1.8 and 1.1 for W87in:Y25A:V260A for one and two exponentials, respectively.
Figure 4
Figure 4
Temperature-dependent solvation correlation decays, c(t), of the time-dependent Stokes shift for all eight mutants studied. The W87in mutant series is shown in the left column and the W167in mutant series is shown in the right column. The temperatures shown are at 10 °C (black), 20 °C (blue), 30 °C (green), 40 °C (orange), and 50 °C (red). All fits are to a single exponential except for W167in. W87in and W167in are reproduced with permission from ref (26).
Figure 5
Figure 5
Arrhenius plots of the temperature-dependent Stokes shift relaxation rates extracted from Figure 4 and Table 4. Each panel contains a comparison between the W87in (red) and the W167in (blue) parent construct (upper left panel); the additional insertions are noted at the top of the panel. The data for the parent constructs are from ref (26).
Figure 6
Figure 6
Arrhenius plots of the temperature-dependent Stern–Volmer collisional quenching rate constants extracted from Figures S9 and S10 in the Supporting Information. Each panel contains the W87in (red) and W167in (blue) parent construct (upper left panel), with the additional mutation noted at the top of the remaining panels.
Figure 7
Figure 7
Residues within ht-ADH shown to be dynamically linked. (Top) Close-up of the network connecting Val260 (blue) in the cofactor-binding domain to Trp87 (red) in the substrate-binding domain. The numbers indicate the distance in angstroms between atoms connected to their respective residues. The modeled substrate, trifluorethanol, is abbreviated TFE. (Middle) Map of intra-subunit protein residues showing the relative distances among the single-Trp probes (red) at positions 87 and 167 relative to the catalytically relevant residues of Tyr25 (plum) and Val260 (blue). (Bottom) Birds-eye view of the tetrameric ht-ADH structure looking down on the crystal structure’s axis of symmetry. The circled Tyr25 π-stacking interaction (red sticks) belongs to both gold monomer subunits. Though intrasubunit distances between Tyr 25 and Trp167 are measured at ca. 44 Å (cf. Middle), the distance to Trp167 (orange sticks) on the opposite blue monomers is roughly 32 Å. For reference, the Trp87 (black sticks) is shown at a distance of 17 Å to Tyr25.
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