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Temperature dependence of protein motions in a thermophilic dihydrofolate reductase and its relationship to catalytic efficiency - PubMed

  • ️Fri Jan 01 2010

Temperature dependence of protein motions in a thermophilic dihydrofolate reductase and its relationship to catalytic efficiency

Olayinka A Oyeyemi et al. Proc Natl Acad Sci U S A. 2010.

Abstract

We report hydrogen deuterium exchange by mass spectrometry (HDX-MS) as a function of temperature in a thermophilic dihydrofolate reductase from Bacillus stearothermophilus (Bs-DHFR). Protein stability, probed with circular dichroism, established an accessible temperature range of 10 degrees C to 55 degrees C for the interrogation of HDX-MS. Although both the rate and extent of HDX are sensitive to temperature, the majority of peptides showed rapid kinetics of exchange, allowing us to focus on plateau values for the maximal extent of exchange at each temperature. Arrhenius plots of the ratio of hydrogens exchanged at 5 h normalized to the number of exchangeable hydrogens vs. 1/T provides an estimate for the apparent enthalpic change of local unfolding, DeltaH degrees (unf(avg)). Most regions in the enzyme show DeltaH degrees (unf(avg)) < or = 2.0 kcal/mol, close to the value of kT; by contrast, significantly elevated values for DeltaH degrees (unf(avg)) are observed in regions within the core of protein that contain the cofactor and substrate-binding sites. Our technique introduces a new strategy for probing the temperature dependence of local protein unfolding within native proteins. These findings are discussed in the context of the demonstrated role for nuclear tunneling in hydride transfer from NADPH to dihydrofolate, and relate the observed enthalpic changes to two classes of motion, preorganization and reorganization, that have been proposed to control the efficiency of hydrogenic wave function overlap. Our findings suggest that the enthalpic contribution to the heavy atom environmental reorganizations controlling the hydrogenic wave function overlap will be dominated by regions of the protein proximal to the bound cofactor and substrate.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Peptic fragments of Bs-DHFR. (A) Eleven nonoverlapping fragments of Bs-DHFR generated after proteolysis by pepsin at pH 2.4. (B) The pepsin-generated fragments are mapped onto the X-ray structure of Bs-DHFR (PDBID:1ZDR), according to colors in (A). (C) X-ray structure of Ec-DHFR, indicating bound dihydrofolate (DHF) (rose) and cofactor NADPH (blue).

Fig. 2.
Fig. 2.

HDX time courses of Bs-DHFR peptides as a function of temperature. Extent of deuteration in peptides of Bs-DHFR, after incubating protein in D2O for 10 s–5 h at 10 (green), 25 (orange), 35 (black), 40 (purple), 50 (red), and 55 (blue). The top of each graph indicates the maximal number of exchangeable amides corrected to 100% D2O.

Fig. 3.
Fig. 3.

Regional variations in HDX temperature effects. (A) HDX at t = 300 min and 10 °C shows high solvent exchange in the adenosine-binding subdomain and αB of Bs-DHFR. In contrast, most of the loop subdomain including M20, FG and GH loops shows intermediate accessibility to solvent, and there is strong protection from exchange in the protein core (βA and βF). (B) At 55 °C, deuteration increases, reaching maximal levels in parts of the loop subdomain, although still submaximal in the M20, FG, and GH loops. The largest temperature dependence of deuteration occurs in βA and αF.

Scheme 1.
Scheme 1.

Schematic illustrating the temperature-dependent disruption of local secondary structures within a native folded protein. According to this model, temperature affects not only the equilibrium constant, Kopen, but also the structure of the opened state of protein. T1 is the lowest initial temperature and T2, an elevated temperature.

Fig. 4.
Fig. 4.

Regional variations in the enthalpy of HDX. Plots of ln (Nt,t=300 min/N∞) vs. 1/T show the linear dependence, with formula image indicated in the frame of each peptide.

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