Designing thermal stability via non-equilibrium simulations

The design of protein thermal stability is appealing for practical uses. In a recent work [1] by Tian, Woodard, Whitney and Shakhnovich [see here] non-equilibrium Monte Carlo simulations were effectively used to explore mutations of the Dihydrofolate Reductase (DHFR) and their impact on both the stability and functionality of the enzyme. 

The key point of the work is the use of non-equilibrium Monte Carlo (MC) simulations. A protein is excited at different temperatures and its "unfolding " as function of MC steps is recorded. 

For each temperature, the average value of a given observable or order parameter that describes the state of the protein, ie the root mean square displacement with respect to the native state, the energy, the gyration radius, depends on the simulation length -in the specific case the number of MC steps. This relates to the fact that the transition from the folded to the unfolded state, for a given temperature, is rate limited by the free energy barrier dividing the two states. How this dependence can be washed up when considering the effect of mutations? 

The authors proved a nice recipe: first, a mutation affects the thermodynamics of the system, formally the free energy difference between folded and unfolded state, but also the kinetics for the folded/unfolded transition, aka the free energy barrier dividing the two state. It is possible to image that the thermodynamic effect is mirrored on the change of free energy barrier via a scaling factor that measures how the mutation influences the transition state of the folding/unfolding process. Secondly, when considering the non-equilibrium MC simulations for both the wild type and the mutant, the shift of the apparent melting temperature (the temperature leading unfolding) of the mutated system with respect to WT results independent from the simulation length. This can be formally showed, and the reader is invited to dig the work.

Using this strategy several mutations stabilizing the protein and that maintain functionality were identified. I wonder whether this approach can be used straightforwardly also for estimating the effect of mutations on mechanical stability.

Schematic view of the free energy profile for the folded (N) unfolded (U) states as it is  pictured in Fig. 1 of Ref. 1

[1] J. Tian, JC Woodard, A. Whitney, EI Shakhnovich, Plos Comp Bio (2015) 11, e1004207.

A conserved structural element is a kinetic modulator of nucleotide exchange in the EF-Tu

A recent publication [1] reports that the P-loop, a conserved structural element in the catalytic domain of many different NTP-ases, might participate in modulating the nucleotide exchange rates for a broad class of NTP-ases.

The study was performed in detail on the EF-Tu protein, which catalyzes the GTP/GDP hydrolysis. Results show that the internal dynamics of the P-loop does not affect the nucleotide exchange rates, but rather P-loop forms a P-loop anchor via hydrogen bonds with another structural element of the protein, helix C. The P-loop anchor contributes to the activation entropy of the nucleotide exchange,  and consequently modulates the nucleotide-binding kinetics. 

Presently, two classes of P-anchors have been identified, depending on the nature of stabilization of the anchor, one class is hydrogen-bond stabilized, while the other is stabilized by the hydrophobic effect. The finding is consistent with the natural mechanism of the nucleotide exchange on the EF-Tu, where the nucleotide is exchanged upon the binding of another protein (EF-Ts) that disrupts the stability of the P-anchor, making the P-loop more flexible. The larger flexbility of the P-loop increases the entropic contribution to the activation free energy, resulting in faster nucleotide dissociation.

[1] Mercier E., Girodat D., Wieden H.-J., A conserved P-loop anchor limits the structural dynamics that mediate nucleotide dissociation in EF-Tu, JA  - Sci. Rep., 2015.

And yet it functions!

A nice paper adressing the issue of how mesophilic/thermophilic enzymes functions at differents temperatures is just out in Biochemistry [see here].

The work by the group of EA Eisenmesser focuses on the behavior of the cyclophilin enzyme from Geobacillus kaustophililus (GeoCyp), a bacterium found in the the deep see sediment of the Mariana Trench,  and compared to its mesophilic homologous from humans (CypA). The study demonstrates that, at variance with other mesophilic/thermophilic pair, here, the thermophile maintains up to 70% of its catalytic power at low temperature where most of thermophiles do not function or have very limited activity. We have already posted on the "corresponding state principle", introduced to explain why most of thermophiles lack activity at ambient conditions. According to this view, the lack of activity is due to the enhanced rigidity of the protein matrix which compromises mobility essential to the catalytic turn-over. At the same time mechanical rigidity is postulated as the source of the enhanced stability of the protein and its resistance to thermal stress. The universality of this principle has been questioned by showing that in many cases thermophiles can be as flexible as their mesophilic variants at the same thermodynamic conditions, thus stability is the results of a smaller entropy penalty between folded (less entropic) and unfolded (more entropic) states. 

According to the work by Eisenmesser and coworkers, the thermophilic GeoCyp is highly similar from the structural point of view to the human CypA, and its dynamics at different timescales is comparable even if its mobility seems more sensitive to temperature increases. What probably causes the 30% drop of activity at low temperature (10°C) with respect to activity at its optimal temperature (60°C) is a reduced local motion of binding-site loop, which gating is affected by the presence of a charged amino-acid, and a slightly less strong electric field measured at the level of the catalytic site and supposed to ease the isomerization of the peptide bond. In summary, this study shows us another deviation from the common believe based on the observation of reduced thermophilic activity at low temperatures. Nice work!