Selection for Protein Kinetic Stability Connects Denaturation Temperatures to Organismal Temperatures and Provides Clues to Archaean Life

<div><p>The relationship between the denaturation temperatures of proteins (T_m values) and the living temperatures of their host organisms (environmental temperatures: T_ENV values) is poorly understood. Since different proteins in the same organism may show widely different T_m’s, no simple universal relationship between T_m and T_ENV should hold, other than T_m≥T_ENV. Yet, when analyzing a set of homologous proteins from different hosts, T_m’s are oftentimes found to correlate with T_ENV’s but this correlation is shifted upward on the T_m axis. Supporting this trend, we recently reported T_m’s for resurrected Precambrian thioredoxins that mirror a proposed environmental cooling over long geological time, while remaining a shocking ~50°C above the proposed ancestral ocean temperatures. Here, we show that natural selection for protein kinetic stability (denaturation rate) can produce a T_m↔T_ENV correlation with a large upward shift in T_m. A model for protein stability evolution suggests a link between the T_m shift and the <i>in vivo</i> lifetime of a protein and, more specifically, allows us to estimate ancestral environmental temperatures from experimental denaturation rates for resurrected Precambrian thioredoxins. The T_ENV values thus obtained match the proposed ancestral ocean cooling, support comparatively high Archaean temperatures, and are consistent with a recent proposal for the environmental temperature (above 75°C) that hosted the last universal common ancestor. More generally, this work provides a framework for understanding how features of protein stability reflect the environmental temperatures of the host organisms.</p></div

Estimating ancestral environment temperatures from the unfolding rates of laboratory resurrected Precambrian thioredoxins.

<p>Environment temperatures are estimated from the unfolding rate profiles of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156657#pone.0156657.g009" target="_blank">Fig 9C</a> by assuming specific values for the denaturation half-life (τ = 1/k_U). (A) A τ value (3.8 days) equal to the denaturation half-life of <i>E</i>. <i>coli</i> thioredoxin is assumed. (B) A one day—two month interval is assumed for τ and the corresponding intervals for the calculated environmental temperature are shown as vertical lines. (C) A one hour—one year interval is assumed for τ and the corresponding intervals for the calculated environmental temperature are shown as vertical lines. In the three panels, the denaturation temperatures for elongation factors and thioredoxins are included, as well as the known estimates of the environmental temperatures derived from the oxygen isotopic composition of cherts. The dashed horizontal line at about 4 billion years (labeled “LUCA”) represents a recent minimum estimate (~75°C) for the environment temperature of the last universal common ancestor [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156657#pone.0156657.ref001" target="_blank">1</a>].</p

Correlation between the denaturation temperature and the logarithm of the unfolding rate constant at 37°C for all thioredoxins from our current and previous [18] work.

<p>Correlation between the denaturation temperature and the logarithm of the unfolding rate constant at 37°C for all thioredoxins from our current and previous [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156657#pone.0156657.ref018" target="_blank">18</a>] work.</p

Kinetic parameters for the <i>in vitro</i> denaturation of <i>E</i>. <i>coli</i> thioredoxin and several resurrected Precambrian thioredoxins.

<p>Kinetic parameters for the <i>in vitro</i> denaturation of <i>E</i>. <i>coli</i> thioredoxin and several resurrected Precambrian thioredoxins.</p

Simulations on the response of protein denaturation temperature to changes in environmental temperatures under conditions of threshold selection for kinetic stability.

<p>The simulation starts with the final state of the T_ENV = 37°C and τ* = 10⁴ min of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156657#pone.0156657.g005" target="_blank">Fig 5</a>. However, after 5000 simulation steps, the T_ENV value is increased to 90°C and, after 15000 simulation steps, it is decreased to 70°C. Profiles of denaturation temperature, degradation half-life, organismal fitness and unfolding rate constant are shown.</p

Thermal denaturation of the laboratory representation of the thioredoxin corresponding to the LPBCA node (see Fig 1) as determined by differential scanning calorimetry (DSC).

<p>(A) DSC profiles for LPBCA denaturation at several scan rates. Dotted lines represent the profiles obtained in the reheating runs of the experiments at 240, 120 and 60 degrees per hour. (B) Plot of calorimetric to van’t Hoff enthalpy ratio versus one over scan rate. (C) Plot of denaturation temperature (defined here as that corresponding to the maximum of the heat capacity profile) versus one over scan rate. The lines in panels B and C are the best fits of second-order polynomials and are used to extrapolate to infinite scan rate (1/v = 0).</p

Experimental determination of the temperature-dependent unfolding rate constants for <i>E</i>. <i>coli</i> thioredoxin and several laboratory resurrections of Precambrian thioredoxins (Fig 1).

<p>Guanidine-induced denaturation experiments were performed at several temperatures and guanidine concentration for each thioredoxin variant. Values of the unfolding rate constants (k_U) were derived from the fits of exponential functions to the corresponding fluorescence profiles. (A) Linear extrapolation to zero guanidine concentration to obtain the values of rate constants at lower temperatures. (B) Constant-ΔG extrapolation to obtain the values of the rate constants at higher temperatures. For illustration, we have included in panels A and B data for <i>E</i>. <i>coli</i> thioredoxin and LBCA thioredoxin. Data for the other thioredoxins studied are included in the Supporting Information. (C) Profiles of unfolding rate constants (values obtained using the extrapolation procedures illustrated in panels A and B) versus temperature for all the thioredoxins studied in this work. Continuous lines are the best fits of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156657#pone.0156657.e013" target="_blank">Eq 13</a> to the data.</p

Simulations on the evolution of protein denaturation temperature under selection for thermodynamic stability only.

<p>All simulations assume an environmental temperature of 37°C and use as starting denaturation parameters those corresponding to <i>E</i>. <i>coli</i> thioredoxin (including a denaturation temperature value of 89°C which is about 50 degrees higher than the assumed environmental temperature). Upper panel: simulations performed using different values for the threshold value of the unfolding equilibrium constant. Lower panel: final values of the denaturation temperature obtained in the simulations shown in the upper panel versus the threshold K* values used.</p

Simple estimation of ancestral environmental temperatures from the denaturation temperatures of resurrected Precambrian proteins.

<p>The color of the data symbols refers to the different protein families studied and is the same as that used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156657#pone.0156657.g001" target="_blank">Fig 1</a>.</p

Illustrative example of a simulation on the evolution of protein denaturation temperature under threshold selection for both thermodynamic stability and kinetic stability.

<p>The simulation shown assumes an environmental temperature of 37°C and uses as starting state the final state of the K* = 10⁻² and τ* = 0 simulation in the upper panel of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156657#pone.0156657.g003" target="_blank">Fig 3</a>. (A) τ* is kept at a zero value for 5000 simulation steps and then it is increased to impose a threshold for degradation half-life of 10⁴ min. (B) Upon τ* increase to 10⁴ min, mutations that decrease the unfolding rate quickly accumulate to bring the degradation half-life above the threshold. (C) The mutations that decrease the unfolding rate, also decrease the unfolding equilibrium constant which, as a result, falls order of magnitude low the (assumed constant in this simulation) K* threshold; (D) The decrease in unfolding equilibrium constant is reflected in an increase in equilibrium denaturation temperature, as expected from the van’t Hoff equation and the endothermic character of protein unfolding. This increase in denaturation temperature reflects exclusively the threshold for kinetic stabilization, as K remains orders of magnitude below K* once τ* = 10⁴ min has been imposed (therefore, changing K* within reasonable limits would not affect the denaturation temperature value).</p