Most biochemists have had the “pleasure” of working with proteins that require cool atmospheres and a comfy solvent to keep them temporarily happy (until they randomly decide to aggregate into protein snot). Rubredoxin from the organism Pyrococcus furiosus, on the other hand, is like an old Jeep that keeps on working despite repeated abuse and temperature fluctuations.
P. furiosus was initially discovered near a deep sea volcanic vent. This hyperthermophilic archae is an anaerobe that grows optimally around 370K (or 100 °C). Rubredoxin, as seen in Figure 1, is a small 53 amino acid protein and has a high spin state iron that is coordinated between four cysteine residues. It is responsible for electron transfer reactions, although the specific reactions it participates in is still unknown. Early research found that rubredoxin maintains its globular or tertiary structure up to 473K.
Clostridium pasteurianum, a mesophilic organism, also contains a rubredoxin (CpRd) that is highly similar to PfRd. CpRd and PfRd are studied side-by-side usually to understand folding/unfolding mechanisms and how enzymes attain thermostable characteristics. Since the sequence similarity is high and folding is synonymous, this must mean that PfRd’s hyperthermostability is attained through other forces.
Cavagnero and coworkers took on the initial task of understanding what factor(s) would lead to PfRd unfolding. A second question they tried to answer was whether ion pairs (where negatively and positively charged amino acids interact) were vital for thermostability. Tryptophan fluorescence, circular dichroism (CD), 1D nuclear magnetic resonance (NMR), and UV-Vis were used to determine if secondary and/or tertiary structural changes occurred. There were no observed differences in PfRd when the pH was dropped from 7 to 2 or when a high level of salt was present in solution at room termperature, which normally disrupts important hydrogen bonds or charge-charge interactions. An ANS (1-anilinonaphthalene-8-sulfonic acid) assay probes for solvent exposed hydrophobic residues, showing that the protein is about to begin the unfolding process. Interestingly, at pH 2 there is a high amount of ANS binding, showing that very small internal changes occur at low pH. Thermal denaturation of PfRd at pH 2.0 was performed next to see if refolding occurs. The melting temperature (Tm) had been decreased from 473K to 340K, and they found that refolding after unfolding was unable to happen (that is, unfolding was not reversible). Using GRASP, surface potentials of rubredoxin were calculated to observe differences between the hyperthermophilic PfRd and mesophilic CpRd, which are seen in Figure 2. Both structures showed the presence of ion pairs, but the CpRd displayed a more even distribution of negative charge. While they were unable to pin point what factors allowed for high temperature stability, they were able to eliminate ion pairing as a key component to hyperthermostability.
This group shifted gears in a later paper to understand the unfolding mechanism of PfRd. Using tryptophan fluorescence, PfRd unfolding at temperatures from 63.3 °C to 77.8 °C was studied and then compared to the unfolding of CpRd. CpRd’s data modeled a standard monoexponential curve where only a single intermediate would be plausible. PfRd’s curves modeled an initial sharp fluorescence increase followed by a slow decrease, which presents the idea for a biexponential mechanism of unfolding. CD data confirmed the biexponential model of unfolding for PfRd. Utilizing these fluorescence, CD, as well as previously published UV-Vis data, Cavagnero and coworkers propose the following mechanism of PfRd unfolding:
Native state <–> Light relaxation of secondary structure (I1) <–> Loss of Fe ion (I2) <–> Exposure of hydrophobic core to the solvent (I3) <–> Unfolded state –> Aggregation
We now have a working theory as to how the protein unfolds, but this does not tell us what is allowing for the thermostability of PfRd. Borreguero and colleagues explore the motions of amino acids in PfRd as a correlation of temperature utilizing quasielastic neutron scattering (QENS) and molecular dynamics (MD) simulations. In their first experiment, they notice that as temperature increases, elastic intensities (indicative of high frequency, localized vibrations) decrease and quasielastic intensities (representative of low frequency motions) increase. This shift occurs around the dynamic transition temperature (TDT). These data were then used to determine the radius of the methyl groups. Their calculations displayed an expected increase once temperatures were above the TDT, but were much smaller than previously studied structures. They concluded that this might either be a characteristic of or the cause of thermal stability in PfRd. Figure 3 shows important residues at the active site of PfRd. Ile7 and Ile40 are responsible for modulating iron’s redox potential by shielding the iron from the solvent and providing backbone hydrogens to the iron-coordinated sulfur atoms in the four cysteines. This observation led to the MD simulation of the electrostatic force vectors exerted on PfRd’s iron ion. When temperatures were below the TDT, the vectors point to the left towards Ile7 and Cys41. Once temperatures are above the TDT, the force vectors point 180° in the opposite direction towards Tyr10 and Ile40. This shift is proposed to be due to anharmonic modes, which contain a high amount of atomic displacement. Data from boson peaks were then used to calculate mean square displacement (MSD) of each hydrogen in PfRd. Ile7 and Ile40 were confirmed to be important residues from their contribution to the boson peak and by their calculated MSD, which identifies a new method for identifying key amino acids to protein function.
Even though the main question has not been directly answered, many other problems have been answered. A previous belief that ion pairs were the reason for thermostability has been dismantled; unfolding intermediates for PfRd were proposed; Ile7 and Ile40 have been shown to have key roles in PfRd’s continued function at high temperatures; a novel method for locating critical residues in a protein has been found. This brief overview on P. furiosus rubredoxin is proof that an inability to answer a primary question does not mean vital, new information can not be distilled from collected data.
Cavagnero S., Zhou Z.H., Adams M.W.W. & Chan S.I. (1995). Response of Rubredoxin from Pyrococcus furiosus to Environmental Changes: Implications for the Origin of Hyperthermostability, Biochemistry, 34 (31) 9865-9873. DOI: 10.1021/bi00031a007
Cavagnero S., Zhou Z.H., Adams M.W.W. & Chan S.I. (1998). Unfolding Mechanism of Rubredoxin from Pyrococcus furiosus, Biochemistry, 37 (10) 3377-3385. DOI: 10.1021/bi9721804
Borreguero J.M., He J., Meilleur F., Weiss K.L., Brown C.M., Myles D.A., Herwig K.W. & Agarwal P.K. (2011). Redox-Promoting Protein Motions in Rubredoxin, The Journal of Physical Chemistry B, 115 (28) 8925-8936. DOI: 10.1021/jp201346x