Theoretical and experimental studies have firmly established that protein folding can be described by a funneled energy landscape. This funneled energy landscape is the result of foldable protein sequences evolving following the principle of minimal frustration, which allows proteins to rapidly fold to their native biologically functional conformations. For a protein family with a given functional fold, the principle of minimal frustration suggests that, independent of sequence, all proteins within this family should fold with similar rates. However, depending on the optimal living temperature of the organism, proteins also need to modulate their thermodynamic stability. Consequently, the difference in thermodynamic stability should be primarily caused by differences in the unfolding rates. To test this hypothesis experimentally, we performed comprehensive thermodynamic and kinetic analyses of 15 different proteins from the thioredoxin family. Eight of these thioredoxins were extant proteins from psychrophilic, mesophilic, or thermophilic organisms. The other seven protein sequences were obtained using ancestral sequence reconstruction and can be dated back over 4 billion years. We found that all studied proteins fold with very similar rates but unfold with rates that differ up to three orders of magnitude. The unfolding rates correlate well with the thermodynamic stability of the proteins. Moreover, proteins that unfold slower are more resistant to proteolysis. These results provide direct experimental support to the principle of minimal frustration hypothesis.protein folding | protein stability | protein evolution T he energy landscape theory provides a conceptual physicochemical framework for understanding protein folding. This theory is based on the principle of minimal frustration that ". . .quantifies the dominance of interactions stabilizing the specific native structure over other interactions that would favor nonnative, topologically distinct traps" (1). A consequence of this is that the folding energy landscape of naturally occurring proteins is funnel-shaped (1-22). The shape of this funnel depends on two main factors that can introduce frustration and roughness: topology and the extent of nonnative interactions. Topological frustration can occur when certain native interactions are formed too early and need to be undone to allow for other interactions to form first, leading to backtracking and/or cracking (23-28). Weak nonnative interactions can have complex effects on the folding landscape (29-31): small amounts of weak nonnative interactions can assist folding, whereas larger amounts can create internal friction that will slow folding (32-35). For a given protein fold, both topological and energetic frustrations will depend on the amino acid sequence. Theoretical studies have suggested that naturally occurring proteins have selected sequences that are compatible with the principle of minimal frustration (36).The goal of this study is to experimentally test the evolutionary validity of the principle...

Gene transcription is catalyzed by DNA-dependent RNA polymerases (RNAPs) in a multistep regulated process (1)(2)(3)(4). Multi-subunit and single-subunit RNAPs catalyze transcription initiation by a series of events -(i) promoter-specific recognition, (ii) formation of a transcription bubble, (iii) RNA synthesis accompanied by DNA scrunching, and (iv) aborted synthesis of short RNA transcripts or transition to the elongation phase with promoter release. Understanding the mechanism of transcription initiation requires capturing key intermediate states of RNAP complexes and characterizing them biochemically and structurally. Mitochondrial DNA is transcribed by single-subunit RNAPs (mtRNAP), which unlike their phage counterparts, depend on one or more transcription factors for promoter-specific transcription initiation. Much of our understanding of mitochondrial DNA transcription comes from studies of yeast (S. cerevisiae) and human mtRNAPs (2,3,5). The yeast mtRNAP transcription initiation complex (y-mtIC) is comprised of the catalytic subunit RPO41 and a transcription factor MTF1. The human mtRNAP transcription initiation complex (h-mtIC) comprises of POLRMT and two transcription factors. The h-mtIC has been structurally characterized by crystallography (6), but the structure was captured in an inactive fingersclenched state with a major part of the transcription bubble disordered. Hence, the structural basis for promoter melting, DNA scrunching, and transcription initiation remains largely unknown for the mtRNAPs. RPO41 (∆N100) and MTF1 were assembled on a pre-melted promoter (-21 to +12, 15S yeast mtDNA promoter; Fig. 1A) to generate the yeast mitochondrial transcription preinitiation complex (y-mtPIC) (Fig. S1). The y-mtPIC was incubated with pppGpG RNA and a . CC-BY-NC: bioRxiv preprint 4 non-hydrolysable UTPaS to generate the y-mtIC poised to incorporate the +3 NTP. Singleparticle cryo-EM data analysis of the quinary y-mtIC revealed a surprising coexistence of PIC and IC states in equilibrium (Fig. S2). The y-mtIC structure had bound RNA and NTP, and y-mtPIC structure had no RNA or NTP. Another dataset collected from a PIC-only grid extends the resolution to 3.1 Å. Key steps guiding transcription initiation are revealed from the 3.1 Å y-mtPIC and 3.7 Å y-mtIC structures.In y-mtPIC structure ( Fig. 1B & C), the MTF1 is traced from 2 to 336 out of 341 amino acid residues, RPO41 is traced from 386 to the end residue 1351 with few disordered regions, and unambiguously traced DNA (Fig. S3A). A stable transcription core is composed of RPO41, MTF1, and transcription bubble (Fig. 1C). RPO41 interacts with MTF1 at multiple locations. Two RPO41 b-hairpins -the intercalating hairpin (ICH) and the MTF1supporting hairpin (K613-P632) form a crescent-shaped platform that accommodates the C-terminal domain of MTF1 (252-325) (Fig. 1D). The N-terminal domain of MTF1 (2-251) contacts the tip of the RPO41 thumb helix (Fig. 1E); biochemically, we show the interaction stabilizes RPO41-MTF1 complex ( Fig. S3C-D)(7). The MTF1-supp...

Highlights d Structure of a partially melted intermediate reveals the promoter melting mechanism d RNA synthesis scrunches the non-template DNA strand into a loop structure d The flexible C-tail of the transcription factor MTF1 stabilizes the scrunched DNA d The co-existence of scrunched and unscrunched states explains abortive synthesis

Mitochondrial RNA polymerase (mtRNAP) is crucial in cellular energy production yet, understanding of mitochondrial DNA transcription initiation lags that of bacterial and nuclear DNA transcription. We report structures of two transcription initiation intermediate states of yeast mtRNAP that explain promoter melting, template alignment, DNA scrunching, abortive synthesis, and transition into elongation. In partially-melted initiation complex (PmIC), transcription factor MTF1 makes base-specific interactions with flipped non-template (NT) -4 to -1 nucleotides 'AAGT' of the DNA promoter. In the initiation complex (IC), the template in the expanded 7-mer bubble positions the RNA and NTP analog, UTP S, while NT scrunches into an NT-loop. The scrunched NT-loop is stabilized by centrally positioned MTF1 C-tail. The IC and PmIC states coexist in solution revealing a dynamic equilibrium between two functional states. Frequent scrunching/unscruching transitions, and the imminent steric clashes of inflating NT-loop and growing RNA:DNA with the C-tail explain abortive synthesis and transition into elongation.

Hydrostatic pressure has a vital role in the biological adaptation of the piezophiles, organisms that live under high hydrostatic pressure. However, the mechanisms by which piezophiles are able to adapt their proteins to high hydrostatic pressure is not well understood. One proposed hypothesis is that the volume changes of unfolding (ΔVTot) for proteins from piezophiles is distinct from those of nonpiezophilic organisms. Since ΔVTot defines pressure dependence of stability, we performed a comprehensive computational analysis of this property for proteins from piezophilic and nonpiezophilic organisms. In addition, we experimentally measured the ΔVTot of acylphosphatases and thioredoxins belonging to piezophilic and nonpiezophilic organisms. Based on this analysis we concluded that there is no difference in ΔVTot for proteins from piezophilic and nonpiezophilic organisms. Finally, we put forward the hypothesis that increased concentrations of osmolytes can provide a systemic increase in pressure stability of proteins from piezophilic organisms and provide experimental thermodynamic evidence in support of this hypothesis.

There is a growing interest in understanding how hydrostatic pressure (P) impacts the thermodynamic stability (ΔG) of globular proteins. The pressure dependence of stability is defined by the change in volume upon denaturation, ΔV = (∂ΔG/∂P)T. The temperature dependence of change in volume upon denaturation itself is defined by the changes in thermal expansivity (ΔE), ΔE = (∂ΔV/∂T)P. The pressure perturbation calorimetry (PPC) allows direct experimental measurement of the thermal expansion coefficient, α = E/V, of a protein in the native, αN(T), and unfolded, αU(T), states as a function of temperature. We have shown previously that αU(T) is a nonlinear function of temperature but can be predicted well from the amino acid sequence using α(T) values for individual amino acids (J. Phys. Chem. B 2010, 114, 16166-16170). In this work, we report PPC results on a diverse set of nine proteins and discuss molecular factors that can potentially influence the thermal expansion coefficient, αN(T), and the thermal expansivity, EN(T), of proteins in the native state. Direct experimental measurements by PPC show that αN(T) and EN(T) functions vary significantly for different proteins. Using comparative analysis and site-directed mutagenesis, we have eliminated the role of various structural or thermodynamic properties of these proteins such as the number of amino acid residues, secondary structure content, packing density, electrostriction, dynamics, or thermostability. We have also shown that αN(T) and EN,sp(T) functions for a given protein are rather insensitive to the small changes in the amino acid sequence, suggesting that αN(T) and EN(T) functions might be defined by a topology of a given protein fold. This conclusion is supported by the similarity of αN(T) and EN(T) functions for six resurrected ancestral thioredoxins that vary in sequence but have very similar tertiary structure.

Summary In yeast mitochondria, transcription initiation requires assembly of mitochondrial RNA polymerase and transcription initiation factor MTF1 at the DNA promoter initiation site. This protocol describes the purification of the component proteins and assembly of partially melted and fully melted initiation complex states. Both states co-exist in equilibrium in the same sample as seen by cryoelectron microscopy (cryo-EM) and allow elucidation of MTF1’s structural roles in controlling the transition into elongation. We further outline how analysis of the complex by light scattering, thermal shift assay, and ultrafiltration assay exhibits reproducible results. For complete details on the use and execution of this protocol, please refer to De Wijngaert et al. (2021) .

The letter by Candel et al. (1) does not address a potential problem with their own experimental set-up. In particular, the use of guanidinium hydrochloride (GdnHCl) is not the best choice of denaturant for studying a folding reaction. There is an ample amount of evidence to suggest that the variations in the ionic strength (with GdnHCl being an ionic compound) can lead to unexpected effects (2), including an increase in protein stability at low denaturant concentration (3-6) and changes in apparent kinetics of folding (7,8). Therefore, we performed our studies using urea (9).Moreover, Candel et al. (1) do not mention their previously published results with the Escheria coli thioredoxin, using urea as a denaturant (see figure 7 of ref. 10), which does not show rollover behavior. This provides additional compelling evidence that the use of GdnHCl can lead to erroneous results.Whereas indeed our folding-unfolding experiments were performed at pH 2 (to observe the transition in urea-induced unfolding for thioredoxins with higher stability), we have provided sufficient evidence that the general features observed at pH 2 are consistent with those at neutral pH. The first piece of evidence is that the stabilities obtained from kinetic and equilibrium urea-induced folding-unfolding experiments are very similar [see figure 3 in Tzul et al. (9)]. The second piece of evidence is that the transition temperatures at pH 2 and pH 7 are highly correlated, suggesting that pH has similar effects on the stabilities of these proteins [see figure S1 in Tzul et al. (9)]. The third piece of evidence is that the proteolytic resistance, which can be used as a proxy for the unfolding rate, of thioredoxins at pH 2 and pH 7, correlates well with the unfolding rates [see figure 4 in Tzul et al. (9)]. Taken together, these data suggest that the behavior observed at pH 2 can be extrapolated to pH 7.The thorough experimental data of our report (9) is fully consistent with the hypothesis of the principle of minimal frustration. We have provided a substantial dataset of thioredoxin variants other than the two variants Candel et al. (1) have called into question. These additional 13 thioredoxin variants strongly support the trend seen from both extant and ancestral thioredoxin groupings. The main focus of our publication (9) is the evolutionary kinetic behavior of the thioredoxin protein family. Although we acknowledge there may be alternative explanations to our data, the principle of minimal frustration is a most consistent explanation and we fully stand by this conclusion.