Stay flexible, Stay stable...

For some time thermophilic proteins have been considered more rigid than their mesophilic homologues. The "rigidity paradigm" was introduce to explain both the extreme stability of thermophiles as well as their lack of activity at ambient conditions. According to this view functionality is then recovered at the high optimal growth temperature because of the activation of the protein flexibility. Experimentally, one of the strongest support to this "corresponding states" picture comes from H/D exchange experiments, see this beautiful comment from R. Jaenicke in PNAS(2000). However, recent works using the H/D exchange, Neutron Scattering and NMR techniques have questioned the paradigm. Inspired by this querelle we have used extensive MD simulations to play with the concept for a model system, a pair of homologous G-domains of different stability content. The paper is just out in JPCB(2013). Quite surprisingly for our system we see that the hyperthermophilic protein show comparable and even enhanced flexibility than the mesophilic less stable variant. The more intriguing feature pops up when the global flexibility is considered. The conformational spaces sampled by the proteins were projected on a reduced network of linked kinetic separated states; and the hyperthermophiles is systematically characterised by a larger number of conformational substates! This flexibility (conformational entropy) is proposed to stabilise the protein by broadening the stability curve and consequently raising the melting temperature. I will get back on this........

Graphical Abstract, JPCB (2013), 117 (44), pp 13775–13785. 

Ideal proteins?

How do proteins form or maintain a unique fold, stable and biologically preferred when at the same time the unfolded or misfolded states are the vast majority of the possible conformations? We know that the information of the three dimensional structure of a protein is encoded in its amino acid sequence (Anfinsen's dogma) [1]. In particular, the funneled energy landscape approach prescribes that amino-acid sequences tend to choose the three dimensional structure that minimizes their free energy. But not everybody embraces the folding-funnel approach, especially since it has been well known that for most proteins the free energy difference between the folded and the unfolded states is only marginally negative [2]. At the same time the protein folding problem has been suggested as an NP-complete one [3], or otherwise no fast solution to it is known, while at the same time nature copes with it very efficiently in biological systems.

So the question remains. Or doesn’t it?
It seems that, through the field of protein design, the recent approach of Baker and colleagues [4] gave a big push towards the answer. The ansatz was: forget about the sequence; there must be a mapping or a relation between specific secondary structure patterns and tertiary structure motifs. Indeed, the authors formulated concrete, unambiguous rules connecting the alternation and length of 2 or 3 secondary structure elements with their supersecondary structure. Specifically, they first defined the notion of chirality (left or right) for the motif βlβ and the notion of orientation (parallel or antiparallel) for the motifs βlα and αlβ, where β, α and l stand for beta, alpha and loop respectively. They then gave the three following fundamental rules. 
1) βlβ rule: the chirality of β-hairpins is determined by the length of the loop between the two strands. Two- and three-residue loops almost always give rise to left-hairpins, whereas five-residue loops give rise primarily to right-hairpins. 
2) βlα rule: The preferred orientation of βlα-units is parallel for two-residue loops and antiparallel for three-residue loops.
3) αlβ rule: The preferred orientation of αlβ-units is parallel. 
At a next level of complexity, from these 3 fundamental rules follow 4 emergent rules concerning βlβlα-, αlβlβ- or βlαlβ-units. Their validation included both Rosetta folding simulations of sequence-independent backbone models as well as analysis of motifs in known protein structures. Both approaches were in agreement with each other. More notably, the authors, following strictly these rules, designed ab initio five different protein folds that exhibited extraordinary thermal stability reaching melting temperatures greater than 95 C. They thus called these models “ideal”.

What triggered a subsequent, recent work by L. Vitagliano and coworkers [5], was the fact that naturally occurring proteins might not follow the rules as strictly as in the above designing, they are however - even if marginally - stable. The overall analysis of crystal structures from the thermophiles Thermotoga maritima, the genus Pyrococcus and the genus Sulfolobus, revealed an adherence to the above rules, with the notable over-representation of the βlβ-l2 (i.e. two-residue loop), a state with exclusive preference for left-chirality.So could the adherence to those rules be driving an evolutionary selection for thermostable proteins? It is possible. Let’s not forget at this point that there is no divine hand defining these rules. As the authors of [4] note, they follow from either minimization of torsional strain or backbone bendability. And this is also the reason why proteins that follow these rules not only have stable native states but also unstable non-native states, a fact partially responsible for the funnel-shaped resulting energy landscapes. A question that naturally arises from Baker’s group's result concerns the functionality of the designed proteins at ambient temperature or not. It is either outside the scope their work or it is implicitly assumed that since one can drive the fold (and decide of course on the sequence) he can design proteins with the desired function. But protein function requires the appropriate, or let’s say the perfect, amount of flexibility. It was very nicely demonstrated by Hans Frauenfelder and co-workers [6] that protein dynamics is slaved by both the hydration shell and the bulk solvent, it is thus controlled by the solvent viscosity which in turn depends on the temperature. As the authors in [5] note, “exceptions to the (above) rules are not rare in naturally thermostable proteins. This observation suggests that in these cases a certain level of “frustration” is likely essential for proteins to carry out their biological functions.”

Bottom line, could we ever succeed in mimicking nature exactly? All it takes after all is to mimic its perfect deviation from the rules.


[1] C.B. Anfinsen, The formation and stabilization of protein structure. Biochem. J. 1992, 128(4), 737-749.
[2] A.D. Robertson and K.P. Murphy, Protein Structure and the Energetics of Protein Stability. Chem. Rev. 1997, 97, 1251−1268.
[3] B. Berger and T. Leighton. Protein folding in the hydrophobic-hydrophilic (hp) is np-complete. In Proceedings of the second annual international conference on Computational molecular biology, RECOMB ’98, pages 30–39, New York, NY, USA, 1998. ACM
[4] Koga N, Tatsumi-Koga R, Liu G, Xiao R, Acton TB, Montelione GT, Baker D (2012) Principles for designing ideal protein structures. Nature 491:222–227.
[5] Balasco N, Esposito L, De Simone A, Vitagliano L., "Role of loops connecting secondary structure elements in the stabilization of proteins isolated from thermophilic organisms", Protein Sci. 2013 Jul;22(7):1016-23. doi: 10.1002/pro.2279.
[6] Hans Frauenfelder, Guo Chena, Joel Berendzena, Paul W. Fenimorea, Helén Janssonb, Benjamin H. McMahona, Izabela R. Stroec, Jan Swensond and Robert D. Younge, A unified model of protein dynamics, vol. 106 no. 13, 5129–5134 (2008)