Tracing a protein’s folding pathway over evolutionary time using ancestral sequence reconstruction and hydrogen exchange

Abstract

The conformations populated during protein folding have been studied for decades; yet, their evolutionary importance remains largely unexplored. Ancestral sequence reconstruction allows access to proteins across evolutionary time, and new methods such as pulsed-labeling hydrogen exchange coupled with mass spectrometry allow determination of folding intermediate structures at near amino-acid resolution. Here, we combine these techniques to monitor the folding of the ribonuclease H family along the evolutionary lineages of T. thermophilus and E. coli RNase H. All homologs and ancestral proteins studied populate a similar folding intermediate despite being separated by billions of years of evolution. Even though this conformation is conserved, the pathway leading to it has diverged over evolutionary time, and rational mutations can alter this trajectory. Our results demonstrate that evolutionary processes can affect the energy landscape to preserve or alter specific features of a protein’s folding pathway.

Evolution, folding, and design of TIM barrels and related proteins

“…Also involving α/β proteins, Kukic et al explored how the folding rates of Procarboxypeptidase A2 can be modulated during evolution by modification of the so-called nucleation-condensation mechanism [ 59 ]. Moreover, the Marqusee lab has made a substantial effort to understand how evolutionary pressures modify folding landscapes and tune kinetic and thermodynamic stability by characterizing one of the oldest protein folds, the RNase H-like superfamily [ 60 , 61 , 62 , 63 ]. Other interesting works are the analysis of the influence of folding energies on the fitness of β-lactamases [ 64 ], the study of protein folding and fitness landscapes of amidases [ 65 ], the analysis of cotranslational folding and fitness of an integral membrane protein [ 66 ], and the evolutionary history of myoglobins [ 67 ].…”

Salt Dependence Conformational Stability of the Dimeric SAM Domain of MAPKKK Ste11 from Budding Yeast: A Native-State H/D Exchange NMR Study

“…Additionally, various studies also ascertain that these substructures or foldons influence and guide the functional and evolutionary properties of the protein in which they occur. 28,55 ■ ASSOCIATED CONTENT * sı Supporting Information…”

“…As per the Boltzmann distribution, under native-state conditions, any protein molecule has a small population of transient state intermediates occupying a higher than ground state energy level, called the partially unfolded forms (PUFs) and even under mildly denaturing condition protein molecules will cycle through these globally unfolded states. , The prolonged hydrogen exchange (HX) behavior thus can be utilized to study the global protein unfolding pathway; the identification and study of these intermediates, therefore, pave the way for elucidating the mechanism of protein cooperativity as well protein folding and unfolding. − The hydrogen exchange study of native protein under mild denaturing conditions is realized through different procedures. − It reveals the potential metastable, partially unfolded intermediates, their interaction (exchange) behavior with the solvent, as well as the denaturant, that occur in the steps leading up to the completely unfolded state. − Such transitions are mostly cooperative and may involve a sequential unfolding of the structural units in tandem with their underlying counterparts. , …”

Opening of a Cryptic Pocket in β-lactamase Increases Penicillinase Activity

“…[2,3] In opposition to this common assumption, there are several compelling examples of functionally relevant excited states. [4][5][6][7][8][9] However, it is still unclear if excited states in general play a role in function.…”