Evolutionary trend toward kinetic stability in the folding trajectory of RNases H

Abstract

Proper folding of proteins is critical to producing the biological machinery essential for cellular function. The rates and energetics of a protein's folding process, which is described by its energy landscape, are encoded in the amino acid sequence. Over the course of evolution, this landscape must be maintained such that the protein folds and remains folded over a biologically relevant time scale. How exactly a protein's energy landscape is maintained or altered throughout evolution is unclear. To study how a protein's energy landscape changed over time, we characterized the folding trajectories of ancestral proteins of the ribonuclease H (RNase H) family using ancestral sequence reconstruction to access the evolutionary history between RNases H from mesophilic and thermophilic bacteria. We found that despite large sequence divergence, the overall folding pathway is conserved over billions of years of evolution. There are robust trends in the rates of protein folding and unfolding; both modern RNases H evolved to be more kinetically stable than their most recent common ancestor. Finally, our study demonstrates how a partially folded intermediate provides a readily adaptable folding landscape by allowing the independent tuning of kinetics and thermodynamics.protein folding | energy landscape | protein evolution | ancestral sequence reconstruction B ecause most proteins need to fold to function, there must be selective pressures for efficient folding and maintenance of structure. Although many studies have investigated the evolution of protein function, a detailed understanding of the evolutionary constraints on protein folding is lacking.The amino acid sequence of a protein encodes its energy landscape, which determines its folding trajectory-its mechanism, rates, and energetics (1). Thus, the energy landscape defines a protein's ability to fold in a biologically reasonable time scale, avoid misfolding, and prevent frequent unfolding-all of which are likely important for the overall fitness of an organism. Indeed, numerous studies have proposed how protein folding might affect molecular evolution (2-8), and there is growing evidence that folding pathways and their associated kinetics and energetics are evolutionarily constrained. To date, however, there is little experimental evidence detailing how folding properties have changed throughout evolution. Are there trends in the folding mechanism or rates? Which aspects of the folding mechanism have been conserved, and how have they played a role in the evolution of modern-day homologs? We might naively expect folding to optimize, so that modern proteins generally fold faster than their ancestors. Or perhaps the energetics of the folding landscape evolved to avoid kinetic traps or misfolded states. If maintaining the folded state is important for fitness, then we might expect an improvement in kinetic stability over time, although a folded state that is too stable might be detrimental for conformational dynamics. These insights, and others, are critical for our und...

DeCoDe: degenerate codon design for complete protein-coding DNA libraries

“…These networks can often be disrupted by even a single amino acid change if that change is non-conservative in a necessary physical property (11). Several research groups have exploited the preservation of these networks over time to reconstruct ancestral protein lineages and better understand the link between protein sequence structure and function (22,36,43). Due to the highly correlated, chemically conserved sequence patterns present in these reconstructed lineages, DeCoDe offers an attractive solution to the problem of synthesizing the complete protein family for functional testing simultaneously.…”

Folding Rate Optimization Promotes Frustrated Interactions in Entangled Protein Structures

“…All sequences are assumed to fold to a fixed native structure according to the Go-like model defined in Eq. (2). The native structure can be chosen as either the entangled structure in Figure 1(a), or the twin non-entangled structure in Figure 1(b).…”

“…INTRODUCTIONThe biological function of most proteins requires them to fold into a well-defined native state, implying that both structure maintenance and efficient folding are kept under selective pressure by evolutionary processes [1]. In particular, a direct experimental evidence, pointing to some degree of folding rate optimization throughout evolution, was recently provided for ribonuclease H, using ancestral sequence reconstruction [2]. Bio-informatics analyses had also uncovered similar evolutionary signals already two decades ago for several folds [3], and more recently for a large catalog of protein domains [4].The latter study was based on the well known empirical correlation between experimentally measured folding rates of proteins and simple descriptors of the structural organization of the native state [5].…”

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

“…ASR has been applied to a variety of protein families and in addition to revealing the evolutionary history, these ancestral proteins can act as intermediates in sequence space to uncover mechanisms underlying protein properties ( Starr et al, 2017 ; Gaucher et al, 2008 ; Hobbs et al, 2012 ; Perez-Jimenez et al, 2011 ; Risso et al, 2013 ; Smock et al, 2016 ; Akanuma et al, 2013 ; Siddiq et al, 2017 ). Recently, ancestral sequence reconstruction was applied to the RNase H family and the thermodynamic and kinetic properties of seven ancestral proteins connecting the lineages of E. coli and T. thermophilus RNase H (ecRNH* and ttRNH*) were characterized ( Hart et al, 2014 ; Lim et al, 2016 ; Lim and Marqusee, 2017 ). Stopped-flow kinetics monitored by circular dichroism (CD) demonstrate that all seven ancestral proteins populate a folding intermediate before the rate-limiting step.…”