The Folding Trajectory of RNase H Is Dominated by Its Topology and Not Local Stability: A Protein Engineering Study of Variants that Fold via Two-State and Three-State Mechanisms

Connell, Katelyn; Miller, Erik J.; Marqusee, Susan

doi:10.1016/j.jmb.2009.05.085

Cited by 23 publications

(28 citation statements)

References 46 publications

(50 reference statements)

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“…All of the RNase H proteins in our study retain the canonical RNase H fold (16) and fold through a three-state pathway. This is consistent with suggestions that topology, rather than sequence, is likely the determinant of the folding trajectory for this protein (49) and possibly proteins in general (50). In addition, the robust folding of the RNase H ancestors is consistent with evidence indicating that ASR generates proteins of evolutionary significance, unlike consensus proteins, which, despite being likened to ancestral proteins, have been found to fold poorly in some instances (18,51).…”

Section: Discussionsupporting

confidence: 89%

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

Lim

Hart

Harms

2016

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussionsupporting

confidence: 89%

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

Lim

Hart

Harms

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Potentially, partial unfolding of an obligate intermediate also applies to RNase H1 folding because the two- and three state folding behavior can be manipulated by point mutants yet folding occurs with the same order [126]. For both a three- [127] and four-helix bundle [102], an intermediate with the majority of the native structure forms in microseconds.…”

Section: Non-native Structuresmentioning

confidence: 99%

The folding of single domain proteins—have we reached a consensus?

Sosnick

Barrick

2011

Current Opinion in Structural Biology

135

130

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Rather than stressing the most recent advances in the field, this review highlights the fundamental topics where disagreement remains and where adequate experimental data are lacking. These topics include properties of the denatured state and the role of residual structure, the nature of the fundamental steps and barriers, the extent of pathway heterogeneity and non-native interactions, recent comparisons between theory and experiment, and finally, dynamical properties of the folding reaction.

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“…HX pulse-labeling and equilibrium native-state HX experiments monitored by NMR showed that I core comprises a continuous region of the protein between helix A and strand 5 and that β-strands 1, 2, and 3 and helix E acquire protection much later, consistent with mutational analysis (2)(3)(4). Single-molecule and mutational studies indicated that the intermediate is obligatory, on-pathway, and folds first even when I core is not observably populated (6,7).…”

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confidence: 99%

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

Hu¹,

Walters

Kan³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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217

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The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a "foldon." Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed "sequential stabilization" based on nativelike interfoldon interactions orders the pathway.D o proteins fold through varied and multiple tracks, or do they fold through predetermined intermediates according to understandable biophysical principles (1)? This question is fundamental for the interpretation of a large amount of biophysical and biological research. The question could be resolved if it were possible to define the intermediate structures and pathways that unfolded proteins move through on their way to the native state. Unfortunately, transient intermediates cannot be studied by the usual crystallographic and NMR methods. The range of kinetic and spectroscopic methods has been applied to many proteins, but these methods do not yield the necessary structural information.We used a developing technology, hydrogen exchange pulse labeling measured by MS (HX MS), to study the folding of a cysteine-free variant of Escherichia coli ribonuclease H1 (RNase H), a mixed α/β protein that has served as a major proteinfolding model (2-5). Previous studies showed that RNase H folds in a fast, unresolved burst phase (15 ms dead time) to an intermediate termed "I core " and then much more slowly (in seconds) to the native state (3). HX pulse-labeling and equilibrium native-state HX experiments monitored by NMR showed that I core comprises a continuous region of the protein between helix A and strand 5 and that β-strands 1, 2, and 3 and helix E acquire protection much later, consistent with mutational analysis (2-4). Single-molecule and mutational studies indicated that the intermediate is obligatory, on-pathway, and folds first even when I core is not observably populated (6, 7).The HX MS technique used here is able to follow the entire folding trajectory of RNase H in considerable structural and temporal detail. The analysis monitors every amide site, evaluates the folding coopera...

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The Folding Trajectory of RNase H Is Dominated by Its Topology and Not Local Stability: A Protein Engineering Study of Variants that Fold via Two-State and Three-State Mechanisms

Cited by 23 publications

References 46 publications

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

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

The folding of single domain proteins—have we reached a consensus?

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

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