Equilibrium unfolding of <i>Escherichia coli</i> ribonuclease H: Characterization of a partially folded state

Dabora, Jonathan M.; Marqusee, Susan

doi:10.1002/pro.5560030906

Cited by 77 publications

(90 citation statements)

References 22 publications

(19 reference statements)

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“…The version of E. coli RNase H [Protein Data Bank (PDB) ID: 1F21] used here is the wild-type protein with its three cysteines changed to alanine. It was expressed and purified as described previously (66).…”

Section: Methodsmentioning

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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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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Section: Methodsmentioning

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.

Self Cite

164

217

View full text Add to dashboard Cite

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“…All genes were cloned into high copy plasmids under a T7 promoter (pAED4 or pET27). The proteins were purified using their respective published protocols with the difference that 1 mM DTT was present during all purification steps (Dabora and Marqusee 1994;Llinas and Marqusee 1998;Robic et al 2002).…”

Section: Protein Preparationmentioning

confidence: 99%

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

et al. 2008

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Here we report on a method that extends the study of the mechanical behavior of single proteins to the low force regime of optical tweezers. This experimental approach relies on the use of DNA handles to specifically attach the protein to polystyrene beads and minimize the non-specific interactions between the tethering surfaces. The handles can be attached to any exposed pair of cysteine residues. Handles of different lengths were employed to mechanically manipulate both monomeric and polymeric proteins. The low spring constant of the optical tweezers enabled us to monitor directly refolding events and fluctuations between different molecular structures in quasiequilibrium conditions. This approach, which has already yielded important results on the refolding process of the protein RNase H (Cecconi et al. in Science 309: 2057-2060-2005, appears robust and widely applicable to any protein engineered to contain a pair of reactive cysteine residues. It represents a new strategy to study protein folding at the single molecule level, and should be applicable to a range of problems requiring tethering of protein molecules.

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“…Aqualitative measure for good packing is the accessibility of the core to the hydrophobic dye 8-anilino-1 -naphthalenesulfonic acid (ANS) (Mulqueen & Kronman, 1982;Semisotnov et al, 1991). These data are shown in Figure 5 along with the fluorescence spectrum of ANS in the presence of the acid state form of ribonuclease H (Dabora & Marqusee, 1994), used here as a positive control. In contrast to ribonuclease H, all ubiquitin variants do not bind ANS, suggesting they are more well-packed than many previously designed proteins.…”

Section: Ans Bindingmentioning

confidence: 99%

De novo design of the hydrophobic core of ubiquitin

1997

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We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that P-sheet structures have more stringent packing requirements than a-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold.Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.Keywords: computational; genetic algorithm; hydrophobic core packing; protein design; ubiquitin Protein design is an extremely powerful method for critically testing the relationship between the primary sequence and threedimensional structure of proteins. One goal of protein design is to reconstruct known three-dimensional folds from completely novel amino acid sequences. The concepts used to design the amino acid sequences constitute a hypothesis of what interactions are necessary for a protein to adopt a given three-dimensional structure with the properties of natural proteins, and at what level of detail these interactions must be considered.The hypotheses of early protein design efforts were based on the assumption that simple empirical rules would be sufficient. These designs included, foremost, patterns of hydrophobic and hydrophilic residues, and to a lesser extent, secondary structure propensities and poten...

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Equilibrium unfolding of Escherichia coli ribonuclease H: Characterization of a partially folded state

Cited by 77 publications

References 22 publications

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

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

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

De novo design of the hydrophobic core of ubiquitin

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