Journal of Molecular Biology
Regular articleNative-state energetics of a thermostabilized variant of ribonuclease HI1
Introduction
The cooperative nature of protein folding enables mutations that stabilize a protein’s structure to affect not only residues that they directly contact, but also the stability and flexibility of those distal from the site of the mutation. The manner in which stabilizing interactions are propagated throughout a protein molecule is important both for understanding the properties of existing enzymes and in the design of more useful protein variants. Because unfolded regions of proteins are better substrates for proteases and other degradation enzymes, the thermodynamic stability of a protein is a crucial determinant for its functional lifetime in the cellular environment. 1 The manner in which stabilizing interactions are transmitted throughout the folded structure is also relevant to understanding the flexibility of dynamic movement necessary for enzymatic catalysis. Furthermore, stability and cooperativity likely play a critical role in protein misfolding diseases such as Creutzfeld-Jakob and Alzheimer’s diseases. 2, 3, 4, 5
In vitro, proteins can be engineered to be more stable by the careful introduction of mutations. Examples of stabilizing mutations include adding disulfide linkages, 6, 7, 8 substituting residues that increase the secondary-structural propensity of α-helices and β-sheets, 9, 10, 11 and the elimination of unfavorable charge interactions. 12, 13 Such alterations of the primary sequence of proteins can allow the production of enzymes that function at temperatures, or under other conditions, that are usually denaturing. Furthermore, misfolding events leading to prion and amyloid diseases 2, 3, 4, 5 might be avoided by additional interactions that stabilize the native fold of susceptible proteins. Therefore the elucidation of how stabilizing mutations alter the Boltzmann distribution of conformations should enable a better description of existing enzymes and lead to clues in the construction of protein variants with desirable properties.
Ribonuclease HI from Escherichia coli provides a good model for the investigation of changes in the energy landscape. This single-domain enzyme has a mixed α/β structure 14, 15 and non-specifically degrades the RNA strand of RNA·DNA hybrids. Previous studies have shown that E. coli RNase HI is moderately stable (∼9 kcal/mol) and unfolds in an apparent two-state manner at equilibrium when viewed by gross spectroscopic techniques such as circular dichroism (CD) and fluorescence. 16 Kinetic studies have demonstrated that a central “core region” of two helices (A and D) fold first, forming a populated kinetic intermediate that serves as a scaffold directing the folding of the remainder of the enzyme. 17, 18, 19
The equilibrium landscape of RNase H has also been probed by native-state hydrogen exchange (HX), which provides high-resolution information regarding the patterns of stability in proteins. 20 The degree to which individual amide protons exchange with solvent deuterons is affected by protein structure, and the results of HX experiments for a given protein at various denaturant concentrations can be used to calculate residue-specific free energies. Such experiments on E. coli RNase HI have demonstrated that the same “core region” that is structured in the kinetic intermediate is the most stable element at equilibrium under native conditions. 21
RNase H has an active site containing four carboxylate side-chains 14, 15 that bind divalent metal ions (Figure 1), the required cofactors for nuclease activity. A mutation in one of these residues (Asp10 → Ala) is known to stabilize the apo-protein by eliminating one of the proximal negative charges in this region. 19, 22 Asp10 resides in strand I of the molecule just outside of the most stable core region. Kinetic studies on the folding of this variant are consistent with the hierarchical model of folding. 18, 19 The global stability of this mutant protein (D10A) is similar to that of the thermophilic enzyme from Thermus thermophilus. 19, 23, 24, 25 In this study, we examined the D10A variant of E. coli RNase H with native-state exchange to determine how the ensemble of forms populated under native conditions is perturbed by the stabilizing mutation.
Section snippets
Results
In the active site of RNase H, there are four negatively charged carboxylate side-chains forming a pocket that binds divalent metal ions. One of these is Asp10, which is absolutely required for enzymatic activity. 22, 26 In the absence of divalent metal, the pKa value of Asp10 is unusually high (∼6). 27 This ∼2 unit pKa shift reflects the destabilizing effect of placing an additional negative charge in the environment of the active site. Removal of the carboxylate group by mutation (D10A)
Discussion
When monitored by spectroscopic techniques such as circular dichroism or UV fluorescence, the unfolding of single-domain proteins usually appears to be two-state, consisting of a population of fully folded or fully unfolded molecules. Native state hydrogen exchange (HX), however, provides a means to detect the rarely populated partially unfolded forms (PUFs) of proteins in conditions in which the fully folded form of the protein is the dominant species. Native state HX has revealed PUFs for
Protein expression and purification
Unlabeled D10A RNase H∗ protein was overexpressed in BL21/DE3 pLysS E. coli cells in LB medium. 18 (RNase H∗ is a variant of E. coli RNase HI with its three free cysteine residues replaced with alanine. 16, 18, 39) 15N-labeled D10A RNase H∗ protein was overexpressed in M9 medium with 15N-labeled ammonium chloride. Protein purification was as described. 40
X-ray crystallography
Crystals of D10A were produced by the hanging-drop vapor diffusion method by microseeding with RNase H∗ crystals; 25 conditions were 20 mM
Acknowledgements
We gratefully acknowledge Giulietta Spudich for assistance with hydrogen exchange and NMR, Manuel Llinás, Charley Ross, and Tanya Raschke for aid in protein production, Sarah McWhirter, James Holton, Professors Tom Alber and James Berger for assistance with X-ray crystallography, Julie Hollien for helpful discussions and Srebrenka Robic for the critical reading of the manuscript. This work was supported by a grant from the NIH (GM50945).
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Edited by P. E. Wright