Predicting extreme p<i>K</i><sub>a</sub> shifts in staphylococcal nuclease mutants with constant pH molecular dynamics

Arthur, Evan J.; Yesselman, Joseph D.; Brooks, Charles L.

doi:10.1002/prot.23195

Cited by 47 publications

(71 citation statements)

References 58 publications

(138 reference statements)

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“…S5-S8). pK a shifts upon mutation are sensitive reporters of changes in the electrostatic environment (21)(22)(23)(24)(25)(26). In WT HP36, the pK a values of D44, E45, and D46 are lower than model compound values by 0.4-1 pK a units, indicating that these residues make favorable NSE electrostatic interactions; E72 and the C terminus have pK a values near model compound values ( Table 2).…”

Section: Resultsmentioning

confidence: 99%

Rational modification of protein stability by targeting surface sites leads to complicated results

Xiao¹,

Patsalo

Shan³

et al. 2013

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

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The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions.atomistic simulations | pH-dependent stability | protein pKa measurements | nuclear magnetic resonance | protein biophysics T he mutation of charged surface residues to enhance electrostatics is a popular approach in protein engineering (1). Target residues are often selected using estimates of the protein electrostatic potential (2). This approach relies on identifying residues that are involved in unfavorable electrostatic interactions, typically with residues of the same charge, or on identifying sites where new favorable electrostatic interactions can be introduced (3). Solvent-exposed charged residues are thought to not be involved in critical packing interactions in the native state, to not suffer large desolvation penalties upon protein folding, nor to make significant interactions in the denatured-state ensemble (DSE). Thus, residues to be targeted are typically chosen on the basis of calculations of protein native-state ensemble (NSE) electrostatics. Methods ranging from simple inspection of the protein surface, modified Tanford-Kirkwood approaches (3, 4) and Poisson-Boltzmann (PB) calculations have been used (5-7). Irrespective of the details, the general strategy is based on the assumption that surface electrostatic interactions can be modified without altering other native-state interactions and without altering DSE energetics. An attractive feature of these approaches is that they are computationally inexpensive and, unlike selectionbased methods or directed evolution, involve the generation of a limited number of mutants. Any increase in stability is generally assumed to result from modification of NSE electrostatics. We show that this approach leads to complicated and unanticipated results, even for very simple proteins.We use the villin headpiece subdomain HP36 as our model system. HP36 is a small three-helix protein that has become an extremely popular model system for experimental and computational studies of protein folding, owing to its ...

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Section: Resultsmentioning

confidence: 99%

Rational modification of protein stability by targeting surface sites leads to complicated results

Xiao¹,

Patsalo

Shan³

et al. 2013

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

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“…The same CpHMD method was also tested by the Brooks group using a variant of wild-type SNase as well as several mutants yielding an average unsigned error of 1.5 units [40]. These data revealed that, while the p K a values are converged due to the enhanced sampling protocol, outliers are due to the limitations of the underlying GB model, i.e., less accurate desolvation free energy for deeply buried sites and distorted local conformational environment of sidechains close to the protein-water interface.…”

Section: Recent Applications Of Constant Ph Molecular Dynamicsmentioning

confidence: 99%

Recent development and application of constant pH molecular dynamics

Chen

Shi

Shen

2014

Molecular Simulation

114

128

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Solution pH is a critical environmental factor for chemical and biological processes. Over the last decade, significant efforts have been made in the development of constant pH molecular dynamics (pHMD) techniques for gaining detailed insights into pH-coupled dynamical phenomena. In this article we review the advancement of this field in the past five years, placing a special emphasis on the development of the all-atom continuous pHMD technique. We discuss various applications, including the prediction of pKa shifts for proteins, nucleic acids and surfactant assemblies, elucidation of pH-dependent population shifts, protein-protein and protein-RNA binding, as well as the mechanisms of pH-dependent self-assembly and phase transitions of surfactants and peptides. We also discuss future directions for the further improvement of the pHMD techniques.

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“…[47][48][49] Recent studies have shown that CPHMD is a reliable and robust method that is capable of predicting pK a values in a variety of biomolecular systems. [50][51][52][53][54][55][56] Thus, to uncover the pHdependent residues in the CIRV p19 protein involved in siRNA binding stability, we have carried out CPHMD simulations [44][45][46] of the p19 protein dimer in both holo (siRNA-bound) and apo (siRNAfree) forms and determined the pK a values for all titratable residues. These results were then used to calculate the pH-dependent siRNA binding stability profile and corresponding pH-dissociation constant profile.…”

Section: Introductionmentioning

confidence: 99%

pH‐sensitive residues in the p19 RNA silencing suppressor protein from carnation Italian ringspot virus affect siRNA binding stability

2013

Self Cite

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: Tombusviruses, such as Carnation Italian ringspot virus (CIRV), encode a protein homodimer called p19 that is capable of suppressing RNA silencing in their infected hosts by binding to and sequestering short-interfering RNA (siRNA) away from the RNA silencing pathway. P19 binding stability has been shown to be sensitive to changes in pH but the specific amino acid residues involved have remained unclear. Using constant pH molecular dynamics simulations, we have identified key pH-dependent residues that affect CIRV p19-siRNA binding stability at various pH ranges based on calculated changes in the free energy contribution from each titratable residue. At high pH, the deprotonation of Lys60, Lys67, Lys71, and Cys134 has the largest effect on the binding stability. Similarly, deprotonation of several acidic residues (Asp9, Glu12, Asp20, Glu35, and/or Glu41) at low pH results in a decrease in binding stability. At neutral pH, residues Glu17 and His132 provide a small increase in the binding stability and we find that the optimal pH range for siRNA binding is between 7.0 and 10.0. Overall, our findings further inform recent experiments and are in excellent agreement with data on the pH-dependent binding profile.

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Predicting extreme pK_a shifts in staphylococcal nuclease mutants with constant pH molecular dynamics

Cited by 47 publications

References 58 publications

Rational modification of protein stability by targeting surface sites leads to complicated results

Rational modification of protein stability by targeting surface sites leads to complicated results

Recent development and application of constant pH molecular dynamics

pH‐sensitive residues in the p19 RNA silencing suppressor protein from carnation Italian ringspot virus affect siRNA binding stability

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Predicting extreme pKa shifts in staphylococcal nuclease mutants with constant pH molecular dynamics

Cited by 47 publications

References 58 publications

Rational modification of protein stability by targeting surface sites leads to complicated results

Rational modification of protein stability by targeting surface sites leads to complicated results

Recent development and application of constant pH molecular dynamics

pH‐sensitive residues in the p19 RNA silencing suppressor protein from carnation Italian ringspot virus affect siRNA binding stability

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Predicting extreme pK_a shifts in staphylococcal nuclease mutants with constant pH molecular dynamics