Contribution of Surface Salt Bridges to Protein Stability: Guidelines for Protein Engineering

Makhatadze, George I.; Loladze, Vakhtang V.; Ermolenko, Dmitri N.; Chen, XiaoFen; Thomas, Susan T.

doi:10.1016/s0022-2836(03)00233-x

Cited by 222 publications

(200 citation statements)

References 102 publications

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“…The effects on unbinding (k off1 ) for ␤ 2 D163A and ␣ 1 R120A were coupled with an energy of 0.63 kcal/mol. This energy is slightly lower than, but consistent with, energies reported for confirmed surface salt bridges in other proteins (0.86 and 0.95 kcal/mol) (Horovitz et al, 1990;Makhatadze et al, 2003). Low coupling energies were expected because salt bridges at solvent-exposed surfaces of a protein have significantly weaker interactions compared with those buried in the hydrophobic interior.…”

Section: Discussionsupporting

confidence: 84%

A State-Dependent Salt-Bridge Interaction Exists across the β/α Intersubunit Interface of the GABAA Receptor

Laha

Wagner²

2011

Molecular Pharmacology

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The GABA A receptor is a multisubunit protein that transduces the binding of a neurotransmitter at an intersubunit interface into the opening of a central ion channel. The structural components that mediate the steps involved in this action are poorly defined. A large amount of work has focused on clarifying the specific functions and interactions of residues believed to surround the GABA binding pocket. Here, we explored two charged residues (␤ 2 Asp163 and ␣ 1 Arg120), which have been suggested by homology models to participate in a salt-bridge interaction. When mutated to alanine, both single mutants, as well as the double mutant, increase EC 50-GABA , decrease the GABA binding rate, and accelerate deactivation and GABA unbinding rates. Double-mutant cycle analysis demonstrates that the effects of each alanine mutation on the GABA binding rate were additive and independent. In contrast, a significant coupling energy was found during an analysis of deactivation time constants. Using kinetic modeling, we further demonstrated that the GABA unbinding rates, in particular, are strongly coupled. These data suggest that ␤ 2 Asp163 and ␣ 1 Arg120 form a state-dependent salt bridge, interacting when GABA is bound to the receptor but not when the receptor is in the unbound state.

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Section: Discussionsupporting

confidence: 84%

A State-Dependent Salt-Bridge Interaction Exists across the β/α Intersubunit Interface of the GABAA Receptor

Laha

Wagner²

2011

Molecular Pharmacology

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“…In such sequences, electrostatic interactions between solvated side chains of opposite sign make important contributions to globule stability (50). This effect is reminiscent of work by Makhatadze and coworkers who showed that through-solvent electrostatic attractions between opposite charges on protein surfaces play an important role in stabilizing globular structures of water-soluble proteins (51,52). To test the validity of the strong polyampholyte classification, which was not represented in the protamine inventory, we performed simulations for eight polyampholytic sequences.…”

Section: Sequence-specificity In Conformational Properties Of Protamimentioning

confidence: 99%

Net charge per residue modulates conformational ensembles of intrinsically disordered proteins

Mao

Crick

Vitalis

et al. 2010

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

541

644

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Intrinsically disordered proteins (IDPs) adopt heterogeneous ensembles of conformations under physiological conditions. Understanding the relationship between amino acid sequence and conformational ensembles of IDPs can help clarify the role of disorder in physiological function. Recent studies revealed that polar IDPs favor collapsed ensembles in water despite the absence of hydrophobic groups-a result that holds for polypeptide backbones as well. By studying highly charged polypeptides, a different archetype of IDPs, we assess how charge content modulates the intrinsic preference of polypeptide backbones for collapsed structures. We characterized conformational ensembles for a set of protamines in aqueous milieus using molecular simulations and fluorescence measurements. Protamines are arginine-rich IDPs involved in the condensation of chromatin during spermatogenesis. Simulations based on the ABSINTH implicit solvation model predict the existence of a globule-to-coil transition, with net charge per residue serving as the discriminating order parameter. The transition is supported by quantitative agreement between simulation and experiment. Local conformational preferences partially explain the observed trends of polymeric properties. Our results lead to the proposal of a schematic protein phase diagram that should enable prediction of polymeric attributes for IDP conformational ensembles using easily calculated physicochemical properties of amino acid sequences. Although sequence composition allows the prediction of polymeric properties, interresidue contact preferences of protamines with similar polymeric attributes suggest that certain details of conformational ensembles depend on the sequence. This provides a plausible mechanism for specificity in the functions of IDPs.Monte Carlo | polyampholyte | polyelectrolyte I ntrinsically disordered proteins (IDPs) are a class of proteins that fail to fold autonomously in aqueous solutions to welldefined three-dimensional structures (1, 2). This "intrinsic disorder" has been implicated in a range of regulatory functions that require IDPs to interact with other macromolecular ligands (3-11). Many of these interactions promote disorder-to-order transitions within IDPs (3, 9), and different mechanistic models (3, 12, 13) have been proposed for coupled folding and binding. To develop a better understanding of how disorder is used in function (2), we have pursued quantitative, polymer-physics-based descriptions (14-16) for conformational ensembles of .Low hydrophobicity is a defining characteristic of IDP sequences (21,22). This suggests that IDPs cannot collapse to form compact, globular conformations in aqueous solutions (21). However, spectroscopic (23-27) and computational investigations (17, 28) have shown that polar tracts form heterogeneous ensembles of collapsed structures in aqueous solutions. These sequences are rich in uncharged, polar amino acids and are devoid of canonical hydrophobic residues. Collapse of polar tracts has been observed for polyglutamine (17...

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“…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).…”

mentioning

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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Contribution of Surface Salt Bridges to Protein Stability: Guidelines for Protein Engineering

Cited by 222 publications

References 102 publications

A State-Dependent Salt-Bridge Interaction Exists across the β/α Intersubunit Interface of the GABAA Receptor

A State-Dependent Salt-Bridge Interaction Exists across the β/α Intersubunit Interface of the GABAA Receptor

Net charge per residue modulates conformational ensembles of intrinsically disordered proteins

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

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