Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

In studies of the ensembles of unfolded structures of a four-helix bundle protein, we have detected the presence of potential precursors of native tertiary structures. These observations were based on the perturbation of NMR chemical shifts of the protein backbone atoms by single site mutations. Some mutations change the chemical shifts of residues remote from the site of mutation indicating the presence of an interaction between the mutated and the remote residues, suggesting that the formation of helix segments and helix-helix interactions is cooperative. We can begin to track down the folding mechanism of this protein using only experimental data by combining the information available for the rate limiting structure formation during the folding process with measurements of the site specific hydrogen bond formation in the burst phase, and with the existence prior to the folding reaction of tertiary structures in the ensemble of otherwise unfolded structures observed in the present study.protein folding | transient structure formation P rotein folding is one of the fundamental processes in living organisms. Genes have been selected in evolution to code for peptide chains with amino acid sequences that fold into well-defined functional structures. Although selection and evolution have ensured that amino acid sequences and the protein folding processes are optimized in any organism, the nature of these mechanisms remains to be fully understood (1).Protein folding has been intensively studied for several decades (2), but so far the atomic details of the folding processes have not been determined experimentally for any protein. The accumulation of detailed knowledge about the mechanisms of protein folding pathways is the tedious and perhaps only way toward understanding the origin of the wealth of protein architectures, which are being formed in the courses of protein folding events. It is relevant, therefore, to develop methods that may describe both the kinetics and the equilibrium thermodynamics of the processes of protein folding. The kinetics of the global protein folding processes are known for many proteins to be very fast, and it has been studied primarily using rapid mixing techniques (3). However, studies of global protein folding processes do not provide any information about those underlying processes, which hold the key to understanding why and how a unique amino acid sequence can form regular protein architecture. Because many proteins do fold spontaneously, there must be an imbedded autonomy in the amino acid sequence directing the course and the result of folding.Here we are concerned with the detection of structure formation in the initial stage of a protein folding event. For this purpose we are using NMR spectroscopy to study the formation of interactions in the unfolded state of acyl coenzyme A binding protein (ACBP) between residues that are separated by more than 10 residues in the amino acid sequence. A number of studies suggest that the transiently populated structures in the unfolded state ...

Section: Resultsmentioning

confidence: 99%

Cooperative formation of native-like tertiary contacts in the ensemble of unfolded states of a four-helix protein

Bruun

Iešmantavičius

Danielsson

et al. 2010

“…DSE electrostatic interactions have been detected in a range of proteins of diverse structure (30,(36)(37)(38)(39)(40)(41)(42), and there are examples of mutations that alter DSE energetics (43). There are also numerous examples in which mutation of charged surface residues leads to effects that differ in magnitude or even directionality from the expected results.…”

Section: Discussionmentioning

confidence: 99%

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

Xiao¹,

Patsalo

Shan³

et al. 2013

Self Cite

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 ...

“…A thorough thermodynamic analysis revealed that the main origin for the stabilization introduced by glycosylation is the unwinding of the residual native structure formed in the unfolded state, which results in a higher enthalpy. This effect serves as another indication of the importance of the unfolded state in controlling and shifting the thermodynamics and kinetics of protein folding (36)(37)(38).…”

Section: Discussionmentioning

confidence: 99%

Effect of glycosylation on protein folding: A close look at thermodynamic stabilization

Shental-Bechor

Levy

2008

528

438

Glycosylation is one of the most common posttranslational modifications to occur in protein biosynthesis, yet its effect on the thermodynamics and kinetics of proteins is poorly understood. A minimalist model based on the native protein topology, in which each amino acid and sugar ring was represented by a single bead, was used to study the effect of glycosylation on protein folding. We studied in silico the folding of 63 engineered SH3 domain variants that had been glycosylated with different numbers of conjugated polysaccharide chains at different sites on the protein's surface. Thermal stabilization of the protein by the polysaccharide chains was observed in proportion to the number of attached chains. Consistent with recent experimental data, the degree of thermal stabilization depended on the position of the glycosylation sites, but only very weakly on the size of the glycans. A thermodynamic analysis showed that the origin of the enhanced protein stabilization by glycosylation is destabilization of the unfolded state rather than stabilization of the folded state. The higher free energy of the unfolded state is enthalpic in origin because the bulky polysaccharide chains force the unfolded ensemble to adopt more extended conformations by prohibiting formation of a residual structure. The thermodynamic stabilization induced by glycosylation is coupled with kinetic stabilization. The effects introduced by the glycans on the biophysical properties of proteins are likely to be relevant to other protein polymeric conjugate systems that regularly occur in the cell as posttranslational modifications or for biotechnological purposes.energy landscape ͉ posttranslational ͉ modification ͉ unfolded state