High Apparent Dielectric Constant Inside a Protein Reflects Structural Reorganization Coupled to the Ionization of an Internal Asp

394

542

Anion effects on the cloud-point temperature for the liquid؊liquid phase transition of lysozyme were investigated by temperature gradient microfluidics under a dark field microscope. It was found that protein aggregation in salt solutions followed 2 distinct Hofmeister series depending on salt concentration. Namely, under low salt conditions the association of anions with the positively charged lysozyme surface dominated the process and the phase transition temperature followed an inverse Hofmeister series. This inverse series could be directly correlated to the size and hydration thermodynamics of the anions. At higher salt concentrations, the liquid-liquid phase transition displayed a direct Hofmeister series that correlated with the polarizability of the anions. A simple model was derived to take both charge screening and surface tension effects into account at the protein/water interface. Fitting the thermodynamic data to this model equation demonstrated its validity in both the high and low salt regimes. These results suggest that in general positively charged macromolecular systems should show inverse Hofmeister behavior only at relatively low salt concentrations, but revert to a direct Hofmeister series as the salt concentration is increased.liquid-liquid phase transition ͉ protein aggregation P rotein-protein interactions can lead to aggregation. Such intermolecular contacts underlie the mechanistic basis for a variety of diseases (1-4) and are key to protein crystallization (5-7). An effective way to determine the strength of biomacromolecular interactions is to study the temperature-induced phase separation that occurs in concentrated protein solutions (8). When a concentrated protein solution is cooled below its cloud-point temperature, the system can separate into 2 coexisting liquid phases: 1 rich in protein and 1 poor. As coacervate droplets of the protein-rich phase grow, the solution turns cloudy (white) as a result of the scattering of visible light. Upon standing, the solution may completely separate by gravity into a protein-rich phase and a nearly pure aqueous phase above it. The temperature at which the initial cloud point occurs provides a simple physical measurement of the forces acting among biomacromolecules. Specifically, the higher the temperature at which the initial cloud point occurs, the stronger the putative attractive forces between the protein molecules should be.Dissolved salts in aqueous solutions have a strong influence on protein-protein interactions and the subsequent aggregated states which are formed. In fact, cloud-point temperatures typically follow a specific ion order according to the Hofmeister series (5,6,(9)(10)(11)(12)(13)(14). The relative effectiveness of anions to induce protein aggregation typically follows 1 of 2 trends depending on the pH of the solution (15, 16). Above a protein's isoelectric point, the macromolecule bears a net negative charge and a direct Hofmeister series is normally observed. In this case, chaotropes such as I Ϫ , ClO 4 Ϫ , and SCN Ϫ he...

Section: Salt Concentration (M)mentioning

confidence: 79%

The inverse and direct Hofmeister series for lysozyme

Zhang

Cremer

2009

394

542

“…When an ionizable group is removed from water and immersed in a less polar and polarizable material, such as the interior of a protein, its pK a value shifts in the direction that promotes the neutral state unless other factors (e.g., hydrogen bonds, pairing with a charge of the opposite sign, and interactions with internal water molecules) compensate for the loss of hydration (11)(12)(13)(14). Ionizable groups with highly perturbed pK a values have significant effects on the pH dependence of the thermodynamic stability (⌬G°H 2 O ) of proteins (11)(12)(13). This effect is illustrated graphically in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Were this the case, the relative paucity of internal ionizable groups could be explained in terms of evolutionary pressure to enhance the stability of proteins by eliminating internal ionizable groups that were not needed for specific structural or functional purposes. However, it is necessary to consider the possibility that the internal ionizable groups are actually not in the charged state at physiological pH (11)(12)(13)(14) and are therefore not as destabilizing as suggested by continuum electrostatics models.…”

mentioning

confidence: 99%

High tolerance for ionizable residues in the hydrophobic interior of proteins

Isom

Cannon

Castañeda

et al. 2008

Self Cite

129

193

Internal ionizable groups are quite rare in water-soluble globular proteins. Presumably, this reflects the incompatibility between charges and the hydrophobic environment in the protein interior. Here we show that proteins can have an inherently high tolerance for internal ionizable groups. The 25 internal positions in staphylococcal nuclease were substituted one at a time with Lys, Glu, or Asp without abolishing enzymatic activity and without detectable changes in the conformation of the protein. Similar results with substitutions of 6 randomly chosen internal positions in ribonuclease H with Lys and Glu suggest that the ability of proteins to tolerate internal ionizable groups might be a property common to many proteins. Eighty-six of the 87 substitutions made were destabilizing, but in all but one case the proteins remained in the native state at neutral pH. By comparing the stability of each variant protein at two different pH values it was established that the pKa values of most of the internal ionizable groups are shifted; many of the internal ionizable groups are probably neutral at physiological pH values. These studies demonstrate that special structural adaptations are not needed for ionizable groups to exist stably in the hydrophobic interior of proteins. The studies suggest that enzymes and other proteins that use internal ionizable groups for functional purposes could have evolved through the random accumulation of mutations that introduced ionizable groups at internal positions, followed by evolutionary adaptation and optimization to modulate stability, dynamics, and other factors necessary for function.electrostatics ͉ internal ionizable groups ͉ pKa values ͉ dehydration

“…For clarity, the transferred proton is shown in green and the D103-phenol hydrogen bond has been omitted. lations (3)(4)(5). Our study provides an example in which a more controlled system with minimal structural rearrangements and incisive knowledge of ionization states may more cleanly isolate electrostatic effects and substantially improve computational accuracy.…”

Section: Electrostatic Effects Of Charge Rearrangement Within the Actmentioning

confidence: 91%

“…Furthermore, it remains extremely challenging to study the electrostatic consequences of charge rearrangements that accompany hydrogen bond-mediated proton transfers. Few experimental methods exist to vary the ionization properties of discrete protein groups incrementally, and structural rearrangements within the protein matrix that typically accompany charge rearrangements complicate computational modeling and the straightforward interpretation of the electrostatic properties of protein active sites and interiors (3)(4)(5).…”

mentioning

confidence: 99%

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

Sigala¹,

Fafarman²,

Schwans³

et al. 2013

Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated sitespecific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol-Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations.computational modeling | enzyme catalysis | protein electrostatics | protein semisynthesis | active site environment H ydrogen bond networks are ubiquitous structural features within proteins, and they play key roles linking secondary and tertiary structural elements and spanning protein-protein interfaces. Such networks are especially common within enzyme active sites, where they position protein and substrate groups for catalysis, stabilize charge rearrangements during chemical transformations, and mediate proton transfers (1). Despite the prevalence and critical structural and functional roles of hydrogen bond networks, incisive dissection of their physical properties within the idiosyncratic interior of folded proteins remains difficult.Hydrogen-bonded protons are not observed in the vast majority of protein X-ray structures due to the low X-ray scattering power of hydrogen atoms (2). Thus, the presence of hydrogen bond networks is typically inferred from the proximity and orientation of hydrogen bond donor and acceptor groups within refined protein structural models. The inherent inability of most X-ray diffraction studies to monitor proton positions imposes additional challenges for dissecting the physical features that influence the equilibrium protonation states of specific residues along a hydrogen-bonded proton transfer network. Furthermore, it remains extremely challenging t...