Daniel G. Isom

Large shifts in pKa values of lysine residues buried inside a protein

Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine molecular determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pKa values, some as low as 5.3, and 20 titrate without triggering any detectable conformational reorganization. Apparently, simply by being buried in the protein interior, these Lys residues acquired pKa values comparable to those of naturally occurring internal ionizable groups involved in catalysis and biological H+ transport. The pKa values of some of the internal Lys residues were affected by interactions with surface carboxylic groups. The apparent polarizability reported by the pKa values varied significantly from location to location inside the protein. These data will enable an unprecedented examination of the positional dependence of the dielectric response of a protein. This study also shows that the ability of proteins to withstand the presence of charges in their hydrophobic interior is a fundamental property inherent to all stable proteins, not a specialized adaptation unique to proteins that evolved to depend on internal charges for function.

Charges in the hydrophobic interior of proteins

Charges are inherently incompatible with hydrophobic environments. Presumably for this reason, ionizable residues are usually excluded from the hydrophobic interior of proteins and are found instead at the surface, where they can interact with bulk water. Paradoxically, ionizable groups buried in the hydrophobic interior of proteins play essential roles, especially in biological energy transduction. To examine the unusual properties of internal ionizable groups we measured the pKa of glutamic acid residues at 25 internal positions in a stable form of staphylococcal nuclease. Two of 25 Glu residues titrated with normal pKa near 4.5; the other 23 titrated with elevated pKa values ranging from 5.2–9.4, with an average value of 7.7. Trp fluorescence and far-UV circular dichroism were used to monitor the effects of internal charges on conformation. These data demonstrate that although charges buried in proteins are indeed destabilizing, charged side chains can be buried readily in the hydrophobic core of stable proteins without the need for specialized structural adaptations to stabilize them, and without inducing any major conformational reorganization. The apparent dielectric effect experienced by the internal charges is considerably higher than the low dielectric constants of hydrophobic matter used to represent the protein interior in electrostatic continuum models of proteins. The high thermodynamic stability required for proteins to withstand the presence of buried charges suggests a pathway for the evolution of enzymes, and it underscores the need to mind thermodynamic stability in any strategy for engineering novel or altered enzymatic active sites in proteins.

High tolerance for ionizable residues in the hydrophobic interior of proteins

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.

Local secondary structure content predicts folding rates for simple, two-state proteins.

Many single-domain proteins exhibit two-state folding kinetics, with folding rates that span more than six orders of magnitude. A quantity of much recent interest for such proteins is their contact order, the average separation in sequence between contacting residue pairs. Numerous studies have reached the surprising conclusion that contact order is well-correlated with the logarithm of the folding rate for these small, well-characterized molecules. Here, we investigate the physico-chemical basis for this finding by asking whether contact order is actually a composite number that measures the fraction of local secondary structure in the protein; viz. turns, helices, and hairpins. To pursue this question, we calculated the secondary structure content for 24 two-state proteins and obtained coefficients that predict their folding rates. The predicted rates correlate strongly with experimentally determined rates, comparable to the correlation with contact order. Further, these predicted folding rates are correlated strongly with contact order. Our results suggest that the folding rate of two-state proteins is a function of their local secondary structure content, consistent with the hierarchic model of protein folding. Accordingly, it should be possible to utilize secondary structure prediction methods to predict folding rates from sequence alone.

The pKa values of acidic and basic residues buried at the same internal location in a protein are governed by different factors

The pK(a) values of internal ionizable groups are usually very different from the normal pK(a) values of ionizable groups in water. To examine the molecular determinants of pK(a) values of internal groups, we compared the properties of Lys, Asp, and Glu at internal position 38 in staphylococcal nuclease. Lys38 titrates with a normal or elevated pK(a), whereas Asp38 and Glu38 titrate with elevated pK(a) values of 7.0 and 7.2, respectively. In the structure of the L38K variant, the buried amino group of the Lys38 side chain makes an ion pair with Glu122, whereas in the structure of the L38E variant, the buried carboxyl group of Glu38 interacts with two backbone amides and has several nearby carboxyl oxygen atoms. Previously, we showed that the pK(a) of Lys38 is normal owing to structural reorganization and water penetration concomitant with ionization of the Lys side chain. In contrast, the pK(a) values of Asp38 and Glu38 are perturbed significantly owing to an imbalance between favorable polar interactions and unfavorable contributions from dehydration and from Coulomb interactions with surface carboxylic groups. Their ionization is also coupled to subtle structural reorganization. These results illustrate the complex interplay between local polarity, Coulomb interactions, and structural reorganization as determinants of pK(a) values of internal groups in proteins. This study suggests that improvements to computational methods for pK(a) calculations will require explicit treatment of the conformational reorganization that can occur when internal groups ionize.

Protons as Second Messenger Regulators of G Protein Signaling

In response to environmental stress, cells often generate pH signals that serve to protect vital cellular components and reprogram gene expression for survival. A major barrier to our understanding of this process has been the identification of signaling proteins that detect changes in intracellular pH. To identify candidate pH sensors, we developed a computer algorithm that searches proteins for networks of proton-binding sidechains. This analysis indicates that Gα subunits, the principal transducers of G protein-coupled receptor (GPCR) signals, are pH sensors. Our structure-based calculations and biophysical investigations reveal that Gα subunits contain networks of pH-sensing sidechains buried between their Ras and helical domains. Further, we show that proton binding induces changes in conformation that promote Gα phosphorylation and suppress receptor-initiated signaling. Together, our computational, biophysical, and cellular analyses reveal an unexpected function for G proteins as mediators of stress-response signaling.

Differences in the Regulation of K-Ras and H-Ras Isoforms by Monoubiquitination

Background: Ras proteins are critical regulators of cellular growth and are differentially modified by ubiquitination. Results: Chemical ubiquitination and immunoprecipitation assays demonstrate that monoubiquitination causes sustained H-Ras activation in the absence of oncogenic mutations. Conclusion: The mechanism by which H-Ras is activated by monoubiquitination is both isoform-specific and site-specific. Significance: Monoubiquitination adds a new level of regulation and complexity to isoform-specific Ras signaling. Ras GTPases are signaling switches that control critical cellular processes including gene expression, differentiation, and apoptosis. The major Ras isoforms (K, H, and N) contain a conserved core GTPase domain, but have distinct biological functions. Among the three Ras isoforms there are clear differences in post-translational regulation, which contribute to differences in localization and signaling output. Modification by ubiquitination was recently reported to activate Ras signaling in cells, but the mechanisms of activation are not well understood. Here, we show that H-Ras is activated by monoubiquitination and that ubiquitination at Lys-117 accelerates intrinsic nucleotide exchange, thereby promoting GTP loading. This mechanism of Ras activation is distinct from K-Ras monoubiquitination at Lys-147, which leads to impaired regulator-mediated GTP hydrolysis. These findings reveal that different Ras isoforms are monoubiquitinated at distinct sites, with distinct mechanisms of action, but with a common ability to chronically activate the protein in the absence of a receptor signal or oncogenic mutation.

A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity

Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High‐throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity, which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol‐reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand‐binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose‐binding protein engineered to contain single, protected cysteines. These straightforward, information‐rich experiments are likely to find applications in protein engineering and functional genomics. Proteins 2011.

Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity

The Gibbs free energy difference between native and unfolded states (“stability”) is one of the fundamental characteristics of a protein. By exploiting the thermodynamic linkage between ligand binding and stability, interactions of a protein with small molecules, nucleic acids, or other proteins can be detected and quantified. Determination of protein stability can therefore provide a universal monitor of biochemical function. Yet, the use of stability measurements as a functional probe is underutilized, because such experiments traditionally require large amounts of protein and special instrumentation. Here we present the quantitative cysteine reactivity (QCR) technique to determine protein stabilities rapidly and accurately using only picomole quantities of material and readily accessible laboratory equipment. We demonstrate that QCR-derived stabilities can be used to measure ligand binding over a wide range of ligand concentrations and affinities. We anticipate that this technique will have broad applications in high-throughput protein engineering experiments and functional genomics.

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation.

Significance In the early 1980s, scientists began searching for cell-surface receptors that bind to hormones and neurotransmitters. Among the first was the β-adrenergic receptor, a G protein-coupled receptor (GPCR) that is activated by norepinephrine and epinephrine. Recent breakthroughs have provided more than 100 new GPCR structures, including several in activated conformations. This new structural information presents an opportunity to identify features that distinguish unactivated and activated receptors. Here we use a computational approach to identify structural signatures unique to activated GPCRs. Remarkably, we find that these signatures also are present in distantly related receptors from archaea and bacteria. We propose that these new structural indicators are central to GPCR function and are indicative of GPCR activation. Seven-transmembrane receptors (7TMRs) have evolved in prokaryotes and eukaryotes over hundreds of millions of years. Comparative structural analysis suggests that these receptors may share a remote evolutionary origin, despite their lack of sequence similarity. Here we used structure-based computations to compare 221 7TMRs from all domains of life. Unexpectedly, we discovered that these receptors contain spatially conserved networks of buried ionizable groups. In microbial 7TMRs these networks are used to pump ions across the cell membrane in response to light. In animal 7TMRs, which include light- and ligand-activated G protein-coupled receptors (GPCRs), homologous networks were found to be characteristic of activated receptor conformations. These networks are likely relevant to receptor function because they connect the ligand-binding pocket of the receptor to the nucleotide-binding pocket of the G protein. We propose that agonist and G protein binding facilitate the formation of these electrostatic networks and promote important structural rearrangements such as the displacement of transmembrane helix-6. We anticipate that robust classification of activated GPCR structures will aid the identification of ligands that target activated GPCR structural states.