Toward the computational design of protein crystals with improved resolution.

Substantial advances have been made in the computational design of protein interfaces over the last 20 years. However, the interfaces targeted by design have typically been stable and high-affinity. Here, we report the development of a generic computational design method to stabilize the weak interactions at crystallographic interfaces. Initially, we analyzed structures reported in the Protein Data Bank to determine whether crystals with more stable interfaces result in higher resolution structures. We found that for 22 variants of a single protein crystallized by a single individual, the Rosetta-calculated `crystal score' correlates with the reported diffraction resolution. We next developed and tested a computational design protocol, seeking to identify point mutations that would improve resolution in a highly stable variant of staphylococcal nuclease (SNase). Using a protocol based on fixed protein backbones, only one of the 11 initial designs crystallized, indicating modeling inaccuracies and forcing us to re-evaluate our strategy. To compensate for slight changes in the local backbone and side-chain environment, we subsequently designed on an ensemble of minimally perturbed protein backbones. Using this strategy, four of the seven designed proteins crystallized. By collecting diffraction data from multiple crystals per design and solving crystal structures, we found that the designed crystals improved the resolution modestly and in unpredictable ways, including altering the crystal space group. Post hoc, in silico analysis of the three observed space groups for SNase showed that the native space group was the lowest scoring for four of six variants (including the wild type), but that resolution did not correlate with crystal score, as it did in the preliminary results. Collectively, our results show that calculated crystal scores can correlate with reported resolution, but that the correlation is absent when the problem is inverted. This outcome suggests that more comprehensive modeling of the crystallographic state is necessary to design high-resolution protein crystals from poorly diffracting crystals.

Dielectric Properties of a Protein Probed by Reversal of a Buried Ion Pair.

Thirty years ago, Hwang and Warshel suggested that a microenvironment preorganized to stabilize an ion pair would be incapable of reorganizing to stabilize the reverse ion pair. The implications were that (1) proteins have a limited capacity to reorganize, even under the influence of strong interactions, such as those present when ionizable groups are buried in the hydrophobic interior of a protein, and (2) the inability of proteins to tolerate the reversal of buried ion pairs demonstrates the limitations inherent to continuum electrostatic models of proteins. Previously we showed that when buried individually in the interior of staphylococcal nuclease, Glu23 and Lys36 have p Ka values near pH 7, but when buried simultaneously, they establish a strong interaction of ∼5 kcal/mol and have p Ka values shifted toward more normal values. Here, using equilibrium thermodynamic measurements, crystal structures, and NMR spectroscopy experiments, we show that although the reversed, individual substitutions-Lys23 and Glu36-also have p Ka values near 7, when buried together, they neither establish a strong interaction nor promote reorganization of their microenvironment. These experiments both confirm Warshel's original hypothesis and expand it by showing that it applies to reorganization, as demonstrated by our artificial ion pairs, as well as to preorganization as is commonly argued for motifs that stabilize naturally occurring ion pairs in polar microenvironments. These data constitute a challenging benchmark useful to test the ability of structure-based algorithms to reproduce the compensation between self-energy, Coulomb and polar interactions in hydrophobic environments of proteins.

Anomalous Properties of Lys Residues Buried in the Hydrophobic Interior of a Protein Revealed with 15N-Detect NMR Spectroscopy.

Ionizable residues buried in hydrophobic environments in proteins are essential for many fundamental biochemical processes. These residues titrate with anomalous pKa values that are challenging to reproduce with structure-based calculations owing to the conformational reorganization coupled to their ionization. Detailed characterization of this conformational reorganization is of interest; unfortunately, the properties of buried Lys residues are difficult to study experimentally. Here we demonstrate the utility of 15N NMR spectroscopy to gain insight into the protonation state, state of hydration and conformational dynamics of the Nζ amino group of buried Lys residues. The experiments were applied to five variants of staphylococcal nuclease, with internal Lys residues that titrate with pKa values ranging from 6.2 to 8.1. Direct detection of buried Lys residues with these NMR spectroscopy methods will enable correlation between thermodynamic and structural data as well as unprecedented examination of how conformational transitions coupled to their ionization affect their pKa values.

Local Backbone Flexibility as a Determinant of the Apparent pKa Values of Buried Ionizable Groups in Proteins.

Ionizable groups buried in the hydrophobic interior of proteins are essential for energy transduction. These groups can have highly anomalous pKa values that reflect the incompatibility between charges and dehydrated environments. A systematic study of pKa values of buried ionizable groups in staphylococcal nuclease (SNase) suggests that these pKa values are determined in part by conformational reorganization of the protein. Lys-66 is one of the most deeply buried residues in SNase. We show that its apparent pKa of 5.7 reflects the average of the pKa values of Lys-66 in different conformational states of the protein. In the fully folded state, Lys-66 is deeply buried in the hydrophobic core of SNase and must titrate with a pKa of ≪5.7. In other states, the side chain of Lys-66 is fully solvent-exposed and has a normal pKa of ≈10.4. We show that the pKa of Lys-66 can be shifted from 5.7 toward a more normal value of 7.1 via the insertion of flanking Gly residues at positions 64 and 67 to promote an "open" conformation of SNase. Crystal structures and nuclear magnetic resonance spectroscopy show that in these Gly-containing variants Lys-66 can access bulk water as a consequence of overwinding of the C-terminal end of helix 1. These data illustrate that the apparent pKa values of buried groups in proteins are governed in part by the difference in free energy between different conformational states of the protein and by differences in the pKa values of the buried groups in the different conformations.

Sharp-Tailed Grouse Nest Survival and Nest Predator Habitat Use in North Dakota's Bakken Oil Field.

Recent advancements in extraction technologies have resulted in rapid increases of gas and oil development across the United States and specifically in western North Dakota. This expansion of energy development has unknown influences on local wildlife populations and the ecological interactions within and among species. Our objectives for this study were to evaluate nest success and nest predator dynamics of sharp-tailed grouse (Tympanuchus phasianellus) in two study sites that represented areas of high and low energy development intensities in North Dakota. During the summers of 2012 and 2013, we monitored 163 grouse nests using radio telemetry. Of these, 90 nests also were monitored using miniature cameras to accurately determine nest fates and identify nest predators. We simultaneously conducted predator surveys using camera scent stations and occupancy modeling to estimate nest predator occurrence at each site. American badgers (Taxidea taxus) and striped skunks (Mephitis mephitis) were the primary nest predators, accounting for 56.7% of all video recorded nest depredations. Nests in our high intensity gas and oil area were 1.95 times more likely to succeed compared to our minimal intensity area. Camera monitored nests were 2.03 times more likely to succeed than non-camera monitored nests. Occupancy of mammalian nest predators was 6.9 times more likely in our study area of minimal gas and oil intensity compared to the high intensity area. Although only a correlative study, our results suggest energy development may alter the predator community, thereby increasing nest success for sharp-tailed grouse in areas of intense development, while adjacent areas may have increased predator occurrence and reduced nest success. Our study illustrates the potential influences of energy development on the nest predator-prey dynamics of sharp-tailed grouse in western North Dakota and the complexity of evaluating such impacts on wildlife.

Charges in Hydrophobic Environments: A Strategy for Identifying Alternative States in Proteins.

In the V23E variant of staphylococcal nuclease, Glu-23 has a pKa of 7.5. At low pH, Glu-23 is neutral and buried in the hydrophobic interior of the protein. Crystal structures and NMR spectroscopy experiments show that when Glu-23 becomes charged, the protein switches into an open state in which strands ß1 and ß2 separate from the ß-barrel; the remaining structure is unaffected. In the open state the hydrophobic interior of the protein is exposed to bulk water, allowing Glu-23 to become hydrated. This illustrates several key aspects of protein electrostatics: (1) The apparent pKa of an internal ionizable group can reflect the average of the very different pKa values (open ≈4.5, closed ≫7.5) sampled in the different conformational states. (2) The high apparent dielectric constant reported by the pKa value of internal ionizable group reflects conformational reorganization. (3) The apparent pKa of internal groups can be governed by large conformational changes. (4) A single charge buried in the hydrophobic interior of a protein is sufficient to convert what might have been a transient, partially unfolded state into the dominant state in solution. This suggests a general strategy for examining inaccessible regions of the folding landscape and for engineering conformational switches driven by small changes in pH. These data also constitute a benchmark for stringent testing of the ability of computational algorithms to predict pKa values of internal residues and to reproduce pH-driven conformational transitions of proteins.

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein.

An artificial charge pair buried in the hydrophobic core of staphylococcal nuclease was engineered by making the V23E and L36K substitutions. Buried individually, Glu-23 and Lys-36 both titrate with pKa values near 7. When buried together their pKa values appear to be normal. The ionizable moieties of the buried Glu-Lys pair are 2.6 Å apart. The interaction between them at pH 7 is worth 5 kcal/mol. Despite this strong interaction, the buried Glu-Lys pair destabilizes the protein significantly because the apparent Coulomb interaction is sufficient to offset the dehydration of only one of the two buried charges. Save for minor reorganization of dipoles and water penetration consistent with the relatively high dielectric constant reported by the buried ion pair, there is no evidence that the presence of two charges in the hydrophobic interior of the protein induces any significant structural reorganization. The successful engineering of an artificial ion pair in a highly hydrophobic environment suggests that buried Glu-Lys pairs in dehydrated environments can be charged and that it is possible to engineer charge clusters that loosely resemble catalytic sites in a scaffold protein with high thermodynamic stability, without the need for specialized structural adaptations.

Structural reorganization triggered by charging of Lys residues in the hydrophobic interior of a protein.

Structural consequences of ionization of residues buried in the hydrophobic interior of proteins were examined systematically in 25 proteins with internal Lys residues. Crystal structures showed that the ionizable groups are buried. NMR spectroscopy showed that in 2 of 25 cases studied, the ionization of an internal Lys unfolded the protein globally. In five cases, the internal charge triggered localized changes in structure and dynamics, and in three cases, it promoted partial or local unfolding. Remarkably, in 15 proteins, the ionization of the internal Lys had no detectable structural consequences. Highly stable proteins appear to be inherently capable of withstanding the presence of charge in their hydrophobic interior, without the need for specialized structural adaptations. The extent of structural reorganization paralleled loosely with global thermodynamic stability, suggesting that structure-based pK(a) calculations for buried residues could be improved by calculation of thermodynamic stability and by enhanced conformational sampling.

Thermodynamic principles for the engineering of pH-driven conformational switches and acid insensitive proteins.

The general thermodynamic principles behind pH driven conformational transitions of biological macromolecules are well understood. What is less obvious is how they can be used to engineer pH switches in proteins. The acid unfolding of staphylococcal nuclease (SNase) was used to illustrate different factors that can affect pH-driven conformational transitions. Acid unfolding is a structural transition driven by preferential H(+) binding to the acid unfolded state (U) over the native (N) state of a protein. It is the result of carboxylic groups that titrate with more normal pK(a) values in the U state than in the N state. Acid unfolding profiles of proteins reflect a balance between electrostatic and non-electrostatic contributions to stability. Several strategies were used in attempts to turn SNase into an acid insensitive protein: (1) enhancing global stability of the protein with mutagenesis or with osmolytes, (2) use of high salt concentrations to screen Coulomb interactions, (3) stabilizing the N state through specific anion effects, (4) removing Asp or Glu residues that titrate with depressed pK(a) values in the N state, and (5) removing basic residues that might have strong repulsive interactions in the N state at low pH. The only effective way to engineer acid resistance in SNase is not through modulation of pK(a) values of Asp/Glu but by enhancing the global stability of the protein. Modulation of pH-driven conformational transitions by selective manipulation of the electrostatic component of the switch is an extremely difficult undertaking.

An efficient data format for mass spectrometry-based proteomics.

The diverse range of mass spectrometry (MS) instrumentation along with corresponding proprietary and nonproprietary data formats has generated a proteomics community driven call for a standardized format to facilitate management, processing, storing, visualization, and exchange of both experimental and processed data. To date, significant efforts have been extended towards standardizing XML-based formats for mass spectrometry data representation, despite the recognized inefficiencies associated with storing large numeric datasets in XML. The proteomics community has periodically entertained alternate strategies for data exchange, e.g., using a common application programming interface or a database-derived format. However, these efforts have yet to gain significant attention, mostly because they have not demonstrated significant performance benefits over existing standards, but also due to issues such as extensibility to multidimensional separation systems, robustness of operation, and incomplete or mismatched vocabulary. Here, we describe a format based on standard database principles that offers multiple benefits over existing formats in terms of storage size, ease of processing, data retrieval times, and extensibility to accommodate multidimensional separation systems.