Discovery of Thermostable, Fluorescently Responsive Glucose Biosensors by Structure-Assisted Function Extrapolation.

Accurate assignment of protein function from sequence remains a fascinating and difficult challenge. The periplasmic-binding protein (PBP) superfamily present an interesting case of function prediction because they are both ubiquitous in prokaryotes and tend to diversify through gene duplication "explosions" that can lead to large numbers of paralogs in a genome. An engineered version of the moderately thermostable glucose-binding PBP from Escherichia coli has been used successfully as a reagentless fluorescent biosensor both in vitro and in vivo. To develop more robust sensors that meet the challenges of real-world applications, we report the discovery of thermostable homologues that retain a glucose-mediated conformationally coupled fluorescence response. Accurately identifying a glucose-binding PBP homologue among closely related paralogs is challenging. We demonstrate that a structure-based method that filters sequences by residues that bind glucose in an archetype structure is highly effective. Using fully sequenced bacterial genomes, we found that this filter reduced high paralog numbers to single hits in a genome, consistent with the accurate separation of glucose binding from other functions. We expressed engineered proteins for eight homologues, chosen to represent different degrees of sequence identity, and tested their glucose-mediated fluorescence responses. We accurately predicted the presence of glucose binding down to 31% sequence identity. We have also successfully identified suitable candidates for next-generation robust, fluorescent glucose sensors.

Harnessing Environmental Ca2+ for Extracellular Protein Thermostabilization.

Ca2+ is the third-most prevalent metal ion in the environment. EF hands are common Ca2+-binding motifs found in both extracellular and intracellular proteins of eukaryotes and prokaryotes. Cytoplasmic EF hand proteins often mediate allosteric control of signal transduction pathway components in response to intracellular Ca2+ concentration fluctuations by coupling Ca2+ binding to changes in protein structure. We show that an extracellular structural Ca2+-binding site mediates protein thermostabilization by such conformational coupling as well. Binding Ca2+ to the EF hand of the extracellular (periplasmic) Escherichia coli glucose-galactose binding protein thermostabilizes this protein by ∼17 K relative to its Ca2+-free form. Using statistical thermodynamic analysis of a fluorescent conjugate of ecGGBP that reports simultaneously on ligand binding and multiple conformational states, we found that its Ca2+-mediated stabilization is determined by conformational coupling mechanisms in two independent conformational exchange reactions. Binding to folded and unfolded states determines the maximum Ca2+-mediated stability. A disorder → order transition accompanies the formation of the Ca2+ complex in the folded state and dictates the minimum Ca2+ concentration at which the Ca2+-bound state becomes dominant. Similar transitions also encode the structural changes necessary for Ca2+-mediated control elements in signal transduction pathways. Ca2+-mediated thermostabilization and allosteric control, therefore, share a fundamental conformational coupling mechanism, which may have implications for the evolution of EF hands.

Accurate Identification of Periplasmic Urea-binding Proteins by Structure- and Genome Context-assisted Functional Analysis.

ABC transporters are ancient and ubiquitous nutrient transport systems in bacteria and play a central role in defining lifestyles. Periplasmic solute-binding proteins (SBPs) are components that deliver ligands to their translocation machinery. SBPs have diversified to bind a wide range of ligands with high specificity and affinity. However, accurate assignment of cognate ligands remains a challenging problem in SBPs. Urea metabolism plays an important role in the nitrogen cycle; anthropogenic sources account for more than half of global nitrogen fertilizer. We report identification of urea-binding proteins within a large SBP sequence family that encodes diverse functions. By combining genetic linkage between SBPs, ABC transporter components, enzymes or transcription factors, we accurately identified cognate ligands, as we verified experimentally by biophysical characterization of ligand binding and crystallographic determination of the urea complex of a thermostable urea-binding homolog. Using three-dimensional structure information, these functional assignments were extrapolated to other members in the sequence family lacking genetic linkage information, which revealed that only a fraction bind urea. Using the same combined approaches, we also inferred that other family members bind various short-chain amides, aliphatic amino acids (leucine, isoleucine, valine), γ-aminobutyrate, and as yet unknown ligands. Comparative structural analysis revealed structural adaptations that encode diversification in these SBPs. Systematic assignment of ligands to SBP sequence families is key to understanding bacterial lifestyles, and also provides a rich source of biosensors for clinical and environmental analysis, such as the thermostable urea-binding protein identified here.

Chromophore carbonyl twisting in fluorescent biosensors encodes direct readout of protein conformations with multicolor switching.

Fluorescent labeling of proteins is a powerful tool for probing structure-function relationships with many biosensing applications. Structure-based rules for systematically designing fluorescent biosensors require understanding ligand-mediated fluorescent response mechanisms which can be challenging to establish. We installed thiol-reactive derivatives of the naphthalene-based fluorophore Prodan into bacterial periplasmic glucose-binding proteins. Glucose binding elicited paired color exchanges in the excited and ground states of these conjugates. X-ray structures and mutagenesis studies established that glucose-mediated color switching arises from steric interactions that couple protein conformational changes to twisting of the Prodan carbonyl relative to its naphthalene plane. Mutations of residues contacting the carbonyl can optimize color switching by altering fluorophore conformational equilibria in the apo and glucose-bound proteins. A commonly accepted view is that Prodan derivatives report on protein conformations via solvatochromic effects due to changes in the dielectric of their local environment. Here we show that instead Prodan carbonyl twisting controls color switching. These insights enable structure-based biosensor design by coupling ligand-mediated protein conformational changes to internal chromophore twists through specific steric interactions between fluorophore and protein.

Describing Complex Structure-Function Relationships in Biomolecules at Equilibrium.

One of the great ambitions of structural biology is to describe structure-function relationships quantitatively. Statistical thermodynamics is a powerful, general tool for computing the behavior of biological macromolecules at equilibrium because it establishes a direct link between structure and function. Complex behavior emerges as equilibria of multiple reactions are coupled. Analytical treatment of linked equilibria scales poorly with increasing numbers of reactions and states as the algebraic constructs rapidly become unwieldy. We therefore developed a generalizable, but straightforward computational method to handle arbitrarily complex systems. To demonstrate this approach, we collected a multidimensional fluorescence landscape of an engineered fluorescent glucose biosensor and showed that its features could be modeled with ten intricately linked ligand-binding and conformational exchange reactions. This protein represents a minimalist model of sufficient complexity to encompass fundamental biomolecular structure-function relationships: two-state and multistate conformational ensembles, conformational hierarchies, osmolytes, coupling between different binding sites and coupling between ligand binding and conformational change. The successful fit of this complex, multifaceted system demonstrates generality of the method.

Local encoding of computationally designed enzyme activity.

One aim of computational protein design is to introduce novel enzyme activity into proteins of known structure by predicting mutations that stabilize transition states. Previously, we showed that it is possible to introduce triose phosphate isomerase activity into the ribose-binding protein of Escherichia coli by constructing 17 mutations in the first two layers of residues that surround the wild-type ligand-binding site. Here, we report that these mutations can be "transplanted" into a homologous ribose-binding protein, isolated from the hyperthermophilic bacterium Thermoanaerobacter tengcongensis, with retention of catalytic activity, substrate affinity, and reaction pH dependence. The observed 10(5)-10(6)-fold rate enhancement corresponds to 70% of the maximally known transition-state binding energy. The wild-type sequences in these two homologues are almost perfectly conserved in the vicinity of their ribose-binding sites, but diverge significantly at increasing distance from these sites. The results demonstrate that the computationally designed mutations are sufficient to encode the observed enzyme activity, that all the observed activity is encoded locally within the layer of residues directly in contact with the substrate and that, in this case, at least 70% of transition state stabilization energy can be achieved using straightforward considerations of stereochemical complementarity between enzyme and reactants.

The backbone structure of the thermophilic Thermoanaerobacter tengcongensis ribose binding protein is essentially identical to its mesophilic E. coli homolog.

BACKGROUND: Comparison of experimentally determined mesophilic and thermophilic homologous protein structures is an important tool for understanding the mechanisms that contribute to thermal stability. Of particular interest are pairs of homologous structures that are structurally very similar, but differ significantly in thermal stability. RESULTS: We report the X-ray crystal structure of a Thermoanaerobacter tengcongensis ribose binding protein (tteRBP) determined to 1.9 A resolution. We find that tteRBP is significantly more stable (appTm value approximately 102 degrees C) than the mesophilic Escherichia coli ribose binding protein (ecRBP) (appTm value ~56 degrees C). The tteRBP has essentially the identical backbone conformation (0.41 A RMSD of 235/271 Calpha positions and 0.65 A RMSD of 270/271 Calpha positions) as ecRBP. Classification of the amino acid substitutions as a function of structure therefore allows the identification of amino acids which potentially contribute to the observed thermal stability of tteRBP in the absence of large structural heterogeneities. CONCLUSION: The near identity of backbone structures of this pair of proteins entails that the significant differences in their thermal stabilities are encoded exclusively by the identity of the amino acid side-chains. Furthermore, the degree of sequence divergence is strongly correlated with structure; with a high degree of conservation in the core progressing to increased diversity in the boundary and surface regions. Different factors that may possibly contribute to thermal stability appear to be differentially encoded in each of these regions of the protein. The tteRBP/ecRBP pair therefore offers an opportunity to dissect contributions to thermal stability by side-chains alone in the absence of large structural differences.

Multifactorial determinants of protein expression in prokaryotic open reading frames.

A quantitative description of the relationship between protein expression levels and open reading frame (ORF) nucleotide sequences is important for understanding natural systems, designing synthetic systems, and optimizing heterologous expression. Codon identity, mRNA secondary structure, and nucleotide composition within ORFs markedly influence expression levels. Bioinformatic analysis of ORF sequences in 816 bacterial genomes revealed that these features show distinct regional trends. To investigate their effects on protein expression, we designed 285 synthetic genes and determined corresponding expression levels in vitro using Escherichia coli extracts. We developed a mathematical function, parameterized using this synthetic gene data set, which enables computation of protein expression levels from ORF nucleotide sequences. In addition to its practical application in the design of heterologous expression systems, this equation provides mechanistic insight into the factors that control translation efficiency. We found that expression is strongly dependent on the presence of high AU content and low secondary structure in the ORF 5' region. Choice of high-frequency codons contributes to a lesser extent. The 3' terminal AU content makes modest, but detectable contributions. We present a model for the effect of these factors on the three phases of ribosomal function: initiation, elongation, and termination.

Noncovalent binding of a reaction intermediate by a designed helix-loop-helix motif-implications for catalyst design.

In our search for a catalyst for the transamination reaction of aspartic acid to form oxaloacetate, twenty-five forty-two-residue sequences were designed to fold into helix-loop-helix dimers and form binding sites for the key intermediate along the reaction pathway, the aldimine. This intermediate is formed from aspartic acid and the cofactor pyridoxal phosphate. The design of the binding sites followed a strategy in which exclusively noncovalent forces were used for binding the aldimine. Histidine residues were incorporated to catalyse the rate-limiting 1,3 proton transfer reaction that converts the aldimine into the ketimine, an intermediate that is subsequently hydrolysed to form oxaloacetate and pyridoxamine phosphate. The two most efficient catalysts, T-4 and T-16, selected from the pool of sequences by a simple screening procedure, were shown by CD and NMR spectroscopies to bind the aldimine intermediate with dissociation constants in the millimolar range. The mean residue ellipticity of T-4 in aqueous solution at pH 7.4 and a concentration of 0.75 mM was -18500 deg x cm(2) dmol(-1). Upon addition of 6 mm l-aspartic acid and 1.5 mM pyridoxal phosphate to form the aldimine, the mean residue ellipticity changed to -19900 deg x cm(2) dmol(-1). The corresponding mean residue ellipticities of T-16 were -21200 deg x cm(2) dmol(-1) and -24000 deg x cm(2) dmol(-1). These results show that the helical content increased in the presence of the aldimine, and that the folded polypeptides bound the aldimine. The (1)H NMR relaxation time of the imine CH proton of the aldimine was affected by the presence of T-4 as was the (31)P NMR resonance linewidth. The catalytic efficiencies of T-4 and T-16 were compared to that of imidazole and found to be more than three orders of magnitude larger. The designed binding sites were thus shown to be capable of binding the aldimine in close proximity to His residues, by noncovalent forces, into conformations that proved to be catalytically active. The results show for the first time the design of well-defined catalytic sites that bind a reaction intermediate with enzyme-like affinities under equilibrium conditions and represent an important advance in de novo catalyst design.

Setting the stage for new catalytic functions in designed proteins--exploring the imine pathway in the efficient decarboxylation of oxaloacetate by an Arg-Lys site in a four-helix bundle protein scaffold.

Fourteen 42-residue polypeptides have been designed to identify reactive sites for the catalysis of the decarboxylation of oxaloacetate, a chemical transformation that proceeds through the formation of an imine intermediate. The sequences fold into helix-loop-helix motifs and dimerize to four-helix bundles. The catalytically active lysine residues were incorporated in several surface exposed positions, but also in positions characterised by hydrophobic properties to reduce their pKa values. The molecular environments of the Lys residues were systematically varied, to find which residues were able to stabilise and bind the imine intermediate in the decarboxylation reaction. A two-residue Arg-Lys site formed the main component of the reactive site of the helix-loop-helix dimer Decarb-K34_R33, which obeyed saturation kinetics in catalysing the reaction with a kcat/KM of 0.59 M-1S-1. The rate constant measured was nearly three orders of magnitude larger than the second-order rate constant of the butylamine-catalysed reaction (0.0011 M-1S-1), and four orders of magnitude larger than the pseudo first-order rate constant of the uncatalyzed reaction (1.3 x 10(-5) s(-1)). The sequence of Decarb-K34_R33 contained only a single lysine residue. It was flanked by an arginine in the preceding position in the sequence. A flanking Arg residue provided more efficient catalysis than a flanking Lys or Gln residue. Arginines in flanking positions in the helix, in positions four residues before or after the Lys in the sequence, are not as important in catalysis as the Arg of the Arg-Lys pair. The effect of pKa on the catalytic efficiency of the Lys residue in the decarboxylation reaction is well known. The identification of the role of the flanking Arg residue in catalysing decarboxylation, its optimal position, and the importance of conformational stability reported here sets the stage for developing a number of catalytic systems that depend on the formation of imine intermediates, but that lead to different reaction products.

Computational design of receptors for an organophosphate surrogate of the nerve agent soman.

We report the computational design of soluble protein receptors for pinacolyl methyl phosphonic acid (PMPA), the predominant hydrolytic product of the nerve agent soman. Using recently developed computational protein design techniques, the ligand-binding pockets of two periplasmic binding proteins, glucose-binding protein and ribose-binding protein, were converted to bind PMPA instead of their cognate sugars. The designs introduce 9-12 mutations in the parent proteins. Twelve of 20 designs tested exhibited PMPA-dependent changes in emission intensity of a fluorescent reporter with affinities between 45 nM and 10 microM. The contributions to ligand binding by individual residues were determined in two designs by alanine-scanning mutagenesis, and are consistent with the molecular models. These results demonstrate that designed receptors with radically altered binding specificities and affinities that rival or exceed those of the parent proteins can be successfully predicted. The designs vary in parent scaffold, sequence diversity, and orientation of docked ligand, suggesting that the number of possible solutions to the design problem is large and degenerate. This observation has implications for the genesis of biological function by random processes. The designed receptors reported here may have utility in the development of fluorescent biosensors for monitoring nerve agents.