Identification of positions in human aldolase a that are neutral for apparent K.

According to evolutionary theory, many naturally-occurring amino acid substitutions are expected to be neutral or near-neutral, with little effect on protein structure or function. Accordingly, most changes observed in human exomes are also expected to be neutral. As such, accurate algorithms for identifying medically-relevant changes must discriminate rare, non-neutral substitutions against a background of neutral substitutions.

Dynamics-based protein network features accurately discriminate neutral and rheostat positions.

In some proteins, a unique class of nonconserved positions is characterized by their ability to generate diverse functional outcomes through single amino acid substitutions. Due to their ability to tune protein function, accurately identifying such "rheostat" positions is crucial for protein design, for understanding the impact of mutations observed in humans, and for predicting the evolution of pathogen drug resistance. However, identifying rheostat positions has been challenging, due-in part-to the absence of a clear structural relationship with binding sites.

Rheostatic contributions to protein stability can obscure a position's functional role.

Rheostat positions, which can be substituted with various amino acids to tune protein function across a range of outcomes, are a developing area for advancing personalized medicine and bioengineering. Current methods cannot accurately predict which proteins contain rheostat positions or their substitution outcomes. To compare the prevalence of rheostat positions in homologs, we previously investigated their occurrence in two pyruvate kinase (PYK) isozymes.

Fructose-1-kinase has pleiotropic roles in Escherichia coli.

In Escherichia coli, the master transcription regulator catabolite repressor activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand.

Rheostats, toggles, and neutrals, Oh my! A new framework for understanding how amino acid changes modulate protein function.

Advances in personalized medicine and protein engineering require accurately predicting outcomes of amino acid substitutions. Many algorithms correctly predict that evolutionarily-conserved positions show "toggle" substitution phenotypes, which is defined when a few substitutions at that position retain function. In contrast, predictions often fail for substitutions at the less-studied "rheostat" positions, which are defined when different amino acid substitutions at a position sample at least half of the possible functional range.

Fructose-1-kinase has pleiotropic roles in .

In , the master transcription regulator Catabolite Repressor Activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand.

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new paradigm?

During protein evolution, some amino acid substitutions modulate protein function ("tuneability"). In most proteins, the tuneable range is wide and can be sampled by a set of protein variants that each contains multiple amino acid substitutions. In other proteins, the full tuneable range can be accessed by a set of variants that each contains a single substitution.

Simulated pressure changes in LacI suggest a link between hydration and functional conformational changes.

The functions of many proteins are associated with interconversions among conformational substates. However, these substates can be difficult to measure experimentally, and determining contributions from hydration changes can be especially difficult. Here, we assessed the use of pressure perturbations to sample the substates accessible to the Escherichia coli lactose repressor protein (LacI) in various liganded forms.

FUS G559A Mutation in a Patient with a Frontotemporal Dementia-Motor Neuron Disease Compatible Syndrome: A Case Report.

Fused in sarcoma (FUS) mutations cause frontotemporal dementia (FTD) and motor neuron disease (MND). Here, we describe a 43-year-old man with progressive behavioral and cognitive change, myelopathy, clinical and electrophysiologic evidence of MND, and a FUS variant of unknown significance (VUS). This VUS, a heterozygous G559A transition (Gly187Ser), was previously reported in a patient with sporadic MND and affects important FUS biophysical properties.

The 2.4 Å structure of Zymomonas mobilis pyruvate kinase: Implications for stability and regulation.

Human liver pyruvate kinase (hlPYK) catalyzes the final step in glycolysis, the formation of pyruvate (PYR) and ATP from phosphoenolpyruvate (PEP) and ADP. Fructose 1,6-bisphosphate (FBP), a pathway intermediate of glycolysis, serves as an allosteric activator of hlPYK. Zymomonas mobilis pyruvate kinase (ZmPYK) performs the final step of the Entner-Doudoroff pathway, which is similar to glycolysis in that energy is harvested from glucose and pyruvate is generated.

Identification of a covert evolutionary pathway between two protein folds.

Although homologous protein sequences are expected to adopt similar structures, some amino acid substitutions can interconvert α-helices and β-sheets. Such fold switching may have occurred over evolutionary history, but supporting evidence has been limited by the: (1) abundance and diversity of sequenced genes, (2) quantity of experimentally determined protein structures, and (3) assumptions underlying the statistical methods used to infer homology. Here, we overcome these barriers by applying multiple statistical methods to a family of ~600,000 bacterial response regulator proteins.

PYK-SubstitutionOME: an integrated database containing allosteric coupling, ligand affinity and mutational, structural, pathological, bioinformatic and computational information about pyruvate kinase isozymes.

Interpreting changes in patient genomes, understanding how viruses evolve and engineering novel protein function all depend on accurately predicting the functional outcomes that arise from amino acid substitutions. To that end, the development of first-generation prediction algorithms was guided by historic experimental datasets. However, these datasets were heavily biased toward substitutions at positions that have not changed much throughout evolution (i.

Odd one out? Functional tuning of Zymomonas mobilis pyruvate kinase is narrower than its allosteric, human counterpart.

Various protein properties are often illuminated using sequence comparisons of protein homologs. For example, in analyses of the pyruvate kinase multiple sequence alignment, the set of positions that changed during speciation ("phylogenetic" positions) were enriched for "rheostat" positions in human liver pyruvate kinase (hLPYK). (Rheostat positions are those which, when substituted with various amino acids, yield a range of functional outcomes).

Structural Plasticity Is a Feature of Rheostat Positions in the Human Na/Taurocholate Cotransporting Polypeptide (NTCP).

In the Na/taurocholate cotransporting polypeptide (NTCP), the clinically relevant S267F polymorphism occurs at a "rheostat position". That is, amino acid substitutions at this position ("S267X") lead to a wide range of functional outcomes. This result was particularly striking because molecular models predicted the S267X side chains are buried, and thus, usually expected to be less tolerant of substitutions.

Substitutions at a rheostat position in human aldolase A cause a shift in the conformational population.

Some protein positions play special roles in determining the magnitude of protein function: at such "rheostat" positions, varied amino acid substitutions give rise to a continuum of functional outcomes, from wild type (or enhanced), to intermediate, to loss of function. This observed range raises interesting questions about the biophysical bases by which changes at single positions have such varied outcomes. Here, we assessed variants at position 98 in human aldolase A ("I98X").

Spectroscopic evidence of tetanus toxin translocation domain bilayer-induced refolding and insertion.

Tetanus neurotoxin (TeNT) is an A-B toxin with three functional domains: endopeptidase, translocation (HCT), and receptor binding. Endosomal acidification triggers HCT to interact with and insert into the membrane, translocating the endopeptidase across the bilayer. Although the function of HCT is well defined, the mechanism by which it accomplishes this task is unknown.

Allosteric regulation within the highly interconnected structural scaffold of AraC/XylS homologs tolerates a wide range of amino acid changes.

To create bacterial transcription "circuits" for biotechnology, one approach is to recombine natural transcription factors, promoters, and operators. Additional novel functions can be engineered from existing transcription factors such as the E. coli AraC transcriptional activator, for which binding to DNA is modulated by binding L-arabinose.

Rheostat functional outcomes occur when substitutions are introduced at nonconserved positions that diverge with speciation.

When amino acids vary during evolution, the outcome can be functionally neutral or biologically-important. We previously found that substituting a subset of nonconserved positions, "rheostat" positions, can have surprising effects on protein function. Since changes at rheostat positions can facilitate functional evolution or cause disease, more examples are needed to understand their unique biophysical characteristics.

A clinically relevant polymorphism in the Na/taurocholate cotransporting polypeptide (NTCP) occurs at a rheostat position.

Conventionally, most amino acid substitutions at "important" protein positions are expected to abolish function. However, in several soluble-globular proteins, we identified a class of nonconserved positions for which various substitutions produced progressive functional changes; we consider these evolutionary "rheostats". Here, we report a strong rheostat position in the integral membrane protein, Na/taurocholate (TCA) cotransporting polypeptide, at the site of a pharmacologically relevant polymorphism (S267F).

Substitutions at Nonconserved Rheostat Positions Modulate Function by Rewiring Long-Range, Dynamic Interactions.

Amino acid substitutions at nonconserved protein positions can have noncanonical and "long-distance" outcomes on protein function. Such outcomes might arise from changes in the internal protein communication network, which is often accompanied by changes in structural flexibility. To test this, we calculated flexibilities and dynamic coupling for positions in the linker region of the lactose repressor protein.

Rheostat positions: A new classification of protein positions relevant to pharmacogenomics.

To achieve the full potential of pharmacogenomics, one must accurately predict the functional out comes that arise from amino acid substitutions in proteins. Classically, researchers have focused on understanding the consequences of individual substitutions. However, literature surveys have shown that most substitutions were created at evolutionarily conserved positions.

Identification of biochemically neutral positions in liver pyruvate kinase.

Understanding how each residue position contributes to protein function has been a long-standing goal in protein science. Substitution studies have historically focused on conserved protein positions. However, substitutions of nonconserved positions can also modify function.

The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules.

Every method used to quantify biomolecular interactions has its own strengths and limitations. To quantify protein-DNA binding affinities, nitrocellulose filter binding assays with P-labeled DNA quantify K values from 10 to 10 M but have several technical limitations. Here, we considered the suitability of biolayer interferometry (BLI), which monitors association and dissociation of a soluble macromolecule to an immobilized species; the ratio k /k determines K .

Functional tunability from a distance: Rheostat positions influence allosteric coupling between two distant binding sites.

For protein mutagenesis, a common expectation is that important positions will behave like on/off "toggle" switches (i.e., a few substitutions act like wildtype, most abolish function).

Homolog comparisons further reconcile in vitro and in vivo correlations of protein activities by revealing over-looked physiological factors.

To bridge biological and biochemical disciplines, the relationship between in vitro protein biochemical function and in vivo activity must be established. Such studies can (a) help determine whether properties measured in simple, dilute solutions extrapolate to the complex in vivo conditions and (b) illuminate cryptic biological factors that are new avenues for study. We have explored the in vivo-in vitro relationship for chimeras built from LacI/GalR transcription regulators.