Incorporating an allosteric regulatory site in an antibody through backbone design

Allosteric regulation underlies living cells' ability to sense changes in nutrient and signaling‐molecule concentrations, but the ability to computationally design allosteric regulation into non‐allosteric proteins has been elusive. Allosteric‐site design is complicated by the requirement to encode the relative stabilities of active and inactive conformations of the same protein in the presence and absence of both ligand and effector. To address this challenge, we used Rosetta to design the backbone of the flexible heavy‐chain complementarity‐determining region 3 (HCDR3), and used geometric matching and sequence optimization to place a Zn²⁺‐coordination site in a fluorescein‐binding antibody. We predicted that due to HCDR3's flexibility, the fluorescein‐binding pocket would configure properly only upon Zn²⁺ application. We found that regulation by Zn²⁺ was reversible and sensitive to the divalent ion's identity, and came at the cost of reduced antibody stability and fluorescein‐binding affinity. Fluorescein bound at an order of magnitude higher affinity in the presence of Zn²⁺ than in its absence, and the increase in fluorescein affinity was due almost entirely to faster fluorescein on‐rate, suggesting that Zn²⁺ preorganized the antibody for fluorescein binding. Mutation analysis demonstrated the extreme sensitivity of Zn²⁺ regulation on the atomic details in and around the metal‐coordination site. The designed antibody could serve to study how allosteric regulation evolved from non‐allosteric binding proteins, and suggests a way to designing molecular sensors for environmental and biomedical targets.     

Constitutive regulatory activity of an evolutionarily excluded riboswitch variant

The exquisite specificity of the adenine-responsive riboswitch toward its cognate metabolite has been shown to arise from the formation of a Watson-Crick interaction between the adenine ligand and residue U65. A recent crystal structure of a U65C adenine aptamer variant has provided a rationale for the phylogenetic conservation observed at position 39 for purine aptamers. The G39-C65 variant adopts a compact ligand-free structure in which G39 is accommodated by the ligand binding site and is base-paired to the cytosine at position 65. Here, we demonstrate using a combination of biochemical and biophysical techniques that the G39-C65 base pair not only severely impairs ligand binding but also disrupts the functioning of the riboswitch in vivo by constitutively activating gene expression. Folding studies using single-molecule FRET revealed that the G39-C65 variant displays a low level of dynamic heterogeneity, a feature reminiscent of ligand-bound wild-type complexes. A restricted conformational freedom together with an ability to significantly fold in monovalent ions are exclusive to the G39-C65 variant. This work provides a mechanistic framework to rationalize the evolutionary exclusion of certain nucleotide combinations in favor of sequences that preserve ligand binding and gene regulation functionalities.     

Introduction of a metal-dependent regulatory switch into an enzyme

A cysteine has been introduced into the hydrophobic binding pocket of staphylococcal nuclease via oligonucleotide-directed mutagenesis. The L89C mutation does not significantly alter the catalytic activity or specificity of the nuclease yet provides a metal-dependent switch for regulating enzymatic activity. The L89C mutant can be inactivated by addition of mercuric or cupric salts and subsequently reactivated by addition of chelating agents. This work may provide a general strategy for regulating the catalytic activity of other enzymes or the binding affinity of proteins to DNA or other proteins.     

Tyrosine phosphorylation: an emerging regulatory device of bacterial physiology

Tyrosine phosphorylation is a key device in numerous cellular functions in eukaryotes, but in bacteria this protein modification was largely ignored until the mid-1990s. The first conclusive evidence of bacterial tyrosine phosphorylation came only a decade ago. Since then, several tyrosine kinases exhibiting unexpected features have been identified in a variety of bacteria. These enzymes use homologues of Walker motifs of nucleotide-binding proteins for their catalytic mechanism, thus defining an idiosyncratic type of bacterial tyrosine kinases. Recently, bacterial tyrosine kinases have been found to phosphorylate an increasing list of endogenous protein substrates. This discovery contributes to the emerging picture that bacterial tyrosine phosphorylation is an important regulatory arsenal of bacterial physiology in addition to the classical serine/threonine kinases, and the 'two-component' and phosphotransferase systems.     

Continuous production of phenylalanine using an Escherichia coli regulatory mutant

Summary Phenylalanine synthesis from glucose and ammonia was studied using a hyperproducing mutant of Escherichia coli. Kinetic parameters (typical values : 8.7 g phenylalanine/l, yield on glucose 0.19 g/g, productivity 0.44 g/l/h) were similar to batch culture values.     

Perspectives on an alternative career path in regulatory science

Perspectives are provided on an alternative career path in regulatory science for those currently involved in basic biology research. This path is compared and contrasted with basic research, and factors to be examined if one is considering such a path are discussed.     

Is there an osmotic regulatory mechanism in algae and higher plants?

Under water-stress conditions the amounts of various polyols and also of the imino acid proline are found to increase significantly in different algae and higher plants. These substances have been interpreted until now to act osmotically by increasing the concentration in the cell, thus causing water reflux and balancing osmotic pressure difference from outside the cell to inside.A new concept is described, which proposes that the regulatory function of these accumulating substances is conducted by two mechanisms quite different from osmotic regulation. It is assumed that these regulatory pathways are connected with the hydrophobic groups of biopolymers in the cell cytoplasm. (1) Polyols can replace water molecules by means of their water like OH-groups and thus participate in the hydrophobically enforced water structure. (2) Proline is postulated to associate via its hydrophobic phot with hydrophobic side chains, thereby converting them into hydrophilic groups by exposure the carboxylic and imino group versus water molecules. The advantage is due to the fact that water associated with hydrophilic groups is bound via hydrogen bonding forces, in contrary to hydrophobic groups. In addition, the number of water molecules adjoining hydrophilic groups is far less than those involved with hydrophobic residues. By means of these two alternative mechanisms complete hydration of the biopolymers is maintained, even with a reduced number of available water molecules.     

Stability analysis of an artificial biomolecular oscillator with non-cooperative regulatory interactions

Oscillators are essential to fuel autonomous behaviours in molecular systems. Artificial oscillators built with programmable biological molecules such as DNA and RNA are generally easy to build and tune, and can serve as timers for biological computation and regulation. We describe a new artificial nucleic acid biochemical reaction network, and we demonstrate its capacity to exhibit oscillatory solutions. This network can be built in vitro using nucleic acids and three bacteriophage enzymes, and has the potential to be implemented in cells. Numerical simulations suggest that oscillations occur in a realistic range of reaction rates and concentrations.     

Sterol regulatory element-binding proteins induce an entire pathway of cholesterol synthesis

To evaluate the effects of sterol regulatory element-binding proteins (SREBPs) on the expression of the individual enzymes in the cholesterol synthetic pathway, we examined expression of these genes in the livers from wild-type and transgenic mice overexpressing nuclear SREBP-1a or -2. As estimated by a Northern blot analysis, overexpression of nuclear SREBP-1a or -2 caused marked increases in mRNA levels of the whole battery of cholesterogenic genes. This SREBP activation covers not only rate-limiting enzymes such as HMG CoA synthase and reductase that have been well established as SREBP targets, but also all the enzyme genes in the cholesterol synthetic pathway tested here. The activated genes include mevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl phosphate isomerase, geranylgeranyl pyrophosphate synthase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, lanosterol synthase, lanosterol demethylase, and 7-dehydro-cholesterol reductase. These results demonstrate that SREBPs activate every step of cholesterol synthetic pathway, contributing to an efficient cholesterol synthesis.