Engineering dynamical control of cell fate switching using synthetic phospho-regulons.

Many cells can sense and respond to time-varying stimuli, selectively triggering changes in cell fate only in response to inputs of a particular duration or frequency. A common motif in dynamically controlled cells is a dual-timescale regulatory network: although long-term fate decisions are ultimately controlled by a slow-timescale switch (e.g.

Aromatic cluster mutations produce focal modulations of β-sheet structure.

Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model β-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain "clusters" at highly solvent-exposed positions in the flat, single-layer β-sheet of Borrelia outer surface protein A (OspA).

High-resolution structure of a self-assembly-competent form of a hydrophobic peptide captured in a soluble beta-sheet scaffold.

beta-Rich self-assembly is a major structural class of polypeptides, but still little is known about its atomic structures and biophysical properties. Major impediments for structural and biophysical studies of peptide self-assemblies include their insolubility and heterogeneous composition. We have developed a model system, termed peptide self-assembly mimic (PSAM), based on the single-layer beta-sheet of Borrelia outer surface protein A.

Using engineered scaffold interactions to reshape MAP kinase pathway signaling dynamics.

Scaffold proteins link signaling molecules into linear pathways by physically assembling them into complexes. Scaffolds may also have a higher-order role as signal-processing hubs, serving as the target of feedback loops that optimize signaling amplitude and timing. We demonstrate that the Ste5 scaffold protein can be used as a platform to systematically reshape output of the yeast mating MAP kinase pathway.

Beta-strand flipping and slipping triggered by turn replacement reveal the opportunistic nature of beta-strand pairing.

We investigated how the register between adjacent beta-strands is specified using a series of mutants of the single-layer beta-sheet (SLB) in Borrelia OspA. The single-layer architecture of this system eliminates structural restraints imposed by a hydrophobic core, enabling us to address this question. A critical turn (turn 9/10) in the SLB was replaced with a segment with an intentional structural mismatch.

Hydrophobic surface burial is the major stability determinant of a flat, single-layer beta-sheet.

Formation of a flat beta-sheet is a fundamental event in beta-sheet-mediated protein self-assembly. To investigate the contributions of various factors to the stability of flat beta-sheets, we performed extensive alanine-scanning mutagenesis experiments on the single-layer beta-sheet segment of Borrelia outer surface protein A (OspA). This beta-sheet segment consists of beta-strands with highly regular geometries that can serve as a building block for self-assembly.

Atomic structures of peptide self-assembly mimics.

Although the beta-rich self-assemblies are a major structural class for polypeptides and the focus of intense research, little is known about their atomic structures and dynamics due to their insoluble and noncrystalline nature. We developed a protein engineering strategy that captures a self-assembly segment in a water-soluble molecule. A predefined number of self-assembling peptide units are linked, and the beta-sheet ends are capped to prevent aggregation, which yields a mono-dispersed soluble protein.

Atomic-resolution crystal structure of Borrelia burgdorferi outer surface protein A via surface engineering.

Outer surface protein A (OspA) from Borrelia burgdorferi has an unusual dumbbell-shaped structure in which two globular domains are connected with a "single-layer" beta-sheet (SLB). The protein is highly soluble, and it has been recalcitrant to crystallization. Only OspA complexes with Fab fragments have been successfully crystallized.

Conformational heterogeneity of an equilibrium folding intermediate quantified and mapped by scanning mutagenesis.

It is challenging to experimentally define an energy landscape for protein folding that comprises multiple partially unfolded states. Experimental results are often ambiguous as to whether a non-native state is conformationally homogeneous. Here, we tested an approach combining systematic mutagenesis and a Brønsted-like analysis to reveal and quantify conformational heterogeneity of folding intermediate states.

Thermodynamic and kinetic exploration of the energy landscape of Borrelia burgdorferi OspA by native-state hydrogen exchange.

We report a native-state hydrogen-exchange (HX) method to simultaneously obtain both thermodynamic and kinetic information on the formation of multiple excited states in a folding energy landscape. Our method exploits the inherent dispersion and pH dependence of the intrinsic HX rates to cover both the EX2 (thermodynamic) and EX1 (kinetic) regimes. At each concentration of denaturant, HX measurements are performed over a range of pH values.