Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats.

Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended ß-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.

Quantitative Affinity Determination by Fluorescence Anisotropy Measurements of Individual Nanoliter Droplets.

Fluorescence anisotropy measurements of reagents compartmentalized into individual nanoliter droplets are shown to yield high-resolution binding curves from which precise dissociation constants (Kd) for protein-peptide interactions can be inferred. With the current platform, four titrations can be obtained per minute (based on ∼100 data points each), with stoichiometries spanning more than 2 orders of magnitude and requiring only tens of microliters of reagents. In addition to affinity measurements with purified components, Kd values for unpurified proteins in crude cell lysates can be obtained without prior knowledge of the concentration of the expressed protein, so that protein purification can be avoided. Finally, we show how a competition assay can be set up to perform focused library screens, so that compound labeling is not required anymore. These data demonstrate the utility of droplet compartments for the quantitative characterization of biomolecular interactions and establish fluorescence anisotropy imaging as a quantitative technique in a miniaturized droplet format, which is shown to be as reliable as its macroscopic test tube equivalent.

Interfacing microwells with nanoliter compartments: a sampler generating high-resolution concentration gradients for quantitative biochemical analyses in droplets.

Analysis of concentration dependencies is key to the quantitative understanding of biological and chemical systems. In experimental tests involving concentration gradients such as inhibitor library screening, the number of data points and the ratio between the stock volume and the volume required in each test determine the quality and efficiency of the information gained. Titerplate assays are currently the most widely used format, even though they require microlitre volumes. Compartmentalization of reactions in pico- to nanoliter water-in-oil droplets in microfluidic devices provides a solution for massive volume reduction. This work addresses the challenge of producing microfluidic-based concentration gradients in a way that every droplet represents one unique reagent combination. We present a simple microcapillary technique able to generate such series of monodisperse water-in-oil droplets (with a frequency of up to 10 Hz) from a sample presented in an open well (e.g., a titerplate). Time-dependent variation of the well content results in microdroplets that represent time capsules of the composition of the source well. By preserving the spatial encoding of the droplets in tubing, each reactor is assigned an accurate concentration value. We used this approach to record kinetic time courses of the haloalkane dehalogenase DbjA and analyzed 150 combinations of enzyme/substrate/inhibitor in less than 5 min, resulting in conclusive Michaelis-Menten and inhibition curves. Avoiding chips and merely requiring two pumps, a magnetic plate with a stirrer, tubing, and a pipet tip, this easy-to-use device rivals the output of much more expensive liquid handling systems using a fraction (∼100-fold less) of the reagents consumed in microwell format.

Affinity maturation of a computationally designed binding protein affords a functional but disordered polypeptide.

Computational methods have been recently applied to the design of protein-protein interfaces. Using this approach, a 61 amino acid long protein called Spider Roll was engineered to recognize the kinase domain of the human p21-activated kinase 1 (PAK1) with good specificity but modest affinity (KD=100µM). Here we show that this artificial protein can be optimized by yeast surface display and fluorescence-activated cell sorting. After three rounds of mutagenesis and screening, a diverse set of tighter binding variants was obtained. A representative binder, MSR7, has a >10(2)-fold higher affinity for PAK1 when displayed on yeast and a 6 to 11-fold advantage when produced free in solution. In contrast to the starting Spider Roll protein, however, MSR7 unexpectedly exhibits characteristics typical of partially disordered proteins, including lower α-helical content, non-cooperative thermal denaturation, and NMR data showing peak broadening and poor signal dispersion. Although conformational disorder is increasingly recognized as an important property of proteins involved in cellular signaling and regulation, it is poorly modeled by current computational methods. Explicit consideration of structural flexibility may improve future protein designs and provide deeper insight into molecular events at protein-protein interfaces.

An N-terminal protein degradation tag enables robust selection of highly active enzymes.

Degradation tags are short peptide sequences that target proteins for destruction by housekeeping proteases. We previously utilized the C-terminal SsrA tag in directed evolution experiments to decrease the intracellular lifetime of a growth-limiting enzyme and thereby facilitate selection of highly active variants. In this study, we examine the N-terminal RepA tag as an alternative degradation signal for laboratory evolution. Although RepA proved to be less effective than SsrA at lowering protein concentrations in the cell, its N-terminal location dramatically reduced the occurrence of truncation and frameshift artifacts in selection experiments. We exploited this improvement to evolve a topologically redesigned chorismate mutase that is intrinsically disordered but already highly active for the conversion of chorismate to prephenate. After three rounds of mutagenesis and high-stringency selection, a robust and more nativelike variant was obtained that exhibited a catalytic efficiency (k(cat)/K(M) = 84000 M(-1) s(-1)) comparable to that of a natural dimeric chorismate mutase. Because of concomitant increases in catalyst yield, the level of intracellular prephenate production increased approximately 30-fold overall over the course of evolution.

A simple selection strategy for evolving highly efficient enzymes.

Combining tunable transcription with an enzyme-degradation tag affords an effective means to reduce intracellular enzyme concentrations from high to very low levels. Such fine-tuned control allows selection pressure to be systematically increased in directed-evolution experiments. This facilitates identification of mutants with wild-type activity, as shown here for an engineered chorismate mutase. Numerous selection formats and cell-based screening methodologies may benefit from the large dynamic range afforded by this easily implemented strategy.

Simultaneous optimization of enzyme activity and quaternary structure by directed evolution.

Natural evolution has produced efficient enzymes of enormous structural diversity. We imitated this natural process in the laboratory to augment the efficiency of an engineered chorismate mutase with low activity and an unusual hexameric topology. By applying two rounds of DNA shuffling and genetic selection, we obtained a 400-fold more efficient enzyme, containing three non-active-site mutations. Detailed biophysical characterization of the evolved variant suggests that it exists predominantly as a trimer in solution, but is otherwise similarly stable as the parent hexamer. The dramatic structural and functional effects achieved by a small number of seemingly innocuous substitutions highlights the utility of directed evolution for modifying protein-protein interactions to produce novel quaternary states with optimized activities.