Computational remodeling of an enzyme conformational landscape for altered substrate selectivity.

Structural plasticity of enzymes dictates their function. Yet, our ability to rationally remodel enzyme conformational landscapes to tailor catalytic properties remains limited. Here, we report a computational procedure for tuning conformational landscapes that is based on multistate design of hinge-mediated domain motions. Using this method, we redesign the conformational landscape of a natural aminotransferase to preferentially stabilize a less populated but reactive conformation and thereby increase catalytic efficiency with a non-native substrate, resulting in altered substrate selectivity. Steady-state kinetics of designed variants reveals activity increases with the non-native substrate of approximately 100-fold and selectivity switches of up to 1900-fold. Structural analyses by room-temperature X-ray crystallography and multitemperature nuclear magnetic resonance spectroscopy confirm that conformational equilibria favor the target conformation. Our computational approach opens the door to targeted alterations of conformational states and equilibria, which should facilitate the design of biocatalysts with customized activity and selectivity.

Generation of bright monomeric red fluorescent proteins via computational design of enhanced chromophore packing.

Red fluorescent proteins (RFPs) have found widespread application in chemical and biological research due to their longer emission wavelengths. Here, we use computational protein design to increase the quantum yield and thereby brightness of a dim monomeric RFP (mRojoA, quantum yield = 0.02) by optimizing chromophore packing with aliphatic residues, which we hypothesized would reduce torsional motions causing non-radiative decay. Experimental characterization of the top 10 designed sequences yielded mSandy1 (λ em = 609 nm, quantum yield = 0.26), a variant with equivalent brightness to mCherry, a widely used RFP. We next used directed evolution to further increase brightness, resulting in mSandy2 (λ em = 606 nm, quantum yield = 0.35), the brightest Discosoma sp. derived monomeric RFP with an emission maximum above 600 nm reported to date. Crystallographic analysis of mSandy2 showed that the chromophore p-hydroxybenzylidene moiety is sandwiched between the side chains of Leu63 and Ile197, a structural motif that has not previously been observed in RFPs, and confirms that aliphatic packing leads to chromophore rigidification. Our results demonstrate that computational protein design can be used to generate bright monomeric RFPs, which can serve as templates for the evolution of novel far-red fluorescent proteins.

Genetically Encoded Fluorescent Biosensor for Rapid Detection of Protein Expression.

Fluorescent proteins are widely used as fusion tags to detect protein expression in vivo. To become fluorescent, these proteins must undergo chromophore maturation, a slow process with a half-time of 5 to >30 min that causes delays in real-time detection of protein expression. Here, we engineer a genetically encoded fluorescent biosensor to enable detection of protein expression within seconds in live bacteria. This sensor for transiently expressed proteins (STEP) is based on a fully matured but dim green fluorescent protein in which pre-existing fluorescence increases 11-fold in vivo following the specific and rapid binding of a protein tag (Kd 120 nM, kon 1.7 × 105 M-1 s-1). In live E. coli cells, our STEP biosensor enables detection of protein expression twice as fast as the use of standard fluorescent protein fusions. Our biosensor opens the door to the real-time study of short timescale processes in live cells with high spatiotemporal resolution.

Structure-guided rational design of red fluorescent proteins: towards designer genetically-encoded fluorophores.

Red fluorescent proteins (RFPs) have become an integral part of modern biological research due to their longer excitation and emission wavelengths. Protein engineering efforts have improved many key properties of RFPs for their practical use in imaging. Even so, continued engineering is required to overcome the shortcomings of the red chromophore and create RFPs with photophysical properties rivalling those of their optimized green and yellow counterparts. Here, we highlight recent examples of structure-guided rational design of RFPs to improve brightness, monomerization, maturation, and photostability, and discuss possible pathways for the future engineering of designer RFPs tailored to specific applications.

Bioanalysis for biocatalysis: multiplexed capillary electrophoresis-mass spectrometry assay for aminotransferase substrate discovery and specificity profiling.

In this work, we introduce an entirely automated enzyme assay based on capillary electrophoresis coupled to electrospray ionization mass spectrometry termed MINISEP-MS for multiple interfluent nanoinjections-incubation-separation-enzyme profiling using mass spectrometry. MINISEP-MS requires only nanoliters of reagent solutions and uses the separation capillary as a microreactor, allowing multiple substrates to be assayed simultaneously. The method can be used to rapidly profile the substrate specificity of any enzyme and to measure steady-state kinetics in an automated fashion. We used the MINISEP-MS assay to profile the substrate specificity of three aminotransferases (E. coli aspartate aminotransferase, E. coli branched-chain amino acid aminotransferase, and Bacillus sp. YM-1 D-amino acid aminotransferase) for 33 potential amino acid substrates and to measure steady-state kinetics. Using MINISEP-MS, we were able to recapitulate the known substrate specificities and to discover new amino acid substrates for these industrially relevant enzymes. Additionally, we were able to measure the apparent K(M) and k(cat) parameters for amino acid donor substrates of these aminotransferases. Because of its many advantages, the MINISEP-MS assay has the potential of becoming a useful tool for researchers aiming to identify or create novel enzymes for specific biocatalytic applications.