Kinetic Reaction Profile Analysis Using Clover‐Ruby2 Fluorescent Fusion Protein Substrates as a Tool for Protease Characterization
Authors: Selby Thomas Lee , Fussell Robert , Sultan Fakhra , Mack Vance 
Published: June 2021

Real‐time imaging of biological reactions in vivo has advanced through the development of photostable fluorescent proteins (FPs) with varied excitation and emission maxima. While the enhanced cyan and yellow fluorescent proteins (ECFP and EYFP) are commonly used for fluorescence resonance energy transfer (FRET) imaging applications, the excitation wavelengths required for these FPs can interfere with biological molecules during in vivo measurements. This interference, along with the need for additional FPs to provide multicolor imaging, has driven advances in the development of red FPs that have excitation and emission maxima in the 550 nm and higher range. To investigate the potential for improving the correlation of in vivo and in vitro protease reactions at longer wavelengths, we have optimized the purification of the Clover (ClFP) and Ruby2 (R2FP) fusion protein as a FRET pair (Cl‐R2) using a number of quality control parameters measured along six different purification paths. These parameters included absorbance and fluorescence measurements, ratios of those values, and kinetic maturation data collected in different buffer conditions from various chromatography columns. These optimization studies were extended to kinetic analysis to better understand how Cl‐R2 substrate quality affects kinetic profiles during protease reactions. Kinetic analysis with different Cl‐R2 substrates resulted in reaction profiles with two distinct phases. A “burst” phase occurs during the first 5 minutes of the reaction where less than 5% of the total substrate has been consumed. A second phase, which is diffusion limited and linear, continues until nearly 85% of the substrate has been consumed. Analysis of the “Burst” and “Diffusion” rates for mutant protease enzymes of the Tobacco Etch Virus (TEV) using the Cl‐R2 substrate resulted in differing profiles in each phase for each mutant enzyme. These differences were concentration dependent, and appear to represent differing affinities of the proteases for the Cl‐R2 substrate during the reaction cycle. In the case of the TEV:S219V and TEV:S219P enzymes the burst‐phase had different rates that were not in the same proportion to the diffusion‐phase rates. While the TEV:219V had a slower burst rate compared to the TEV:219P enzyme, the TEV:219P enzyme had a slower diffusion rate compared to TEV:219V. These differences in burst and diffusion rates indicate the enzyme's ability to locate substrate molecules at higher concentrations, and as the concentration of substrate begins to decrease. This represents an additional parameter for in vitro protease characterization that is relevant to monitoring protease reactions in vivo, and may provide additional information on the ability of a given protease to locate substrates when the substrate molecules are at low concentrations.Support or Funding Information1 R15 GM080691This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.