Peptidyl Prolyl Isomerases (PPIases) accelerate cis-trans isomerization of prolyl peptide bonds. In rice, the PPIase LRT2 is essential for lateral root initiation. LRT2 displays in vitro isomerization of a highly conserved W-P peptide bond ( 104 W-P 105 ) in the natural substrate OsIAA11. OsIAA11 is a transcription repressor that, in response to the plant hormone auxin, is targeted to ubiquitinmediated proteasomal degradation via specific recognition of the cis isomer of its 104 W-P 105 peptide bond. OsIAA11 controls transcription of specific genes, including its own, that are required for lateral root development. This auxin-responsive negative feedback circuit governs patterning and development of lateral roots along the primary root. The ability to tune LRT2 activity via mutagenesis is crucial for understanding and modeling the role of this bimodal switch in the auxin circuit and lateral root development. We present characterization of the thermal stability and isomerization rates of several LRT2 mutants acting on the OsIAA11 substrate. The thermally stable mutants display activities lower than that of wild-type (WT) LRT2. These include binding diminished but catalytically active P125K, binding incompetent W128A, and binding capable but catalytically incompetent H133Q mutations. Additionally, LRT2 homologs hCypA from human, TaCypA from Triticum aestivum (wheat) and PPIB from E. coli were shown to have 110%, 50% and 60% of WT LRT2 activity on the OsIAA11 substrate. These studies identify several thermally stable LRT2 mutants with altered activities that will be useful for establishing relationships between cis-trans isomerization, auxin circuit dynamics, and lateral root development in rice.

The analysis of peak lineshapes in two-dimensional NMR spectra acquired during titration experiments is a powerful technique for quantitative studies of biological interfaces. The theoretical basis for how the thermodynamics (equilibrium populations) and kinetics (rate constants) of a multi-state equilibrium are encoded in the lineshape of an NMR peak is presented. Insights on how an exchange process influences the observed magnetization, how to construct the rate matrix for a given reaction scheme, and the origins of the governing lineshape equation are included. Applications of lineshape analysis to two-state binding and to four-state peptidyl prolyl cis–trans isomerization reactions involved in Alzheimer's disease and lateral root development in rice are presented. These studies illustrate how lineshape analysis of NMR titration experiments provides key mechanistic details for understanding the role of biological interfaces. Importantly, the quantitative model obtained from observations of an exchange reaction in the NMR tube enables predictions of activity to be scaled to cellular concentrations. This method is highly complementary to more recently developed NMR relaxation techniques. The application of lineshape analysis and relaxation methods to the same samples could enable quantitative characterization of even more complex multi-state systems.