Investigating protein-protein interactions in molecular recognition and association processes at the molecular level is essential to understanding the spontaneous self-assembly in biomolecular systems. Ribonuclease S complex (RNase S) is a well-known model system for studying coupled protein folding and binding mechanism and stability. RNase S is a protein complex consisting of non-covalently bonded proteolytic two fragments of RNase A, large S-protein (residues 21-124) and short S-peptide (residues 1-20). The binding of two fragments results in the formation of an enzymatically active RNase S. The binding mechanism of S-protein and S-peptide has been investigated in various biophysical approaches but is still not fully understood. To better understand the non-equilibrium structural dynamics of RNase S with related binding free energies, we have applied laser-induced temperature-jump (T-jump) and temperature-drop (T-drop) of both pump-probe and 2D spectroscopy with infrared detection across from nanoseconds extended to many hundreds of seconds. These transient IR spectroscopy techniques allowed us to track the T-jump/T-drop responses from biomolecules related to amide I spectral changes reported on secondary structure changes. We observed that thermally induced coupled dissociation-unfolding and reassociation-refolding of the RNase S complex involves two exponential kinetic components: milliseconds and seconds timescale, respectively vary with following a temperature perturbation. In comparison, we performed transient 2DIR measurement on isotope-labeled RNase S. With the aid of interpretation from equilibrium FTIR and 2DIR spectra, we find the spectral conformational feature changes of the different secondary structures of the protein complex corresponds to multiple transition kinetics.

Tracking coupled binding and folding dynamics of ribonuclease S with temperature-jump 2DIR spectroscopy

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