Unraveling coupled folding and binding dynamics of ribonuclease S with transient infrared spectroscopy

Molecular interactions of proteins consisting of coupled disorder-to-order transitions and spontaneous binding have a crucial role in understanding their biological function and working mechanism. Here we investigate a well-known globular binding system, ribonuclease S (RNase S), to study the dynamics of protein-peptide interaction. Various biophysical approaches have investigated the binding mechanism of two proteolytic fragments, S-peptide and S-protein, which non-covalently bind to form an active enzyme RNase S. Due to challenges in dissecting the coupled nature of protein binding and folding, molecular level details remain elusive. Here, we use amide-I vibrations as a label-free probe of protein secondary structure and temperature as a control variable. By temperature-jump two-dimensional infrared spectroscopy (t-2DIR), we track the thermally-induced conformational changes associated with relaxation time that span from nanoseconds to many minute timescales. Additionally, we compare the results of an unlabeled complex with one containing a 13C=18O isotope-label of a residue buried within the helical segment of S-peptide. t‑2DIR measurements of labeled- and unlabeled- RNase S and isolated S-protein reveal two kinetic components on millisecond (fast) and second (slow) time scales. Both slow and fast phases are dominated by β-sheet disordering signatures that indicate S-protein unfolding. The isotope-label exhibits significant amplitude in the slow phase, suggesting this time window corresponds to both unbinding and unfolding of the S-peptide. Together, these results indicate that thermally-induced partial unfolding of the S-protein destabilizes the hydrophobic binding pocket containing S-peptide that, in turn, promotes unbinding and unfolding of the S-peptide. Our direct observations from transient spectroscopic data demonstrate the role of a conformational transition state during assembly of the RNase S complex.