We first aimed to characterize the thermodynamic driving forces for the complexation of ProTa and H1. We leveraged single-molecule FRET experiments with labelled ProTa to measure affinities with H1. Our experiments revealed pico- to nanomolar affinity for ProTα-H1 dimer formation near physiological salt concentrations and an exquisite sensitivity of the affinities to salt concentration. Affinity measurements with different salts including those with monovalent anion and cations suggested that the entropy counter-ion release is an important driving force. Next, we carried out temperature-dependent single-molecule FRET which showed increased affinity of the complex at higher temperatures demonstrating the complex formation to be enthalpically unfavorable or endothermic, which was confirmed by isothermal titration calorimetry (ITC). At higher concentrations, with single-molecule FRET we also identified stoichiometrically defined ternary complexes between ProTα and H1 at equilibrium and were able to measure their salt-dependent stabilities. Thus, ProTα and H1 complexation involves not only the dimer but also the ternary complexes, which, if not taken into account, lead to an erroneous estimation of ProTα-H1 dimer affinity from ITC. However, analysis with a proper equilibrium model that includes ternary complexes resolves this apparent discrepancy, highlighting the role of proper modeling and integrative analysis for such complexation processes. Finally, we show that an analytical mean-field polymer theory that explicitly accounts for counterion absorption and release can explain all salt-dependent experimental observables. The picture that emerges from all our experiments, integrative analysis and theory is that counterion release entropy is a key driving force, the overall enthalpy of the complexation is determined by the relative endothermicities of counterion absorption vs interchain ion pairing, in cases where the former is greater the overall complexation will be enthalpically unfavorable.
A direct corollary of the counterion release being a significant driving force is the exquisite salt sensitivity of the affinity, a simple extrapolation would suggest the affinity increase by a stagerring 10^26 fold from ~210 mM salt to 10 mM salt. A key question we wanted to answer is does the complex remain dynamic despite such extreme affinities at low salt concentration or I transforms into a dynamically arrested frozen state. Toward this end we first optimized purification strategies for ProTa and H1 utilizing an intein based system that allowed rapid, near degradation free facile purification of multiple variants. Using 15 inter and intramolecular distances we from smFRET we probed the complex ensemble as function of salt concentrations. We detected a continuous compaction of the complex with loweing salt concentration which suggested a dynamic complex even at extreme affinities. Using nanosecond correlation spectroscopy, we determined the timescales of long range distance dynamics of the complex at low salt to be in 100s of nanoseconds, and only a 2.5 slowdown of the dynamics from high (210 mM) to low (10 mM) salt. This demonstrated a massive decoupling between affinity and dynamics-while the likely affinity increase from high to low salt conditions is 10^26 fold the slowdown of dynamics is only 2.5 fold. We rationalized this decoupling from the differential sensitivities of counterion ion release entropy and slat bridge strengths to salt concentration, the former determines the affinity while the latter determines the dynamics.
In the context of phase separation we characterized phase separation of ProTa and H1 to be maximal at charge balanced stoichiometries-suggesting the coacervate phase composition to be a charge balanced mixture of ProTa and H1. We optimized confocal spectroscopy modalities including fluorescent correlation spectroscopy-based methodology for characterizing dilute and dense phase concentration and characterized the full phase diagram (binodals) for ProTa and H1 phase separation.
