Periodic Reporting for period 1 - DisCharge (Exploring molecular determinants of charged disordered protein interactions, phase separation and function from the test-tube to the cell)
Reporting period: 2021-09-01 to 2023-08-31
The overall objective of the project is to uncover specificity determinants of interaction and phase separation of highly charged IDPs using ProTa/H1 interaction as a model. Only a multidisciplinary study spanning several complexity regimes, which is currently lacking, can uncover such specificity determinants. It is hypothesized that physiologically, ProTa/H1 interaction plays out in LLPS-driven heterochromatin assemblies leading to transcriptional regulation. ProTa/H1 interactions and phase behaviour will be probed with a set of systematically designed variants in incrementally complex milieu, from the test-tube to the cell, to reveal how the sequence dictates interactions and phase behaviour in-vitro and attribute a given function in-cell. This will shed light on specificity determinants of interaction and function for such highly charged IDPs.
In order to achieve this goal first we first uncovered the thermodynamic basis of interaction ProTa and H1 using a combination of single molecule FRET spectroscopy, calorimetery, integrative data analysis and analytical polymer theory. We discovered that a key driving force for this interaction is the entropy of released counter ions akin to synthetic poly-electrolytes. We further found out the complex to be experimentally dynamic at all conditions even when the affinity is expected to be very high, that is there is an affinity dynamics decoupling in such types of complexes, and we rationalized this behavior using simple physical principles. Furthermore, we characterized the full phase diagram of ProTa/H1 and optimized conditions for performing single molecule fluorescence experiments in such condensates. Some technical challenges were identified in experiments on ternary phases of nucleic acids/ProTa/H1 as well as for in-cellulo experiments, and strategies to develop these were addressed. Also, for the all the above mentioned accomplishments robust biochemical strategies for purification and labeling of ProTa and H1 variants, the latter being exceedingly degradation prone and consequently difficult to purify, were established.
In order to achieve this goal first we first uncovered the thermodynamic basis of interaction ProTa and H1 using a combination of single molecule FRET spectroscopy, calorimetery, integrative data analysis and analytical polymer theory. We discovered that a key driving force for this interaction is the entropy of released counter ions akin to synthetic poly-electrolytes. We further found out the complex to be experimentally dynamic at all conditions even when the affinity is expected to be very high, that is there is an affinity dynamics decoupling in such types of complexes, and we rationalized this behavior using simple physical principles. Furthermore, we characterized the full phase diagram of ProTa/H1 and optimized conditions for performing single molecule fluorescence experiments in such condensates. Some technical challenges were identified in experiments on ternary phases of nucleic acids/ProTa/H1 as well as for in-cellulo experiments, and strategies to develop these were addressed. Also, for the all the above mentioned accomplishments robust biochemical strategies for purification and labeling of ProTa and H1 variants, the latter being exceedingly degradation prone and consequently difficult to purify, were established.
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.
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.
To the best of our knowledge, the results of our project represent the most comprehensive picture of polyelectrolyte complexation at the single chain level, that is heterodimer formation by two polyelectrolyte chains that is distinct from coacervate formation. We rationalize our results with concepts from the field of synthetic polyelectrolytes, in particular a minimal mean-field theory that treats a salt-moderated balance between Coulomb energy of bound ion pairs and free-ion entropy, and accounts for counterion adsorption and release. Additionally our experiments reveal the basis of remarkably fast dynamics in these high affinity complexes, suggesting such disordered charge driven complexes can attain high affinity despite paying a dynamic penalty. Our results may further contribute to a quantitative understanding of polyelectrolyte complex coacervation by complementing theories for phase as dilute phase complex formation is the first step towards coacervation. Overall we provide the experimental and theoretical rubric for characterizing the thermodynamics, dynamics and phase diagrams for polyelectrolyte like IDPs, which are rife in cellular physiology; the impact of extends beyond the field of IDPs into polyelectrolyte complexes, polymer physics and phase separation.