Modulation of conformational space and dynamics of unfolded outer membrane proteins by periplasmic chaperones

Beta-barrel outer membrane proteins (OMPs) present on the outer membrane of Gram-negative bacteria are vital to cell survival. Their biogenesis is a challenging process which is tightly regulated by protein-chaperone interactions at various stages. Upon secretion from the inner membrane, OMPs are solubilized by periplasmic chaperones seventeen kilodalton protein (Skp) and survival factor A (SurA) and maintained in a folding competent state until they reach the outer membrane. As periplasm has an energy deficient environment, thermodynamics plays an important role in fine tuning these chaperone-OMP interactions. Thus, a complete understanding of such associations necessitates an investigation into both structural and thermodynamic aspects of the underlying intercommunication. Yet, they have been difficult to discern because of the conformational heterogeneity of the bound substrates, fast chain dynamics and the aggregation prone nature of OMPs. This demands for use of single molecule spectroscopy techniques, specifically, single molecule Förster resonance energy transfer (smFRET). In this thesis, upon leveraging the conformational and temporal resolution offered by smFRET, an exciting insight is obtained into the mechanistic and functional features of unfolded and Skp/SurA - bound states of two differently sized OMPs: OmpX (8 β-strands) and outer membrane phospholipase A (OmpLA – 12 β-strands). First, it was elucidated that the unfolded states of both the proteins exhibit slow interconversion within their sub-populations. Remarkably, upon complexing with chaperones, irrespective of the chosen OMP, the bound substrates expanded with localised chain reconfiguration on a sub-millisecond timescale. Yet, due to the different interaction mechanisms employed by Skp (encapsulation) and SurA (multivalent binding), their clients were found to be characterised by distinct conformational ensembles. Importantly, the extracted thermodynamic parameters of change in enthalpy and entropy exemplified the mechanistically dissimilar functionalities of the two chaperones. Furthermore, both Skp and SurA were found to be capable of disintegrating aggregated OMPs rather cooperatively, highlighting their multifaceted chaperone activity. This work is of significant fundamental value towards understanding the ubiquitous chaperone-protein interactions and opens up the possibility to design drugs targeting the chaperone-OMP complex itself, one step ahead of the OMP assembly on the outer membrane

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology

Modulation of conformational space and dynamics of unfolded outer membrane proteins by periplasmic chaperones

Beta-barrel outer membrane proteins (OMPs) present on the outer membrane of Gram-negative bacteria are vital to cell survival. Their biogenesis is a challenging process which is tightly regulated by protein-chaperone interactions at various stages. Upon secretion from the inner membrane, OMPs are solubilized by periplasmic chaperones seventeen kilodalton protein (Skp) and survival factor A (SurA) and maintained in a folding competent state until they reach the outer membrane. As periplasm has an energy deficient environment, thermodynamics plays an important role in fine tuning these chaperone-OMP interactions. Thus, a complete understanding of such associations necessitates an investigation into both structural and thermodynamic aspects of the underlying intercommunication. Yet, they have been difficult to discern because of the conformational heterogeneity of the bound substrates, fast chain dynamics and the aggregation prone nature of OMPs. This demands for use of single molecule spectroscopy techniques, specifically, single molecule Förster resonance energy transfer (smFRET). In this thesis, upon leveraging the conformational and temporal resolution offered by smFRET, an exciting insight is obtained into the mechanistic and functional features of unfolded and Skp/SurA - bound states of two differently sized OMPs: OmpX (8 β-strands) and outer membrane phospholipase A (OmpLA – 12 β-strands). First, it was elucidated that the unfolded states of both the proteins exhibit slow interconversion within their sub-populations. Remarkably, upon complexing with chaperones, irrespective of the chosen OMP, the bound substrates expanded with localised chain reconfiguration on a sub-millisecond timescale. Yet, due to the different interaction mechanisms employed by Skp (encapsulation) and SurA (multivalent binding), their clients were found to be characterised by distinct conformational ensembles. Importantly, the extracted thermodynamic parameters of change in enthalpy and entropy exemplified the mechanistically dissimilar functionalities of the two chaperones. Furthermore, both Skp and SurA were found to be capable of disintegrating aggregated OMPs rather cooperatively, highlighting their multifaceted chaperone activity. This work is of significant fundamental value towards understanding the ubiquitous chaperone-protein interactions and opens up the possibility to design drugs targeting the chaperone-OMP complex itself, one step ahead of the OMP assembly on the outer membrane

Modulation of conformational space and dynamics of unfolded outer membrane proteins by periplasmic chaperones

Beta-barrel outer membrane proteins (OMPs) present on the outer membrane of Gram-negative bacteria are vital to cell survival. Their biogenesis is a challenging process which is tightly regulated by protein-chaperone interactions at various stages. Upon secretion from the inner membrane, OMPs are solubilized by periplasmic chaperones seventeen kilodalton protein (Skp) and survival factor A (SurA) and maintained in a folding competent state until they reach the outer membrane. As periplasm has an energy deficient environment, thermodynamics plays an important role in fine tuning these chaperone-OMP interactions. Thus, a complete understanding of such associations necessitates an investigation into both structural and thermodynamic aspects of the underlying intercommunication. Yet, they have been difficult to discern because of the conformational heterogeneity of the bound substrates, fast chain dynamics and the aggregation prone nature of OMPs. This demands for use of single molecule spectroscopy techniques, specifically, single molecule Förster resonance energy transfer (smFRET). In this thesis, upon leveraging the conformational and temporal resolution offered by smFRET, an exciting insight is obtained into the mechanistic and functional features of unfolded and Skp/SurA - bound states of two differently sized OMPs: OmpX (8 β-strands) and outer membrane phospholipase A (OmpLA – 12 β-strands). First, it was elucidated that the unfolded states of both the proteins exhibit slow interconversion within their sub-populations. Remarkably, upon complexing with chaperones, irrespective of the chosen OMP, the bound substrates expanded with localised chain reconfiguration on a sub-millisecond timescale. Yet, due to the different interaction mechanisms employed by Skp (encapsulation) and SurA (multivalent binding), their clients were found to be characterised by distinct conformational ensembles. Importantly, the extracted thermodynamic parameters of change in enthalpy and entropy exemplified the mechanistically dissimilar functionalities of the two chaperones. Furthermore, both Skp and SurA were found to be capable of disintegrating aggregated OMPs rather cooperatively, highlighting their multifaceted chaperone activity. This work is of significant fundamental value towards understanding the ubiquitous chaperone-protein interactions and opens up the possibility to design drugs targeting the chaperone-OMP complex itself, one step ahead of the OMP assembly on the outer membrane

Replica Exchange Molecular Dynamics Study of Dimerization in Prion Protein: Multiple Modes of Interaction and Stabilization

The pathological forms of prions are known to be a result of misfolding, oligomerization, and aggregation of the cellular prion. While the mechanism of misfolding and aggregation in prions has been widely studied using both experimental and computational tools, the structural and energetic characterization of the dimer form have not garnered as much attention. On one hand dimerization can be the first step toward a nucleation-like pathway to aggregation, whereas on the other hand it may also increase the conformational stability preventing self-aggregation. In this work, we have used extensive all-atom replica exchange molecular dynamics simulations of both monomer and dimer forms of a mouse prion protein to understand the structural, dynamic, and thermodynamic stability of dimeric prion as compared to the monomeric form. We show that prion proteins can dimerize spontaneously being stabilized by hydrophobic interactions as well as intermolecular hydrogen bonding and salt bridge formation. We have computed the conformational free energy landscapes for both monomer and dimer forms to compare the thermodynamic stability and misfolding pathways. We observe large conformational heterogeneity among the various modes of interactions between the monomers and the strong intermolecular interactions may lead to as high as 20% β-content. The hydrophobic regions in helix-2, surrounding coil regions, terminal regions along with the natively present β-sheet region appear to actively participate in prion–prion intermolecular interactions. Dimerization seems to considerably suppress the inherent dynamic instability observed in monomeric prions, particularly because the regions of structural frustration constitute the dimer interface. Further, we demonstrate an interesting reversible coupling between the Q160-G131 interaction (which leads to inhibition of β-sheet extension) and the G131-V161 H-bond formation

Temperature-Induced Misfolding in Prion Protein: Evidence of Multiple Partially Disordered States Stabilized by Non-Native Hydrogen Bonds

The structural basis of pathways of misfolding of a cellular prion (PrP^C) into the toxic scrapie form (PrP^SC) and identification of possible intermediates (e.g., PrP*) still eludes us. In this work, we have used a cumulative ∼65 μs of replica exchange molecular dynamics simulation data to construct the conformational free energy landscapes and capture the structural and thermodynamic characteristics associated with various stages of the thermal denaturation process in human prion protein. The temperature-dependent free energy surfaces consist of multiple metastable states stabilized by non-native contacts and hydrogen bonds, thus rendering the protein prone to misfolding. We have been able to identify metastable conformational states with high β-content (∼30–40%) and low α-content (∼10–20%) that might be precursors of PrP^SC oligomer formation. These conformations also involve participation of the unstructured N-terminal domain, and its role in misfolding has been investigated. All the misfolded or partially unfolded states are quite compact in nature despite having large deviations from the native structure. Although the number of native contacts decreases dramatically at higher temperatures, the radius of gyration and number of intraprotein hydrogen bonds and contacts remain relatively unchanged, leading to stabilization of the misfolded conformations by non-native interactions. Our results are in good agreement with the established view that the C-terminal regions of the second and third helices (H2 and H3, respectively) of mammal prions might be the Achilles heels of their stability, while separation of B1–H1–B2 and H2–H3 domains seems to play a key role, as well

Chaperones Skp and SurA dynamically expand unfolded OmpX and synergistically disassemble oligomeric aggregates.

Periplasmic chaperones 17-kilodalton protein (Skp) and survival factor A (SurA) are essential players in outer membrane protein (OMP) biogenesis. They prevent unfolded OMPs from misfolding during their passage through the periplasmic space and aid in the disassembly of OMP aggregates under cellular stress conditions. However, functionally important links between interaction mechanisms, structural dynamics, and energetics that underpin both Skp and SurA associations with OMPs have remained largely unresolved. Here, using single-molecule fluorescence spectroscopy, we dissect the conformational dynamics and thermodynamics of Skp and SurA binding to unfolded OmpX and explore their disaggregase activities. We show that both chaperones expand unfolded OmpX distinctly and induce microsecond chain reconfigurations in the client OMP structure. We further reveal that Skp and SurA bind their substrate in a fine-tuned thermodynamic process via enthalpy-entropy compensation. Finally, we observed synergistic activity of both chaperones in the disaggregation of oligomeric OmpX aggregates. Our findings provide an intimate view into the multifaceted functionalities of Skp and SurA and the fine-tuned balance between conformational flexibility and underlying energetics in aiding chaperone action during OMP biogenesis

An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics.

Acknowledgements: We thank all members of the Schlierf lab for lively discussions during the development of this project. This research was funded by TU Dresden core funds (M.S.), the DFG SCHL1896/3-1 (M.S.) and SCHL1896/4-1 (M.S.), the BMBF OptiZeD Grant Z22E511 (M.S.), the European Social Fund and co-financed by tax funds based on the budget approved by the members of the Saxon State Parliament (M.Sche.) and by a grant from Mukoviszidose Institut gGmbH, Bonn, the research and development arm of the German Cystic Fibrosis Association Mukoviszidose e.V. (M.S.). We acknowledge support by the European Research Council (ERC) under the European Union’s Horizon 2020 Framework Programme through the Marie Skłodowska-Curie Grant MicroSPARK (agreement no. 841466; G.K.), the Herchel Smith Funds of the University of Cambridge (G.K.), and the Wolfson College Junior Research Fellowship (G.K.).Funder: Mukoviszidose Institut gGmbHFunder: European Social Fund and Saxon State ParliamentFunder: Herchel Smith Funds of the University of Cambridge Wolfson College Junior Research FellowshipSingle-molecule FRET (smFRET) has become a versatile tool for probing the structure and functional dynamics of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Confocal smFRET measurements are amongst the widely used smFRET assays and are typically performed in a single-well format. Thus, sampling of many experimental parameters is laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality to allow for continuous and automated smFRET measurements. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, competitive and cooperative protein-DNA interactions, and drug-discovery, revealing insights that would be very difficult to achieve with conventional single-well format measurements. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software

An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics

Acknowledgements: We thank all members of the Schlierf lab for lively discussions during the development of this project. This research was funded by TU Dresden core funds (M.S.), the DFG SCHL1896/3-1 (M.S.) and SCHL1896/4-1 (M.S.), the BMBF OptiZeD Grant Z22E511 (M.S.), the European Social Fund and co-financed by tax funds based on the budget approved by the members of the Saxon State Parliament (M.Sche.) and by a grant from Mukoviszidose Institut gGmbH, Bonn, the research and development arm of the German Cystic Fibrosis Association Mukoviszidose e.V. (M.S.). We acknowledge support by the European Research Council (ERC) under the European Union’s Horizon 2020 Framework Programme through the Marie Skłodowska-Curie Grant MicroSPARK (agreement no. 841466; G.K.), the Herchel Smith Funds of the University of Cambridge (G.K.), and the Wolfson College Junior Research Fellowship (G.K.).Funder: Mukoviszidose Institut gGmbHFunder: European Social Fund and Saxon State ParliamentFunder: Herchel Smith Funds of the University of Cambridge Wolfson College Junior Research FellowshipSingle-molecule FRET (smFRET) has become a versatile tool for probing the structure and functional dynamics of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Confocal smFRET measurements are amongst the widely used smFRET assays and are typically performed in a single-well format. Thus, sampling of many experimental parameters is laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality to allow for continuous and automated smFRET measurements. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, competitive and cooperative protein–DNA interactions, and drug-discovery, revealing insights that would be very difficult to achieve with conventional single-well format measurements. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software

Mechanism of Unfolding of Human Prion Protein

Misfolding and aggregation of prion proteins are associated with several neurodegenerative diseases. Therefore, understanding the mechanism of the misfolding process is of enormous interest in the scientific community. It has been speculated and widely discussed that the native cellular prion protein (PrP^C) form needs to undergo substantial unfolding to a more stable PrP^C* state, which may further oligomerize into the toxic scrapie (PrP^Sc) form. Here, we have studied the mechanism of the unfolding of the human prion protein (huPrP) using a set of extensive well-tempered metadynamics simulations. Through multiple microsecond-long metadynamics simulations, we find several possible unfolding pathways. We show that each pathway leads to an unfolded state of lower free energy than the native state. Thus, our study may point to the signature of a PrP^C* form that corresponds to a global minimum on the conformational free-energy landscape. Moreover, we find that these global minima states do not involve an increased β-sheet content, as was assumed to be a signature of PrP^Sc formation in previous simulation studies. We have further analyzed the origin of metastability of the PrP^C form through free-energy surfaces of the chopped helical segments to show that the helices, particularly H2 and H3 of the prion protein, have the tendency to form either a random coil or a β-structure. Therefore, the secondary structural elements of the prion protein are only weakly stabilized by tertiary contacts and solvation forces so that relatively weak perturbations induced by temperature, pressure, pH, and so forth can lead to substantial unfolding with characteristics of intrinsically disordered proteins