Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of ...unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central "hubs". Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Therapeutic antibody discovery often relies on in-vitro display methods to identify lead candidates. Assessing selected output diversity traditionally involves random colony picking and Sanger ...sequencing, which has limitations. Next-generation sequencing (NGS) offers a cost-effective solution with increased read depth, allowing a comprehensive understanding of diversity. Our study establishes NGS guidelines for antibody drug discovery, demonstrating its advantages in expanding the number of unique HCDR3 clusters, broadening the number of high affinity antibodies, expanding the total number of antibodies recognizing different epitopes, and improving lead prioritization. Surprisingly, our investigation into the correlation between NGS-derived frequencies of CDRs and affinity revealed a lack of association, although this limitation could be moderately mitigated by leveraging NGS clustering, enrichment and/or relative abundance across different regions to enhance lead prioritization. This study highlights NGS benefits, offering insights, recommendations, and the most effective approach to leverage NGS in therapeutic antibody discovery.
Intrinsically disordered proteins (IDPs) lack well-defined secondary or tertiary structures in solution but are found to be involved in a wide range of critical cellular processes that highlight ...their functional importance. IDPs usually undergo folding upon binding to their targets. Such binding coupled to folding behavior has widened our perspective on the protein structure-dynamics-function paradigm in molecular biology. However, characterizing the folding upon binding mechanism of IDPs experimentally remains quite challenging. Molecular simulations emerge as a potentially powerful tool that offers information complementary to experiments. Here we present a general computational framework for the molecular simulations of IDP folding upon binding processes that combines all-atom molecular dynamics (MD) and coarse-grained simulations. The classical all-atom molecular dynamics approach using GPU acceleration allows the researcher to explore the properties of the IDP conformational ensemble, whereas coarse-grained structure-based models implemented with parameters carefully calibrated to available experimental measurements can be used to simulate the entire folding upon binding process. We also discuss a set of tools for the analysis of MD trajectories and describe the details of the computational protocol to follow so that it can be adapted by the user to study any IDP in isolation and in complex with partners.
Intrinsically disordered proteins (IDPs) exploit their plasticity to deploy a rich panoply of soft interactions and binding phenomena. Advances in tailoring molecular simulations for IDPs combined ...with experimental cross-validation offer an atomistic view of the mechanisms that control IDP binding, function, and dysfunction. The emerging theme is that unbound IDPs autonomously form transient local structures and self-interactions that determine their binding behavior. Recent results have shed light on whether and how IDPs fold, stay disordered or drive condensation upon binding; how they achieve binding specificity and select among competing partners. The disorder-binding paradigm is now being proactively used by researchers to target IDPs for rational drug design and engineer molecular responsive elements for biosensing applications.
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•IDP binding mechanisms can now be tested with atomistic simulations cross-validated with experimental data.•Unbound IPDs form fleeting self-interactions that control their function and dysfunction.•IDPs barter conformational dynamics for partner interactions to fold, stay disordered, or condense upon binding.•New drug design approaches that use multi-conformer targets are leading to IDP blockers.•IDPs can be employed as molecular responsive elements for biotechnological applications.
Intrinsically disordered proteins (IDPs) fold upon binding to select/recruit multiple partners, morph around the partner's structure, and exhibit allostery. However, we do not know whether these ...properties emerge passively from disorder, or rather are encoded into the IDP's folding mechanisms. A main reason for this gap is the lack of suitable methods to dissect the energetics of IDP conformational landscapes without partners. Here we introduce such an approach that we term molecular LEGO, and apply it to NCBD, a helical, molten globule–like IDP, as proof of concept. The approach entails the experimental and computational characterization of the protein, its separate secondary structure elements (LEGO building blocks), and their supersecondary combinations. Comparative analysis uncovers specific, yet inconspicuous, energetic biases in the conformational/folding landscape of NCBD, including 1) strong local signals that define the three native helices, 2) stabilization of helix–helix interfaces via soft pairwise tertiary interactions, 3) cooperative stabilization of a heterogeneous three-helix bundle fold, and 4) a dynamic exchange between sets of tertiary interactions (native and nonnative) that recapitulate the different structures NCBD adopts in complex with various partners. Crucially, a tug of war between sets of interactions makes NCBD gradually shift between structural subensembles as a conformational rheostat. Such conformational rheostatic behavior provides a built-in mechanism to modulate binding and switch/recruit partners that is likely at the core of NCBD's function as transcriptional coactivator. Hence, the molecular LEGO approach emerges as a powerful tool to dissect the conformational landscapes of unbound IDPs and rationalize their functional mechanisms.
SignificanceIntrinsically disordered proteins have the unique ability to morph in response to multiple partners and thereby process sophisticated inputs and outputs. It is, however, a mystery whether ...their response is passive, that is, entirely determined by the partner, or controlled via an internal, yet unknown, folding mechanism. Here we introduce an approach to examine this key question and demonstrate its potential by dissecting the conformational properties of the partially disordered protein NCBD and obtaining important clues about how it performs its biological function.
In order to execute their biological activities, most proteins fold into their unique, three-dimensional structure. The discovery of intrinsically disordered proteins (IDPs) about two decades ago, ...which are now widely found in eukaryotes, has since challenged the structure-function paradigm. IDPs, which in isolation exist as broad, non-random, conformational ensembles of interconverting states, are centrally involved in many biological processes. The key to their functioning is the ability to fold when bound to ligand partner(s), thus operating as morphing proteins. Despite booming interest in morphing behavior, investigating their structural transitions and mechanism remains extremely difficult because of their distinct characteristics. Previously, we observed a close connection between intrinsically partially disordered proteins (IPDPS) and gradual (un)folding transitions of downhill folders, leading to the hypothesis that many IPDPs work as a conformational rheostat. The scope of this dissertation is to investigate the biological and technological implications of gradual conformational transitions. We first demonstrate the design principles of protein-based scaffolds by utilizing gradual (un)folding coupled to binding for developing rheostatic conformational transducers using computational modeling and experiments. Our engineered transducers showcase >6 orders of magnitude change in analyte concentration (broadband sensitivity) and have practical advantages over extant ones, which conventionally operate as conformational switches. Next, inspired by the LEGO toy, we devised a novel modular approach to dissect the folding cooperativity and the energetic contributions of native interactions in defining the conformational ensemble and binding properties of IPDPs. Using an integrated strategy of computation and experiments, we perform an ensemble-based conformational analysis and find that the approach provides an exciting new tool for analyzing morphing transitions that should generally apply to any IPDP, thereby addressing a fundamental gap in the field. One particularly interesting IPDP is NCBD that binds to multiple structurally diverse ligand partners and recruits the basal transcription machinery. We then explore the concept of NCBD functioning as a conformational rheostat, which allows its promiscuous binding. Finally, using extensive all-atom Molecular Dynamics simulations of NCBD and its biological partners in their free and bound forms, we decipher the hidden conformational biases in the dynamics of the heterogeneous ensemble of NCBD, undergoing gradual morphing transitions hinting at a working conformational rheostat in transcription.
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