scholarly journals Force-dependent switch in protein unfolding pathways and transition-state movements

2016 ◽  
Vol 113 (6) ◽  
pp. E715-E724 ◽  
Author(s):  
Pavel I. Zhuravlev ◽  
Michael Hinczewski ◽  
Shaon Chakrabarti ◽  
Susan Marqusee ◽  
D. Thirumalai
Keyword(s):  
Transition State ◽  
Single Molecule ◽  
Single Point ◽  
Optical Tweezer ◽  
Sh3 Domain ◽  
Single Domain ◽  
Structural Basis ◽  
Single Point Mutation ◽  
Single Chain ◽  
Upward Curvature

Although it is known that single-domain proteins fold and unfold by parallel pathways, demonstration of this expectation has been difficult to establish in experiments. Unfolding rate, ku(f), as a function of force f, obtained in single-molecule pulling experiments on src SH3 domain, exhibits upward curvature on a log⁡ku(f) plot. Similar observations were reported for other proteins for the unfolding rate ku([C]). These findings imply unfolding in these single-domain proteins involves a switch in the pathway as f or [C] is increased from a low to a high value. We provide a unified theory demonstrating that if log⁡ku as a function of a perturbation (f or [C]) exhibits upward curvature then the underlying energy landscape must be strongly multidimensional. Using molecular simulations we provide a structural basis for the switch in the pathways and dramatic shifts in the transition-state ensemble (TSE) in src SH3 domain as f is increased. We show that a single-point mutation shifts the upward curvature in log⁡ku(f) to a lower force, thus establishing the malleability of the underlying folding landscape. Our theory, applicable to any perturbation that affects the free energy of the protein linearly, readily explains movement in the TSE in a β-sandwich (I27) protein and single-chain monellin as the denaturant concentration is varied. We predict that in the force range accessible in laser optical tweezer experiments there should be a switch in the unfolding pathways in I27 or its mutants.

scholarly journals Structural basis for a complex I mutation that blocks pathological ROS production

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhan Yin ◽  
Nils Burger ◽  
Duvaraka Kula-Alwar ◽  
Dunja Aksentijević ◽  
Hannah R. Bridges ◽  
...  
Keyword(s):  
Point Mutation ◽  
Complex I ◽  
Structural Changes ◽  
Ischemia Reperfusion ◽  
Single Point ◽  
Structural Basis ◽  
Single Point Mutation ◽  
Ros Production

AbstractMitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia–reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the “deactive” state, usually formed only after prolonged inactivity. Despite its tendency to adopt the “deactive” state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.

Identification of Allosteric Fingerprints of Alpha-Synuclein Aggregates in Matrix Metalloprotease-1 and Substrate-Specific Virtual Screening with Single Molecule Insights

2021 ◽  
Author(s):  
Sumaer Kamboj ◽  
Chase Harms ◽  
Derek Wright ◽  
Anthony Nash ◽  
Lokender Kumar ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Single Molecule ◽  
Conformational Changes ◽  
Single Point ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Alpha Synuclein ◽  
Matrix Metalloproteases ◽  
Single Point Mutation ◽  
Mmp Inhibitor

Abstract Alpha-synuclein (aSyn) has implications in pathological protein aggregations in neurodegeneration. Matrix metalloproteases (MMPs) are broad-spectrum proteases and cleave aSyn, leading to aggregation. Previously, we showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, activity, and ligands. Here we report quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations that are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics, where the two states and kinetic rates of interconversion between them are obtained from histograms and autocorrelations of FRET values. Since a crystal structure of aSyn-bound MMP1 is not available, we performed molecular docking of MMP1 with aSyn using ClusPro. We simulated MMP1 dynamics using different docking poses and matched the experimental and simulated interdomain dynamics to identify an appropriate pose. We used experimentally validated simulations to define conformational changes at the catalytic site and identify allosteric residues in the hemopexin domain having strong correlations with the catalytic motif residues. We defined Shannon entropy to quantify MMP1 dynamics. We performed virtual screening against a site on selected aSyn-MMP1 binding poses and showed that lead molecules differ between free MMP1 and substrate-bound MMP1. Also, identifying aSyn-specific allosteric residues in MMP1 enabled further selection of lead molecules. In other words, virtual screening needs to take substrates into account for substrate-specific control of MMP1 activity. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading aggregates by targeting MMPs.

scholarly journals Structural effects of the highly protective V127 polymorphism on human prion protein

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Laszlo L. P. Hosszu ◽  
Rebecca Conners ◽  
Daljit Sangar ◽  
Mark Batchelor ◽  
Elizabeth B. Sawyer ◽  
...  
Keyword(s):  
Prion Protein ◽  
Prion Disease ◽  
Structural Changes ◽  
Prion Diseases ◽  
Single Point ◽  
Structural Basis ◽  
Single Point Mutation ◽  
Solution Dynamics ◽  
Human Prion Protein ◽  
Prion Transmission

AbstractPrion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the β2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease.

scholarly journals Characterization and engineering of S100A12–heparan sulfate interactions

Glycobiology ◽  
2020 ◽  
Vol 30 (7) ◽  
pp. 463-473
Author(s):  
Xiaoxiao Zhang ◽  
Chihyean Ong ◽  
Guowei Su ◽  
Jian Liu ◽  
Ding Xu
Keyword(s):  
Heparan Sulfate ◽  
Binding Site ◽  
Calcium Binding ◽  
Divalent Cations ◽  
Single Point ◽  
Cell Types ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Single Point Mutation ◽  
Unexpected Finding

Abstract S100A12, an EF-hand calcium-binding protein, can be secreted by a variety of cell types and plays proinflammatory roles in a number of pathological conditions. Although S100A12 has been shown to interact with heparan sulfate (HS), the molecular detail of the interaction remains unclear. Here we investigate the structural basis of S100A12–HS interaction and how the interaction is regulated by the availability of divalent cations and the oligomeric states of S100A12. We discovered that S100A12–HS interaction requires calcium, while zinc can further enhance binding by inducing S100A12 hexamerization. In contrast, the apo form and zinc-induced tetramer form were unable to bind HS. Guided by the crystal structures of S100A12, we have identified the HS-binding site of S100A12 by site-directed mutagenesis. Characterization of the HS-binding site of S100A12 allowed us to convert the non-HS-binding apo and tetramer forms of S100A12 into a high affinity HS-binding variant by engineering a single-point mutation. Using a HS oligosaccharide microarray, we demonstrated that the N43K mutant displayed markedly enhanced selectivity toward longer HS oligosaccharides compared to the WT S100A12, likely due to the expanded dimension of the reengineered HS-binding site in the mutant. This unexpected finding strongly suggests that HS-binding sites of proteins might be amenable for engineering.

scholarly journals Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

2016 ◽  
Vol 113 (34) ◽  
pp. E4956-E4965 ◽  
Author(s):  
Namita Bisaria ◽  
Max Greenfeld ◽  
Charles Limouse ◽  
Dmitri S. Pavlichin ◽  
Hideo Mabuchi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Structural Changes ◽  
Structural Information ◽  
Equilibrium Constants ◽  
Single Point ◽  
Single Point Mutation ◽  
Single Molecule Fret ◽  
Predictive Understanding ◽  
And Function ◽  
And Behavior

The past decade has seen a wealth of 3D structural information about complex structured RNAs and identification of functional intermediates. Nevertheless, developing a complete and predictive understanding of the folding and function of these RNAs in biology will require connection of individual rate and equilibrium constants to structural changes that occur in individual folding steps and further relating these steps to the properties and behavior of isolated, simplified systems. To accomplish these goals we used the considerable structural knowledge of the folded, unfolded, and intermediate states of P4-P6 RNA. We enumerated structural states and possible folding transitions and determined rate and equilibrium constants for the transitions between these states using single-molecule FRET with a series of mutant P4-P6 variants. Comparisons with simplified constructs containing an isolated tertiary contact suggest that a given tertiary interaction has a stereotyped rate for breaking that may help identify structural transitions within complex RNAs and simplify the prediction of folding kinetics and thermodynamics for structured RNAs from their parts. The preferred folding pathway involves initial formation of the proximal tertiary contact. However, this preference was only ∼10 fold and could be reversed by a single point mutation, indicating that a model akin to a protein-folding contact order model will not suffice to describe RNA folding. Instead, our results suggest a strong analogy with a modified RNA diffusion-collision model in which tertiary elements within preformed secondary structures collide, with the success of these collisions dependent on whether the tertiary elements are in their rare binding-competent conformations.

scholarly journals Energy Landscape of Ubiquitin is Weakly Multidimensional

2021 ◽  
Author(s):  
Balaka Mondal ◽  
D. Thirumalai ◽  
Govardhan Reddy
Keyword(s):  
Transition State ◽  
Single Molecule ◽  
Brownian Dynamics ◽  
Reaction Pathway ◽  
Energy Landscape ◽  
Reaction Coordinate ◽  
Single Domain ◽  
Analytical Models ◽  
Coarse Grained ◽  
One Dimensional

AbstractSingle molecule pulling experiments report time-dependent changes in the extension (X) of a biomolecule as a function of the applied force (f). By fitting the data to one-dimensional analytical models of the energy landscape, the hopping rates between the folded and unfolded states in two-state folders, the height and the location of the transition state (TS) can be extracted. Although this approach is remarkably insightful, there are cases for which the energy landscape is multidimensional (catch bonds being the most prominent). To assess if the unfolding energy landscape in small single domain proteins could be one dimensional, we simulated force-induced unfolding of Ubiquitin (Ub) using the coarse-grained Self-Organized Polymer-Side Chain (SOP-SC) model. Brownian dynamics simulations using the SOP-SC model reveal that the Ub energy landscape is weakly multidimensional (WMD) governed predominantly by a single barrier. The unfolding pathway is confined to a narrow reaction pathway that could be described as diffusion in a quasi 1D X-dependent free energy profile. However, a granular analysis using the Pfold analysis, which does not assume any form for the reaction coordinate, shows that X alone does not account for the height, and more importantly, the location of the TS. The f-dependent TS location moves towards the folded state as f increases, in accord with the Hammond postulate. Our study shows that, in addition to analyzing the f-dependent hopping rates, the transition state ensemble must also be determined without resorting to X as a reaction coordinate in order to describe the unfolding energy landscapes of single domain proteins, especially if they are only WMD.TOC Graphic

scholarly journals Low force unfolding of a single-domain protein by parallel pathways

2020 ◽  
Author(s):  
Pavel I. Zhuravlev ◽  
Michael Hinczewski ◽  
D. Thirumalai
Keyword(s):  
Single Molecule ◽  
Protein Unfolding ◽  
Experimental Studies ◽  
Single Domain ◽  
Wild Type ◽  
Single Domain Protein ◽  
Parallel Pathways ◽  
Denaturant Concentration ◽  
Upward Curvature ◽  
Unfolding Kinetics

AbstractDeviations from linearity in the dependence of the logarithm of protein unfolding rates, log ku(f), as a function of mechanical force, f, measurable in single molecule experiments, can arise for many reasons. In particular, upward curvature in log ku(f) as a function of f implies that the underlying energy landscape must be multidimensional with the possibility that unfolding ensues by parallel pathways. Here, simulations using the SOP-SC model of a wild type β-sandwich protein and several mutants, with immunoglobulin folds, show upward curvature in the unfolding kinetics. There are substantial changes in the structures of the transition state ensembles as force is increased, signaling a switch in the unfolding pathways. Our results, when combined with previous theoretical and experimental studies, show that parallel unfolding of structurally unrelated single domain proteins can be determined from the dependence of log ku(f) as a function of force (or log ku[C] where [C] is the denaturant concentration).

scholarly journals Molecular mechanism of cell-cell adhesion mediated by cadherin-23

2017 ◽  
Author(s):  
G. S. Singaraju ◽  
A. Kumar ◽  
J. S. Samuel ◽  
A. Sagar ◽  
J. P. Hazra ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Point ◽  
Cell Junctions ◽  
Single Point Mutation ◽  
Molecular Architecture ◽  
Binding Interface ◽  
Extracellular Region ◽  
Classical Cadherins ◽  
Cadherin 23 ◽  
Cell Cell

AbstractAdherin-junctions are traditionally described by the homophilic-interactions of classical cadherin-proteins at the extracellular region. However, the role of long-chain non-classical cadherins like cadherin-23(Cdh23) is not explored as yet even though it is implicated in tissue-morphogenesis, cancer, and force-sensing in neuronal tissues. Here, we identified a novel antiparallel-binding interface of Cdh23 homodimer in solution by combining biophysical and computational methods, in-vitro cell-binding, and mutational modifications. The dimer consists of two electrostatic-based interfaces extended up to two terminal domains, atypical to classical-cadherins known so far, and forms the strongest interactions in cadherin-family as measured using single-molecule force-spectroscopy. We further identified single point-mutation, E78K, that completely disrupts this binding. Interestingly, the mutation, S77L, found in skin cancers falls within the binding interface of the antiparallel-dimer. Overall, we provide the molecular architecture of Cdh23 at the cell-cell junctions which are likely to have far-reaching applications in the fields of mechanobiology and cancer.

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