The mechanism by which mechanical force regulates the kinetics of a chemical reaction is unknown. Here, we use single-molecule force–clamp spectroscopy and protein engineering to study effect on thiol/disulfide exchange. Reduction disulfide bonds through exchange crucial in regulating function known occur mechanically stressed proteins. We apply constant stretching single engineered measure their rate reduction DTT. Although linearly dependent concentration DTT, it exponentially applied...

A model-free method is described for constructing time-resolved area-normalized emission spectra (TRANES) using luminescence decays at all wavelengths. An isoemissive point in TRANES indicates that the observed from sample due to two species only, irrespective of origin or excited-state kinetics. Proof existence an given various cases involving emissive species. The qualitatively similar isosbestic absorption (TRAS) kinetic spectrophotometry

It is experimentally challenging to directly obtain structural information of the transition state (TS), high-energy bottleneck en route from reactants products, for solution-phase reactions. Here, we use single-molecule experiments as well high-level quantum chemical calculations probe TS disulfide bond reduction, a bimolecular nucleophilic substitution (SN2) reaction. We an atomic force microscope in force-clamp mode apply mechanical forces protein and force-dependent rate constants...

Time resolved emission spectroscopy (TRES) provides information on the excited state kinetics and heterogeneity of emissive species in a system. area normalized (TRANES), an extension to TRES, is novel, model-free method for analysis intrinsic or extrinsic fluorescence probes complex chemical biophysical systems [Koti, Krishna, Periasamy, J. Phys. Chem. A 105, 1767 (2001)]. Observation single isoemissive point TRANES unambiguous indication presence two The multiple points spectra confirmed...

This communication describes the first observation of J-aggregate formation a porphyrin by polymer template. The chirality is superimposed on J-aggregate. Regular micrometer long rod-shaped structures polymer-templated has been observed AFM. material potentially useful for photonic and electronic molecular devices since absorbs intensely at 490 nm.

Photophysical properties and the rotational relaxation dynamics of a phenazine based dye, neutral red, have been investigated in aqueous solutions presence β-cyclodextrin (β-CD) using ground-state absorption steady-state time-resolved fluorescence measurements. It has observed that while form (NR) dye forms an inclusion complex with β-CD, its protonated (NRH+) does not. In β-CD (10 mM), pKa value is estimated to be about 6.06 ± 0.05, much lower than 6.81 0.05 measured solution. Both...

We used single molecule force spectroscopy to characterize the mechanical stability of enhanced yellow fluorescent protein (EYFP) (a mutant form green (GFP)) and two its circularly permutated variants. In all three constructs, we found main unfolding peaks; first corresponds a transition state placed close termini second halfway through molecule. attribute shear rupture beta1- beta6-strands, which verified by introducing point mutation in this region. Although both peaks were observed EYFP...

This illustration highlights how metal binding can alter protein structure. SUMO1, a small globular protein, forms molten globule state upon with platinum. The Pt ions, shown as fiery nodes, indicate the energy and disruption introduced at specific sites in protein. As binds, SUMO1′s fold softens, transitioning into "molten globule" – partially unfolded, more flexible, yet retaining some structural elements. visual underscores dynamic nature of shapes response to external ion interactions....

Designing functional molecules which can recognize and modify the activity of a specific protein is frequently encountered challenge in biology pharmaceutical chemistry, requires major effort for each target. Here we demonstrate that "self-peptides", parts folded proteins by their nature are recognizable rest protein, provide general route to developing such molecules. Such synthetic peptide with chemically prestabilized conformation incorporate into target during its folding, potentially...

Despite the widespread use of imidazolium-based ionic liquids (ILs) in biotechnology, pharmaceuticals, and green chemistry, their detailed interactions with proteins, particularly affecting structural stability, remain poorly understood. This study examines effects ILs on ubiquitin, a thermodynamically robust protein β-grasp structure. We found that IL-induced destabilization follows consistent order previous findings: [BMIM]+ > [BMPyr]+ [EMIM]+ for cations [BF4]- [MeSO4]- [Cl]- anions....

Protein folding and unfolding are complex phenomena, it is accepted that multidomain proteins generally follow multiple pathways. Maltose-binding protein (MBP) a large (a two-domain, 370-amino acid residue) bacterial periplasmic involved in maltose uptake. Despite the size, has been shown to exhibit an apparent two-state equilibrium bulk experiments. Single-molecule studies can uncover rare events masked by averaging studies. Here, we use single-molecule force spectroscopy study mechanical...

Experimental studies on the folding and unfolding of large multi-domain proteins are challenging, given proteins' complex energy landscapes with multiple intermediates. Here, we report a mechanical study 346-residue, two-domain leucine binding protein (LBP) from bacterial periplasm. Forced LBP is prerequisite for its translocation across cytoplasmic membrane into During stretching LBP, observe that 38% flux followed two-state pathway, giving rise to single force peak at ~70 pN an contour...

Liquid–liquid phase separation (LLPS) plays a crucial role in cellular organization, primarily driven by intrinsically disordered proteins (IDPs) leading to the formation of biomolecular condensates. A folded protein SUMO that post-translationally modifies has recently emerged as regulator LLPS. Given its compact structure and limited flexibility, precise condensate remains be investigated. Here, we show rapid SUMO1 into micrometer-sized liquid-like condensates inert crowders under...

We present an array of force spectroscopy experiments that aim to identify the role solvent hydrogen bonds in protein folding and chemical reactions at single-molecule level. In our we control strength environment by substituting water (H(2)O) with deuterium oxide (D(2)O). Using a combination protocols, demonstrate unfolding, collapse, reaction are affected different ways H(2)O D(2)O. find D(2)O molecules form integral part unfolding transition structure immunoglobulin module human cardiac...

A- and B-type lamins are intermediate filament proteins constituting the nuclear lamina underneath envelope thereby conferring proper shape mechanical rigidity to nucleus. Lamin also shown be related diversely basic processes. More than 400 mutations in human lamin A protein alone have been reported produce at least 11 different disease conditions jointly termed as laminopathies. These scattered throughout its helical rod domain, well C-terminal domain containing conserved Ig-fold region....