Copper-induced spectroscopic and structural changes in short peptides derived from azurin.

The active sites of metalloproteins may be mimicked by designing peptides that bind to their respective metal ions. Studying the binding of protein ligands to metal ions along with the associated structural changes is important in understanding metal uptake, transport and electron transfer functions of proteins. Copper-binding metalloprotein azurin is a 128-residue electron transfer protein with a redox-active copper cofactor. Here, we report the copper-binding associated spectroscopic and structural properties of peptide loops (11 and 13 residues) from the copper-binding site of azurin. These peptides develop a ß-turn upon copper-binding with a 1:1 Cu2+:peptide stoichiometry as seen in circular dichroism and exhibit electronic transitions centered at 340 nm and 540 nm. Further addition of copper develops a helical feature along with a shift in the absorption maxima to ~360 nm and ~580 nm at 2:1 Cu2+:peptide stoichiometry, indicating stoichiometric dependence of copper-binding geometry. Mass spectrometry indicates the copper-binding to cysteine, histidine and methionine in the peptide with 1:1 stoichiometry, and interestingly, dimerization through a disulfide linkage at 2:1 stoichiometry, as observed previously for denatured azurin. Fluorescence quenching studies on peptides with tryptophan further confirm the copper-binding induced changes in the two peptides are bi-phasic.

Small peptide binding stiffens the ubiquitin-like protein SUMO1.

Posttranslational modification by small ubiquitin-like modifiers (SUMOs), known as SUMOylation, is a key regulatory event in many eukaryotic cellular processes in which SUMOs interact with a large number of target proteins. SUMO binding motifs (SBMs) are small peptides derived from these target proteins that interact noncovalently with SUMOs and induce conformational changes. To determine the effect of SBMs on the mechanical properties of SUMO1 (the first member of the human SUMO family), we performed single-molecule force spectroscopy experiments on SUMO1/SBM complexes. The unfolding force of SUMO1 (at a pulling speed of 400 nm/s) increased from ∼ 130 pN to ∼ 170 pN upon binding to SBMs, indicating mechanical stabilization upon complexation. Pulling-speed-dependent experiments and Monte Carlo simulations measured a large decrease in distance to the unfolding transition state for SUMO1 upon SBM binding, which is by far the largest change measured for any ligand binding protein. The stiffness of SUMO1 (measured as a spring constant for the deformation response along the line joining the N- and C-termini) increased upon SBM binding from ∼ 1 N/m to ∼ 3.5 N/m. The relatively higher flexibility of ligand-free SUMO1 might play a role in accessing various conformations before binding to a target.

Cell-size-dependent regulation of Ezrin dictates epithelial resilience to stretch by countering myosin-II-mediated contractility.

The epithelial adaptations to mechanical stress are facilitated by molecular and tissue-scale changes that include the strengthening of junctions, cytoskeletal reorganization, and cell-proliferation-mediated changes in tissue rheology. However, the role of cell size in controlling these properties remains underexplored. Our experiments in the zebrafish embryonic epidermis, guided by theoretical estimations, reveal a link between epithelial mechanics and cell size, demonstrating that an increase in cell size compromises the tissue fracture strength and compliance. We show that an increase in E-cadherin levels in the proliferation-deficient epidermis restores epidermal compliance but not the fracture strength, which is largely regulated by Ezrin-an apical membrane-cytoskeleton crosslinker. We show that Ezrin fortifies the epithelium in a cell-size-dependent manner by countering non-muscle myosin-II-mediated contractility. This work uncovers the importance of cell size maintenance in regulating the mechanical properties of the epithelium and fostering protection against future mechanical stresses.

Single-molecule studies on PolySUMO proteins reveal their mechanical flexibility.

Proteins with ß-sandwich and ß-grasp topologies are resistant to mechanical unfolding as shown by single-molecule force spectroscopy studies. Their high mechanical stability has generally been associated with the mechanical clamp geometry present at the termini. However, there is also evidence for the importance of interactions other than the mechanical clamp in providing mechanical stability, which needs to be tested thoroughly. Here, we report the mechanical unfolding properties of ubiquitin-like proteins (SUMO1 and SUMO2) and their comparison with those of ubiquitin. Although ubiquitin and SUMOs have similar size and structural topology, they differ in their sequences and structural contacts, making them ideal candidates to understand the variations in the mechanical stability of a given protein topology. We observe a two-state unfolding pathway for SUMO1 and SUMO2, similar to that of ubiquitin. Nevertheless, the unfolding forces of SUMO1 (∼130 pN) and SUMO2 (∼120 pN) are lower than that of ubiquitin (∼190 pN) at a pulling speed of 400 nm/s, indicating their lower mechanical stability. The mechanical stabilities of SUMO proteins and ubiquitin are well correlated with the number of interresidue contacts present in their structures. From pulling speed-dependent mechanical unfolding experiments and Monte Carlo simulations, we find that the unfolding potential widths of SUMO1 (∼0.51 nm) and SUMO2 (∼0.33 nm) are much larger than that of ubiquitin (∼0.19 nm), indicating that SUMO1 is six times and SUMO2 is three times mechanically more flexible than ubiquitin. These findings might also be important in understanding the functional differences between ubiquitin and SUMOs.

Reconstruction of the Free Energy Profile for SUMO1 from Nonequilibrium Single-Molecule Pulling Experiments.

Free energy profiles form the cornerstone in the study of protein folding and function. In this study, the free energy profile of SUMO1 protein is directly reconstructed using an extension of the Jarzynski equality from atomic force microscope (AFM) based single-molecule force spectroscopy (SMFS) experiments. SUMO1 is a ubiquitin-like posttranslational modifier protein having a ß clamp motif in its structure, imparting it with mechanical stability. We use the Jarzynski equality to obtain the equilibrium free energy profile from repeated nonequilibrium single-molecule pulling experiments. Indeed, the free energy values determined by the Jarzynski equality are lesser than the normal work average at all extensions. The free energy profiles constructed for the two velocities (100 and 400 nm/s) overlap with each other. The unfolding free energy barrier is estimated to be ∼7.5 kcal/mol. We anticipate that the Jarzynski equality can be applied in a similar manner to other ubiquitin-like proteins to extract their differences in the free energy profile, and hence, the effect of sequence diversity of structurally homologous proteins on the free energy landscape can be studied.

Pyrogallol, Corilagin and Chebulagic acid target the "fuzzy coat" of alpha-synuclein to inhibit the fibrillization of the protein.

The accumulation of the intrinsically disordered protein alpha-synuclein (αSyn) in the form of insoluble fibrillar aggregates in the central nervous system is linked to a variety of neurodegenerative disorders such as Parkinson's disease, Lewy body dementia, and multiple system atrophy. Here we show that Pyrogallol, Corilagin and Chebulagic acid, compounds containing a different number of catechol rings, are independently capable of delaying and reducing the extent of αSyn fibrillization. The efficiency of inhibition was found to correlate with the number of catechol rings. Further, our NMR studies reveal that these compounds interact with the N-terminal region of αSyn which is unstructured even in the fibrillar form of the protein and is known as the "fuzzy coat" of fibrils. Thus, Corilagin and Chebulagic acid target the fuzzy coat of αSyn and not the amyloid core which is a common target for the inhibition of protein fibrillization. Our results indicate that the N-terminus also plays a key role in the fibrillization of αSyn.

Pulling the springs of a cell by single-molecule force spectroscopy.

The fundamental unit of the human body comprises of the cells which remain embedded in a fibrillar network of extracellular matrix proteins which in turn provides necessary anchorage the cells. Tissue repair, regeneration and reprogramming predominantly involve a traction force mediated signalling originating in the ECM and travelling deep into the cell including the nucleus via circuitry of spring-like filamentous proteins like microfilaments or actin, intermediate filaments and microtubules to elicit a response in the form of mechanical movement as well as biochemical changes. The 'springiness' of these proteins is highlighted in their extension-contraction behaviour which is manifested as an effect of differential traction force. Atomic force microscope (AFM) provides the magic eye to visualize and quantify such force-extension/indentation events in these filamentous proteins as well as in whole cells. In this review, we have presented a summary of the current understanding and advancement of such measurements by AFM based single-molecule force spectroscopy in the context of cytoskeletal and nucleoskeletal proteins which act in tandem to facilitate mechanotransduction.

Triphala inhibits alpha-synuclein fibrillization and their interaction study by NMR provides insights into the self-association of the protein.

The process of assembly and accumulation of the intrinsically disordered protein (IDP), alpha-synuclein (αSyn) into amyloid fibrils is a pathogenic process leading to several neurodegenerative disorders such as Parkinson's disease, multiple system atrophy and others. Although several molecules are known to inhibit αSyn fibrillization, the mechanism of inhibition is just beginning to emerge. Here, we report the inhibition of fibrillization of αSyn by Triphala, a herbal preparation in the traditional Indian medical system of Ayurveda. Triphala was found to be a rich source of polyphenols which are known to act as amyloid inhibitors. ThT fluorescence and TEM studies showed that Triphala inhibited the fibrillization of αSyn. However, it was observed that Triphala does not disaggregate preformed αSyn fibrils. Further, native-PAGE showed that Triphala reduces the propensity of αSyn to oligomerize during the lag phase of fibrillization. Our NMR results showed that certain stretches of residues in the N-terminal and NAC regions of αSyn play an anchor role in the self-association process of the protein, thereby providing mechanistic insights into the early events during αSyn fibrillization.

Single-molecule force spectroscopy measurements of bond elongation during a bimolecular reaction.

It is experimentally challenging to directly obtain structural information of the transition state (TS), the high-energy bottleneck en route from reactants to products, for solution-phase reactions. Here, we use single-molecule experiments as well as high-level quantum chemical calculations to probe the TS of disulfide bond reduction, a bimolecular nucleophilic substitution (S N2) reaction. We use an atomic force microscope in force-clamp mode to apply mechanical forces to a protein disulfide bond and obtain force-dependent rate constants of the disulfide bond reduction initiated by a variety of nucleophiles. We measure distances to the TS or bond elongation (Delta x), along a 1-D reaction coordinate imposed by mechanical force, of 0.31 +/- 0.05 and 0.44 +/- 0.03 A for thiol-initiated and phosphine-initiated disulfide bond reductions, respectively. These results are in agreement with quantum chemical calculations, which show that the disulfide bond at the TS is longer in phosphine-initiated reduction than in thiol-initiated reduction. We also investigate the effect of solvent environment on the TS geometry by incorporating glycerol into the aqueous solution. In this case, the Delta x value for the phosphine-initiated reduction is decreased to 0.28 +/- 0.04 A whereas it remains unchanged for thiol-initiated reduction, providing a direct test of theoretical calculations of the role of solvent molecules in the reduction TS of an S N2 reaction. These results demonstrate that single-molecule force spectroscopy represents a novel experimental tool to study mechanochemistry and directly probe the sub-ångström changes in TS structure of solution-phase reactions. Furthermore, this single-molecule method opens new doors to gain molecular level understanding of chemical reactivity when combined with quantum chemical calculations.

A single-molecule perspective on the role of solvent hydrogen bonds in protein folding and chemical reactions.

We present an array of force spectroscopy experiments that aim to identify the role of solvent hydrogen bonds in protein folding and chemical reactions at the single-molecule level. In our experiments we control the strength of hydrogen bonds in the solvent environment by substituting water (H(2)O) with deuterium oxide (D(2)O). Using a combination of force protocols, we demonstrate that protein unfolding, protein collapse, protein folding and a chemical reaction are affected in different ways by substituting H(2)O with D(2)O. We find that D(2)O molecules form an integral part of the unfolding transition structure of the immunoglobulin module of human cardiac titin, I27. Strikingly, we find that D(2)O is a worse solvent than H(2)O for the protein I27, in direct contrast with the behaviour of simple hydrocarbons. We measure the effect of substituting H(2)O with D(2)O on the force dependent rate of reduction of a disulphide bond engineered within a single protein. Altogether, these experiments provide new information on the nature of the underlying interactions in protein folding and chemical reactions and demonstrate the power of single-molecule techniques to identify the changes induced by a small change in hydrogen bond strength.