Probing Aromatic Side Chains Reveals the Site-Specific Melting in the SUMO1 Molten Globule.

The conventional idea that a well-defined protein structure governs its functions is being challenged by the evolving significance of conformational flexibility and disorder in influencing protein activity. Here, we focus on the Small Ubiquitin-like MOdifier 1 (SUMO1) protein, a post-translational modifier, which binds various target proteins during the process of SUMOylation. We present evidence supporting the presence of both folded and "ordered" molten globule (MG) states in SUMO1 under physiological conditions.

Platinum Stabilises a Molten-Globule Conformation of a Small Globular Cytosolic Protein SUMO1.

Proteins are generally resistant to large conformational changes under physiological conditions. Here, we show that platinum (Pt(II)), which is widely-used metal centre in cancer therapeutic drugs, binds to a cytosolic protein, small ubiquitin-like modifier 1 (SUMO1), under physiological conditions and changes its conformation to a molten globule (MG). Mass spectrometry (ICP-MS) studies confirmed stoichiometric Pt(II) binding to SUMO1.

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.

Protein engineering using circular permutation - structure, function, stability, and applications.

Protein engineering is important for creating novel variants from natural proteins, enabling a wide range of applications. Approaches such as rational design and directed evolution are routinely used to make new protein variants. Computational tools like de novo design can introduce new protein folds.

Circular permutation at azurin's active site slows down its folding.

Circular permutation (CP) is a technique by which the primary sequence of a protein is rearranged to create new termini. The connectivity of the protein is altered but the overall protein structure generally remains unperturbed. Understanding the effect of CP can help design robust proteins for numerous applications such as in genetic engineering, optoelectronics, and improving catalytic activity.

Phase Separation and Aggregation of a Globular Folded Protein Small Ubiquitin-like Modifier 1 (SUMO1).

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 cellular proteins has recently emerged as a regulator of LLPS. Given its compact structure and limited flexibility, the precise role of SUMO in condensate formation remains to be investigated.

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.

Metal-binding and circular permutation-dependent thermodynamic and kinetic stability of azurin.

Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order of the secondary structure connectivity while retaining the three-dimensional structure, making it an elegant and powerful approach to altering native topology. Previous studies elucidated the influence of CP in proteins with different folds such as Greek key β-barrel, β-sandwich, β-α-β, and all α-Greek key.

Long-Range Charge Delocalization Mediates the Ultrafast Ligand-to-Metal Charge Transfer Dynamics at the Cu-Active Site in Azurin.

The blue color in metalloprotein azurin has traditionally been attributed to the intense cysteine-to-Cu ligand-to-metal charge transfer transition centered at 628 nm. Although resonance Raman measurements of the Cu active site have implied that the LMCT transition electronically couples to the protein scaffold well beyond its primary metal-ligand coordination shell, the structural extent of this electronic coupling and visualization of the protein-mediated charge transfer dynamics have remained elusive. Here, using femtosecond broadband transient absorption and impulsive Raman spectroscopy, we provide direct evidence for a rapid relaxation between two distinct charge transfer states, having different spatial delocalization, within ∼300 fs followed by recombination of charges in subpicosecond time scales.

Native Salt Bridges Are a Key Regulator of Ubiquitin's Mechanical Stability.

Although it is known that various intramolecular interactions determine protein mechanical stability, a detailed molecular-level understanding of the key regulators of protein mechanical stability is still lacking. Here, we present evidence for salt bridges in ubiquitin as important intramolecular interactions that can affect protein mechanical stability. Ubiquitin has two salt bridges: one relatively surface-exposed (SB1:K11-E34) and the other relatively buried (SB2:K27-D52).

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 β clamp motif in its structure, imparting it with mechanical stability.

Rational Design of Protein-Specific Folding Modifiers.

Protein-folding can go wrong and , with significant consequences for the living organism and the pharmaceutical industry, respectively. Here we propose a design principle for small-peptide-based protein-specific folding modifiers. The principle is based on constructing a "xenonucleus", which is a prefolded peptide that mimics the folding nucleus of a protein.

Disruption of desmosome function leads to increased centrosome clustering in 14-3-3γ-knockout cells with supernumerary centrosomes.

14-3-3 proteins are conserved, dimeric, acidic proteins that regulate multiple cellular pathways. Loss of either 14-3-3ε or 14-3-3γ leads to centrosome amplification. However, we find that while the knockout of 14-3-3ε leads to multipolar mitoses, the knockout of 14-3-3γ results in centrosome clustering and pseudo-bipolar mitoses.

Azurin-Derived Peptides: Comparison of Nickel- and Copper-Binding Properties.

Metalloproteins are an important class of proteins involved in metal uptake, transport, and electron-transfer reactions. Mimicking the active sites of these proteins through miniaturization is an active area of research with applications in biotechnology and medicine. Azurin is a 128-residue copper-binding cupredoxin protein involved in electron-transfer reactions.

Applications of atomic force microscopy in modern biology.

Single-molecule force spectroscopy (SMFS) is an emerging tool to investigate mechanical properties of biomolecules and their responses to mechanical forces, and one of the most-used techniques for mechanical manipulation is the atomic force microscope (AFM). AFM was invented as an imaging tool which can be used to image biomolecules in sub-molecular resolution in physiological conditions. It can also be used as a molecular force probe for applying mechanical forces on biomolecules.

Role of Ligand Binding Site in Modulating the Mechanical Stability of Proteins with β-Grasp Fold.

Despite many studies on ligand-modulated protein mechanics, a comparative analysis of the role of ligand binding site on any specific protein fold is yet to be made. In this study, we explore the role of ligand binding site on the mechanical properties of β-grasp fold proteins, namely, ubiquitin and small ubiquitin related modifier 1 (SUMO1). The terminal segments directly connected through hydrogen bonds constitute the β-clamp geometry (or mechanical clamp), which confers high mechanical resilience to the β-grasp fold.

An evolutionary non-conserved motif in Helicobacter pylori arginase mediates positioning of the loop containing the catalytic residue for catalysis.

The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bacterium in the human stomach. Despite conservation of the catalytic residues, this single Trp enzyme has an insertion sequence (-153ESEEKAWQKLCSL165-) that is extremely crucial to function. This sequence contains the critical residues, which are conserved in the homolog of other Helicobacter gastric pathogens.

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.

The unfolding transition state of ubiquitin with charged residues has higher energy than that with hydrophobic residues.

The native-state structure and folding pathways of a protein are encoded in its amino acid sequence. Ubiquitin, a post-translational modifier, primarily noted for its role in intracellular protein degradation, has two salt bridges: one relatively exposed (SB1:K11-E34) and the other relatively buried (SB2:K27-D52). Here, we study the role of hydrophobic interactions and sequence specificity in protein folding, by mutating the salt-bridge residues in ubiquitin with hydrophobic residues.

A new tension induction paradigm unravels tissue response and the importance of E-cadherin in the developing epidermis.

The epidermis, being the outermost epithelial layer in metazoans, experiences multiple external and self-generated mechanical stimuli. The tissue-scale response to these mechanical stresses has been actively studied in the adult stratified epidermis. However, the response of the developing bi-layered epidermis to differential tension and its molecular regulation has remained poorly characterised.

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.

Variance of Atomic Coordinates as a Dynamical Metric to Distinguish Proteins and Protein-Protein Interactions in Molecular Dynamics Simulations.

Protein dynamics is a manifestation of the complex trajectories of these biomolecules on a multidimensional rugged potential energy surface (PES) driven by thermal energy. At present, computational methods such as atomistic molecular dynamics (MD) simulations can describe thermal protein conformational changes in fully solvated environments over millisecond timescales. Despite these advances, a quantitative assessment of protein dynamics remains a complicated topic, intricately linked to issues such as sampling convergence and the identification of appropriate reaction coordinates/structural features to describe protein conformational states and motions.

Experimental comparison of energy landscape features of ubiquitin family proteins.

Small ubiquitin-related modifiers (SUMO1 and SUMO2) are ubiquitin family proteins, structurally similar to ubiquitin, differing in terms of their amino acid sequence and functions. Therefore, they provide a great platform for investigating sequence-structure-stability-function relationship. Here, we used chemical denaturation in comparing the folding-unfolding pathways of the SUMO proteins with their structural homologue ubiquitin (UF45W-pseudo wild-type [WT] tryptophan variant) with structurally analogous tryptophan mutations (SUMO1 [S1F66W], SUMO2 [S2F62W]).

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.

Insight into the Excitation-Dependent Fluorescence of Carbon Dots.

High quantum yield, photoluminescence tunability, and sensitivity to the environment are a few distinct trademarks that make carbon nanodots (CDs) interesting for fundamental research, with potential to replace the prevalent inorganic semiconductor quantum dots. Currently, application and fundamental understanding of CDs are constrained because it is difficult to make a quantitative comparison among different types of CDs simply because their photoluminescence properties are directly linked to their size distribution, the surface functionalization, the carbon core structures (graphitic or amorphous) and the number of defects. Herein, we report a facile one-step synthesis of mono-dispersed and highly fluorescent nanometre size CDs from a 'family' of glucose-based sugars.