Label-Free Single Protein Dynamics Revealed by Metasurface-Enhanced Raman Spectroscopy

Chain Collapse, Reduced Dielectric, and Water Release Drive Protein Phase Separation.

Biomolecular condensates represent unique microenvironments that organize intracellular biology and promote biochemical reactions. However, the biomolecular interactions driving condensate phase separation are often weak, transient, and heterogeneous. Investigating the structural biology and chemical properties of condensate interiors has therefore proven experimentally challenging, often requiring the use of perturbative probes. To overcome this challenge, we combine label-free optical scattering and vibrational spectroscopy approaches spanning ultraviolet, visible, mid-infrared, and terahertz wavelengths with deep-learning-based ensemble prediction of intrinsically disordered protein conformations. This suite of label-free approaches provides quantitative insights into protein-protein/protein-solvent interactions and the chemical properties of condensate interiors. Investigating the N-terminal domain of the RNA DEAD-box helicase 4 (DDX4), our experimental and computational results support a model of phase separation involving protein chain collapse, reduced dielectric, and water release. These molecular events are expected to enhance the strength of multivalent protein-protein interactions within condensates, creating a positive feedback loop important for condensate growth and phase separation.

Conformational Switching of Proteins on Material Surfaces Enabled by Peptide‐Oriented Adsorption

Conformationally responsive proteins are increasingly being harnessed to build stimulus‐responsive biointerfaces, yet the interconversion dynamics of proteins are routinely lost upon adsorption to inorganic surfaces. Heterogeneous protein attachment, driven by the stochastic presentation of surface‐interactive residues, can occlude active sites, mask allosteric interfaces, and diminish the dynamic properties the protein was recruited to provide. Here, we demonstrate a genetically encoded strategy to preserve conformational switching by biasing surface attachment through the modification of calmodulin (CaM) with a Au binding domain, the AuBP1 peptide, to promote a preferred adsorption geometry at aqueous Au interfaces. Quartz crystal microbalance with dissipation measurements reveal fully reversible viscoelastic changes in the adsorbed layer for CaM fused to AuBP1 across multiple Ca 2+ binding/release cycles with no measurable mass loss, which was absent for wild‐type CaM. Atomic force microscopy independently confirmed conformation‐dependent surface morphologies for the apo and holo states. Molecular dynamics simulations support a dominant AuBP1‐mediated binding configuration that minimizes disruptive surface contacts. These observations identify transferable design principles for switchable biointerfaces and establish that non‐covalent peptide‐mediated orientation can sustain functional protein dynamics in direct contact with a metal without reliance on covalent immobilization.

Correction to "Mapping Free-Energy Landscapes to Decipher Amphiphilic Peptide Multistep Self-Assembly".

Mapping Free-Energy Landscapes to Decipher Amphiphilic Peptide Multistep Self-Assembly.

The formation of fibrous architectures via peptide self-assembly underpins numerous biological functions and biomaterial applications; however, the thermodynamic origins of multistep assembly pathways remain elusive. Here, we map the complete free-energy landscape governing the liquid-liquid phase separation (LLPS)-mediated self-assembly of an amphiphilic peptide by exploiting temperature as a tunable parameter. We discover an unexpected thermodynamic mechanism: the initial LLPS-like clustering is enthalpy-driven but limited by a positive enthalpic barrier (+121 kJ mol-1), arising from the endothermic disruption of intramolecular hydrogen bonds before interpeptide contacts can form. Subsequent nucleation and fibril growth are governed by negative entropic barriers (-56 and -39 kJ mol-1, respectively), reflecting the reorganization cost of partially ordered oligomers. The energy landscape identifies LLPS as the rate-limiting step with the highest Gibbs free-energy barrier (+26 kJ mol-1). Our findings establish a generalizable framework for decoding multistep biomolecular self-organization, with implications for designing adaptive biomaterials and understanding aberrant phase transitions in diseases.

Single-molecule fluorescence spectroscopy of biomolecular condensates.

Single-molecule spectroscopy is emerging as a powerful approach for elucidating the molecular structure and dynamics of biomolecular condensates. Unlike ensemble methods, it can resolve heterogeneous conformations, dynamics, interactions, concentrations, and transport properties across a broad range of length- and timescales, even for minute amounts of biomolecules. Recent applications, especially fluorescence-based methods such as single-molecule Förster resonance energy transfer, fluorescence correlation spectroscopy (FCS), fluorescence anisotropy, and nanosecond FCS, have revealed how proteins and nucleic acids behave within dense phases, and how molecular-scale dynamics relate to mesoscopic properties and biological function. Combined with molecular simulations, these measurements yield mechanistic insight into condensate organization, dynamics, and aging. We highlight recent advances, key applications, and promising directions for probing the properties of condensates with single-molecule spectroscopy.

Polyphenols at the Helm: Excited State Photophysics and Mechanistic Insights Into Protein-Aggregation Inhibition Through Ultrafast Studies.

Understanding how small molecules modulate protein aggregation is pivotal for developing strategies against amyloid-related disorders. Polyphenols are promising modulators, yet the connection between their molecular photophysics and aggregation inhibition remains largely unresolved. In this article, femtosecond transient absorption spectroscopy has been employed to unravel the excited-state dynamics of some commonly used polyphenols, namely, Baicalein, EGCG, and Myricetin across varied environments-neat solvents, micelles and reverse micelles, and in the presence of globular and intrinsically disordered protein (in its native and aggregated forms). Excited-state intramolecular proton transfer (ESIPT) emerges to be highly sensitive to hydrogen-bonding, polarity, and local confinement, serving as a subpicosecond reporter of polyphenol-environment interactions. In protein systems, modulation of ESIPT dynamics signals environment-specific embedding of polyphenols within evolving protein landscapes. A unique tri-parameter correlation-linking binding affinities of the polyphenols to the native proteins, ESIPT lifetimes, and aggregation kinetics-reveals that inhibitory potency arises not from tight native binding but from selective engagement with aggregation-prone species. This work posits ESIPT as a sensitive molecular signature that links ultrafast photophysics to the functional inhibition of protein aggregation, providing new insights into how small molecules navigate complex biomolecular environments to regulate self-assembly pathways.