Structural information such as orientations of interfacial proteins and peptides is important for understanding properties and functions of such biological molecules, which play crucial roles in biological applications and processes such as antimicrobial selectivity, membrane protein activity, biocompatibility, and biosensing performance. The α-helical and β-sheet structures are the most widely encountered secondary structures in peptides and proteins. In this paper, for the first time, a method to quantify the orientation of the interfacial β-sheet structure using a combined Attenuated Total Reflectance Fourier Transformation Infrared Spectroscopic (ATR-FTIR) and Sum Frequency Generation (SFG) vibrational spectroscopic study was developed. As an illustration of the methodology, the orientation of tachyplesin I, a 17-amino acid peptide with an anti-parallel β-sheet, adsorbed to polymer surfaces as well as associated with a lipid bilayer was determined using the regular and chiral SFG spectra, together with polarized ATR-FTIR amide I signals. Both the tilt angle (θ) and the twist angle (ψ) of the β-sheet at interfaces are determined. The developed method in this paper can be used to obtain in situ structural information of β-sheet components in complex molecules. The combination of this method and the existing methodology that is currently used to investigate α-helical structures will greatly broaden the application of optical spectroscopy in physical chemistry, biochemistry, biophysics, and structural biology.

There is considerable evidence for the slaving of biomolecular dynamics to the motions of the surrounding solvent environment, but to date there have been few direct experimental measurements capable of site-selectively probing both the dynamics of the water and the protein with ultrafast time resolution. Here, two-dimensional infrared spectroscopy (2D-IR) is used to study the ultrafast hydration and protein dynamics sensed by a metal carbonyl vibrational probe covalently attached to the surface of hen egg white lysozyme dissolved in D(2)O/glycerol solutions. Surface labeling provides direct access to the dynamics at the protein-water interface, where both the hydration and the protein dynamics can be observed simultaneously through the vibrational probe's frequency-frequency correlation function. In pure D(2)O, the correlation function shows a fast initial 3 ps decay corresponding to fluctuations of the hydration water, followed by a significant static offset attributed to fluctuations of the protein that are not sampled within the <20 ps experimental window. Adding glycerol increases the bulk solvent viscosity while leaving the protein structurally intact and hydrated. The hydration dynamics exhibit a greater than 3-fold slowdown between 0 and 80% glycerol (v/v), and the contribution from the protein's dynamics is found to slow in a nearly identical fashion. In addition, the magnitude of the dynamic slowdown associated with hydrophobic hydration is directly measured and shows quantitative agreement with predictions from molecular dynamics simulations.

Phase separation of intracellular components has been recently realized as a mechanism by which cells achieve membraneless organization. Here, we study the associative liquid-liquid phase separation (LLPS) of DNA upon complexation with cationic polypeptides. Comparing the phase behavior of different single-stranded DNA as well as double-stranded DNA (dsDNA) sequences that differ in persistence lengths, we find that DNA local flexibility, not simply charge density, determines the LLPS. Furthermore, in a nucleotide- and DNA-dependent manner, free nucleotide triphosphates promote LLPS of polypeptide-dsDNA complexes that are otherwise prone to precipitation. Under these conditions, dsDNA undergoes a secondary phase separation forming liquid-crystalline subcompartments inside the droplets. These results point toward a role of local DNA flexibility, encoded in the sequence, in the regulation and selectivity of multicomponent LLPS in membraneless intracellular organization.

Ultrafast two-dimensional infrared (2D-IR) spectroscopy reveals picosecond protein and hydration dynamics of crowded hen egg white lysozyme (HEWL) labeled with a metal-carbonyl vibrational probe covalently attached to a solvent accessible His residue. HEWL is systematically crowded alternatively with polyethylene glycol (PEG) or excess lysozyme in order to distinguish the chemically inert polymer from the complex electrostatic profile of the protein crowder. The results are threefold: (1) A sharp dynamical jamming-like transition is observed in the picosecond protein and hydration dynamics that is attributed to an independent-to-collective hydration transition induced by macromolecular crowding that slows the hydration dynamics up to an order of magnitude relative to bulk water; (2) The interprotein distance at which the transition occurs suggests collective hydration of proteins over distances of 30-40 Å; and (3) Comparing the crowding effects of PEG400 to our previously reported experiments using glycerol exposes fundamental differences between small and macromolecular crowding agents.

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids has emerged as an important phenomenon in membraneless intracellular organization. We demonstrate that the linker histone H1 condenses into liquid-like droplets in the nuclei of HeLa cells. The droplets, observed during the interphase of the cell cycle, are colocalized with DNA-dense regions indicative of heterochromatin. In vitro, H1 readily undergoes LLPS with both DNA and nucleosomes of varying lengths but does not phase separate in the absence of DNA. The nucleosome core particle maintains its structural integrity inside the droplets, as demonstrated by FRET. Unexpectedly, H2A also forms droplets in the presence of DNA and nucleosomes in vitro, whereas the other core histones precipitate. The phase diagram of H1 with nucleosomes is invariant to the nucleosome length at physiological salt concentration, indicating that H1 is capable of partitioning large segments of DNA into liquid-like droplets. Of the proteins tested (H1, core histones, and the heterochromatin protein HP1a), this property is unique to H1. In addition, free nucleotides promote droplet formation of H1 nucleosome in a nucleotide-dependent manner, with droplet formation being most favorable with ATP. Although LLPS of HP1a is known to contribute to the organization of heterochromatin, our results indicate that H1 also plays a role. Based on our study, we propose that H1 and DNA act as scaffolds for phase-separated heterochromatin domains.