Noble metal nanoclusters (NCs) show great promise as nanoprobes for bioanalysis and cellular imaging in biological applications due to ultrasmall size, good photophysical properties, and excellent biocompatibility. In order to achieve a comprehensive understanding of possible biological implications, a series of spectroscopic measurements were conducted under different temperatures to investigate the interactions of Au NCs (∼1.7 nm) with three model plasmatic proteins (human serum albumin (HSA), γ-globulins, and transferrin). It was found that the fluorescence quenching of HSA and γ-globulins triggered by Au NCs was due to dynamic quenching mechanism, while the fluorescence quenching of transferrin by Au NCs was a result of the formation of a Au NC-transferrin complex. The apparent association constants of the Au NCs bound to HSA, γ-globulins, and transferrin demonstrated no obvious difference. Thermodynamic studies demonstrated that the interaction between Au NCs and HSA (or γ-globulins) was driven by hydrophobic forces, while the electrostatic interactions played predominant roles in the adsorption process for transferrin. Furthermore, it was proven that Au NCs had no obvious interference in the secondary structures of these three kinds of proteins. In turn, these three proteins had a minor effect on the fluorescence intensity of Au NCs, which made fluorescent Au NCs promising in biological applications owing to their chemical and photophysical stability. In addition, by comparing the interactions of small molecules, Au NCs, and large nanomaterials with serum albumin, it was found that the binding constants were gradually increased with the increase of particle size. This work has elucidated the interaction mechanisms between nanoclusters and proteins, and shed light on a new interaction mode different from the protein corona on the surface of nanoparticles, which will highly contribute to the better design and applications of fluorescent nanoclusters.

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Sulfur is critical for the correct structure and proper function of proteins. Yet, lacking a sensitive enough isotope, nuclear magnetic resonance (NMR) experiments are unable to deliver for sulfur in proteins the usual wealth of chemical, dynamic, and structural information. This limitation can be circumvented by substituting sulfur with selenium, which has similar physicochemical properties and minimal impact on protein structures but possesses an NMR compatible isotope (77Se). Here we exploit the sensitivity of 77Se NMR to the nucleus’ chemical milieu and use selenomethionine as a probe for its proteinaceous environment. However, such selenium NMR spectra of proteins currently resist a reliable interpretation because systematic connections between variations of system variables and changes in 77Se NMR parameters are still lacking. To start narrowing this knowledge gap, we report here on a biological 77Se magnetic resonance data bank based on a systematically designed library of GB1 variants in which a single selenomethionine was introduced at different locations within the protein. We recorded the resulting isotropic 77Se chemical shifts and relaxation times for six GB1 variants by solution-state 77Se NMR. For four of the GB1 variants we were also able to determine the chemical shift anisotropy tensor of SeM by solid-state 77Se NMR. To enable interpretation of the NMR data, the structures of five of the GB1 variants were solved by X-ray crystallography to a resolution of 1.2 Å, allowing us to unambiguously determine the conformation of the selenomethionine. Finally, we combine our solution- and solid-state NMR data with the structural information to arrive at general insights regarding the execution and interpretation of 77Se NMR experiments that exploit selenomethionine to probe proteins.

Ideally, a single-molecule platform would be simple to implement (in terms of hardware, method, and analysis) and mimic cell-like conditions. This inspired us to develop Convex Lens-induced Confinement (CLiC) microscopy, to enable direct visualization, manipulation, and quantification of biomolecules at the single molecule level. CLiC overcomes the limitations of existing techniques like TIRF (total internal reflection fluorescence), confocal microscopy, and optical/magnetic trapping. By mechanically confining molecules to nanoscale wells (and other features), CLiC enables long observation times (from seconds to hours) of untethered and freely diffusing biomolecules. Looking at hundreds of copies of molecules (or more) at once, it provides high statistics and good signal-to-noise, enabling dissection of complex processes. By enabling reagent exchange and control over confinement geometry, CLiC mimics crowded conditions in cells. In this work, we use CLiC imaging to investigate the role of DNA structure in mediating the binding activity of small molecules (oligonucleotide probes, proteins) to target sites on DNA, building on our recent publication (Scott et. al., Nucleic Acids Research, 2018). We study the impact of temperature and DNA supercoiling upon the binding affinity and kinetics of small molecules to individual, untethered DNA plasmids. By interpreting our microscopy results with supporting simulations, we investigate the role of specific higher-order structures (such as ZDNA) in actively mediating site-unwinding; for instance, we establish that ZDNA is prevalent at low temperature, but suppressed at high temperature. Further, we explore how oligonucleotide sequence, solution salinity, crowding agents, and other cofactors (such as oligo-binding proteins) impact the interaction kinetics and affinities. Looking ahead, we extend our CLiC nucleic acid assay to interrogate and quantify the binding of modified DNA to RNA targets, a fundamental parameter in understanding the mechanisms and efficacies of nucleic acid therapeutics. 2350-PosTemperature Dependence of the Protein-Chromophore Hydrogen Bond Dynamics in the Far-Red Fluorescent Proteins mNeptune1, mNeptune2.5 and mCardinal2 Chandra Dhakal, Prem Chapagain, Xuewen Wang. Physics, Florida International University, Miami, FL, USA. Low temperature experiments on mneptune1, mNeptune2.5 and mcardinal2 show a reduced stokes shift compared to room temperature. This suggests that the increased flexibility of the chromophore environment at higher temperatures along with its ability to reorganize after excitation is related to the larger stokes shift. We used molecular dynamics (MD) simulations to investigate the dynamics of the hydrogen bonds formed in the chromophore regions of the farred fluorescent proteins mNeptune1, mneptune2.5 and mcardinal2. We explored the protein-chromophore hydrogen bond pattern at various temperatures and correlate the hydrogen bond dynamics to the experimentally observed Stoke's shifts. Spider silks form biomaterials including fibres, fil...

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