Session B5: Molecular Biophysics: Structural and Functional Properties

Increasing protein production rates can decrease the rate at which functional protein is produced

The rate at which soluble, functional protein is produced by the ribosome has recently been found to vary in complex and unexplained ways as various translation-associated rates are altered through synonymous codon substitutions. We combine a well-established ribosome-traffic model with a master-equation model of co-translational domain folding to explore the scenarios that are possible for the protein production rate, \textbf{\textit{J}}, and the functional-nascent protein production rate, \textbf{\textit{F}}, as the rates associated with translation are altered. We find that while \textbf{\textit{J}} \quad monotonically increases as the rates of translation-initiation, -elongation and -termination increase, \textbf{\textit{F}} can either increase or decrease. \textbf{\textit{F}} exhibits non-monotonic behavior because increasing these rates can cause a protein to be synthesized more rapidly but provide less time for nascent-protein domains to co-translationally fold thereby producing less functional nascent protein immediately after synthesis. We further demonstrate that these non-monotonic changes in \textbf{\textit{F}}\textbf{ }affect the post-translational, steady-state levels of functional protein in a similar manner. Our results provide a possible explanation for recent experimental observations that the specific activity of enzymatic proteins can decrease with increased synthesis rates and can in principle be used to rationally-design transcripts to maximize the production of functional nascent protein.   

Separation and Characterization of DNA Molecules and Intermolecular Interactions in Pressure-Driven Micro Flow

Pressure-driven flow in micron-sized diameter capillaries can be used to separate DNA molecules by size in a technique called Free Solution Hydrodynamic Separation. By coupling this technique with Cylindrical Illumination Confocal Spectroscopy, we have developed a highly sensitive and quantitative platform capable of separating DNA molecules by length over a large dynamic range (25 bp to 48 kbp) in a single run using only picoliters or femtograms of a DNA sample. The optical detection volume completely spans the capillary cross section, enabling highly efficient single molecule detection for enhanced sensitivity and quantification accuracy via single molecule counting. Because each DNA molecule generates its own fluorescent burst, these burst profiles can be further analyzed to individually characterize each DNA molecule's shape as it passes through the detection region. We exploit these burst profiles to visualize fluctuations in conformation under shear flow in microcapillaries, and utilizing combined mobility shift analysis, explore the complex relationship between molecular properties including length and conformation, hydrodynamic mobility, solution conditions including ion species and concentrations, and separation conditions including flow rate and capillary diameter.   

Optical Properties of Fresh Biomass Burning Aerosols: A comparison of experimental measurements and T-Matrix Method Calculations

Due to their abundance the absorption properties of airborne soot aerosols from biomass burning (BB) may influence directly the atmospheric visibility as well as local and global climate. A major issue with soot particles is that they cannot be considered as spherical and thus, `conventional' scattering theories such as the Mie theory results cannot be compared with the experimental measurements. An adequate numerical treatment is possible with the T-matrix method. We measured the extinction and scattering cross sections of fresh BB aerosols experimentally in our lab. TEM images of filter samples revealed the shape and morphology of the soot particles. Assuming that the particles can be approximated by spheroids, T-matrix method can be used. This is a suitable numerical tool, since it is an exact method based on a solution of Maxwell's equations. We used the FORTRAN code provided by Mishchenko and Travis and calculated the absorption, and scattering cross sections of soot. We present preliminary results comparing the measured and calculated values at selected wavelengths of light.   

Evidence for conformational capture mechanism for damage recognition by NER protein XPC/Rad4.

Altered flexibility of damaged DNA sites is considered to play an important role in damage recognition by DNA repair proteins. Characterizing lesion-induced DNA dynamics has remained a challenge. We have combined ps-resolved fluorescence lifetime measurements with cytosine analog FRET pair uniquely sensitive to local unwinding/twisting to analyze DNA conformational distributions. This innovative approach maps out with unprecedented sensitivity the alternative conformations accessible to a series of DNA constructs containing 3-base-pair mismatch, suitable model lesions for the DNA repair protein xeroderma pigmentosum C (XPC) complex. XPC initiates eukaryotic nucleotide excision repair by recognizing various DNA lesions primarily through DNA deformability. Structural studies show that Rad4 (yeast ortholog of XPC) unwinds DNA at the lesion site and flips out two nucleotide pairs. Our results elucidate a broad range of conformations accessible to mismatched DNA even in the absence of the protein. Notably, the most severely distorted conformations share remarkable resemblance to the deformed conformation seen in the crystal structure of the Rad4-bound ``recognition'' complex supporting for the first time a possible ``conformational capture'' mechanism for damage recognition by XPC/Rad4.   

Dopant-Engineered Wide-Band Gap Semiconductors for Deep Tissue Bioimaging.

Optical spectroscopy promises improved lateral resolution for in vivo imaging but is limited by background fluorescence and photon attenuation. There is clearly an unmet clinical need for new hybrid approaches that use fluorescence to identify cancer margins intraoperatively during the initial operation. An efficient strategy to increase the imaging depth and diagnostic capability, beyond what two-photon absorption (2PA) offers, is to use longer excitation wavelengths outside the water absorption window through three-photon absorption (3PA). Although a variety of existing fluorescent dyes, fluorescent proteins, and calcium indicators could be used in 3PA, they have low or moderate 3PA cross-sections and suffer from photobleaching. The non-linear 3PA coefficient of such fluorescent probes is often low necessitating high excitation powers, which could cause overheating, photodamage, and photo-induced toxicity. To address this demand we have designed dopant-engineered ZnO nanoparticles (d-ZnO NPs) for enabling 3PA with higher penetration depth, lower background noise, and improved spatial resolution (\textless 1 um) at powers below 5 mW.   

Non-perturbative Quantification of Ionic Charge Transfer through Nm-Scale Protein Pores Using Graphene Microelectrodes

Conventional electrical methods for detecting charge transfer through protein pores perturb the electrostatic condition of the solution and chemical reactivity of the pore, and are not suitable to be used for complex biofluids. We developed a non-perturbative methodology (\textasciitilde fW input power) for quantifying trans-pore electrical current and detecting the pore status (i.e., open vs. closes) via graphene microelectrodes. Ferritin was used as a model protein featuring a large interior compartment, well-separated from the exterior solution with discrete pores as charge commuting channels. The charge flowing through the ferritin pores transfers into the graphene microelectrode and is recorded by an electrometer. In this example, our methodology enables the quantification of an inorganic nanoparticle--protein nanopore interaction in complex biofluids.   

An Enhanced Platform for Bioelectrochemical Systems: A Novel Approach to Characterize Lipid Structure on Graphene

Graphene is a two-dimensional material composed of a single carbon layer that offers several appealing properties including high conductivity, large surface area, and flexibility. Its unique properties make graphene an ideal substrate for several applications, including energy storage, optical electronics, and medical devices. Functionalizing graphene with a lipid bilayer both increases its biocompatibility and provides a platform for diverse bioelectrochemical systems. However, characterization of lipids on graphene is challenging since traditional fluorescent methods for characterization of supported lipid structures are ineffective on graphene due to its highly quenching nature. Furthermore, there are multiple conflicting models published for the structure of lipids on graphene. We demonstrate that a novel technique using Raman spectroscopy and photoluminescence (PL) allows for characterization of lipids on graphene while providing additional benefits over conventional setups. We use Raman-PL in conjunction with liquid-AFM and QCM-D to determine the structure, fluidity, and homogeneity of lipids on graphene.   

Role of Transbilayer Distribution of Lipid Molecules on the Structure and Protein-Lipid Interaction of an Amyloidogenic Protein on the Membrane Surface

We used molecular dynamics simulations to examine the effects of transbilayer distribution of lipid molecules, particularly anionic lipids with negatively charged headgroups, on the structure and binding kinetics of an amyloidogenic protein on the membrane surface and subsequent protein-induced structural disruption of the membrane. Our systems consisted of a model beta-sheet rich dimeric protein absorbed on asymmetric bilayers with neutral and anionic lipids and symmetric bilayers with neutral lipids. We observed larger folding, domain aggregation, and tilt angle of the absorbed protein on the asymmetric bilayer surfaces. We also detected more focused bilayer thinning in the asymmetric bilayer due to weak lipid-protein interactions. Our results support the mechanism that the higher lipid packing in the protein-contacting lipid leaflet promotes stronger protein-protein but weaker protein-lipid interactions of an amyloidogenic protein on the membrane surface. We speculate that the observed surface-induced structural and protein-lipid interaction of our model amyloidogenic protein may play a role in the early membrane-associated amyloid cascade pathway that leads to membrane structural damage of neurons in Alzheimer's disease.   

Effective bending rigidity of lipid membranes with coexisting gel and fluid domains

Lipid membranes undergo a wide array of dynamic transformations that are essential to cell function. These hierarchical dynamics span several decades in length and time scales, ranging from the rotation and diffusion of individual lipids to the undulation of micron-sized patches of the membrane. Formation of rigid domains in a fluid lipid matrix not only impacts the local lipid dynamics, but also is predicted to modulate the membrane mechanical properties that govern large-scale membrane deformations. Here we use neutron spin echo spectroscopy (NSE) to show that the effective bending modulus of lipid membranes with coexisting gel and fluid phases directly depends on the area fraction of the rigid gel domains. Our experimental results are in good agreement with theoretical predictions for heterogeneous lipid membranes and have important implications for understanding the effects of rigid inhomogeneities, such as transmembrane proteins or lipid domains, on the elasticity of biological membranes.   

Charge Inversion in semi-permeable membranes

Role of semi-permeable membranes like lipid bilayer is ubiquitous in a myriad of physiological and pathological phenomena. Typically, lipid membranes are impermeable to ions and solutes; however, protein channels embedded in the membrane allow the passage of selective, small ions across the membrane enabling the membrane to adopt a semi-permeable nature. This semi-permeability, in turn, leads to electrostatic potential jump across the membrane, leading to effects such as regulation of intracellular calcium, extracellular-vesicle-membrane interactions, etc. In this study, we theoretically demonstrate that this semi-permeable nature may trigger the most remarkable charge inversion (CI) phenomenon in the cytosol-side of the negatively-charged lipid bilayer membrane that are selectively permeable to only positive ions of a given salt. This CI is manifested as the changing of the sign of the electrostatic potential from negative to positive from the membrane-cytosol interface to deep within the cytosol. We study the impact of the parameters such as the concentration of this salt with selectively permeable ions as well as the concentration of an external salt in the development of this CI phenomenon. We anticipate such CI will profoundly influence the interaction of membrane and intra-cellular moieties (e.g., exosome or multi-cellular vesicles) having implications for a host of biophysical processes.   

Characterization of Alzheimer's Protective and Causative Amyloid-beta Variants Using a Combination of Simulations and Experiments

The aggregation of amyloid-beta (A$\beta )$ peptides plays a crucial role in the etiology of Alzheimer's disease (AD). Recently, it has been reported that an A2T mutation in A$\beta $ can protect from AD. Interestingly, an A2V mutation has been also found to offer protection against AD in the heterozygous state. Structural characterization of these natural A$\beta $ variants thus offers an intriguing approach to understand the molecular mechanism of AD. Toward this goal, we have characterized the conformational landscapes of the intrinsically disordered WT, A2V, and A2T A$\beta $1-42 variant monomers and dimers by using extensive atomistic molecular dynamics (MD) simulations. Simulations reveal markedly different secondary and tertiary structure at the central and C-terminal hydrophobic regions of the peptide, which play a crucial role in A$\beta $ aggregation and related toxicity. For example, an enhanced double $\beta $-hairpin formation was observed in A2V monomer. In contrast, the A2T mutation enhances disorder of the conformational ensemble due to formation of atypical long-range interactions. These structural insights obtained from simulations allow understanding of the differential aggregation, oligomer morphology, and LTP inhibition of the variants observed in the experiments and offer a path toward designing and testing aggregation inhibitors.   

Single-Molecule Studies of Hyaluronic Acid Conformation

Hyaluronic acid (HA) is a charged linear polysaccharide abundant in extracellular spaces. Its solution conformation and mechanical properties help define the environment outside of cells, play key roles in cell motility and adhesion processes, and are of interest for the development of HA biomaterials. Intra-chain hydrogen bonds and electrostatic repulsion contribute to HA’s physical structure, but the nature of this structure, as well as its dependence on solution electrostatics, are not well-understood. To address this problem, we have investigated HA conformation and mechanical properties under a range of solution conditions systematically designed to affect charge screening or hydrogen bonding. We used magnetic tweezers to apply biological-scale stretching forces to individual HA chains under varying solution conditions.