Single-Particle Tracking of Human Lipoproteins.

Lipoproteins, such as high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very-low density lipoprotein (VLDL), play a critical role in heart disease. Lipoproteins vary in size and shape as well as in their apolipoprotein content. Here, we developed a new experimental framework to study freely diffusing lipoproteins from human blood, allowing analysis of even the smallest HDL with a radius of 5 nm. In an easily constructed confinement chamber, individual HDL, LDL, and VLDL particles labeled with three distinct fluorophores were simultaneously tracked by wide-field fluorescence microscopy and their sizes were determined by their motion. This technique enables studies of individual lipoproteins in solution and allows characterization of the heterogeneous properties of lipoproteins which affect their biological function but are difficult to discern in bulk studies.

Single-molecule measurements of the CCR5 mRNA unfolding pathways.

Secondary or tertiary structure in an mRNA, such as a pseudoknot, can create a physical barrier that requires the ribosome to generate additional force to translocate. The presence of such a barrier can dramatically increase the probability that the ribosome will shift into an alternate reading frame, in which a different set of codons is recognized. The detailed biophysical mechanism by which frameshifting is induced remains unknown. Here we employ optical trapping techniques to investigate the structure of a -1 programmed ribosomal frameshift (-1 PRF) sequence element located in the CCR5 mRNA, which encodes a coreceptor for HIV-1 and is, to our knowledge, the first known human -1 PRF signal of nonviral origin. We begin by presenting a set of computationally predicted structures that include pseudoknots. We then employ what we believe to be new analytical techniques for measuring the effective free energy landscapes of biomolecules. We find that the -1 PRF element manifests several distinct unfolding pathways when subject to end-to-end force, one of which is consistent with a proposed pseudoknot conformation, and another of which we have identified as a folding intermediate. The dynamic ensemble of conformations that CCR5 mRNA exhibits in the single-molecule experiments may be a significant feature of the frameshifting mechanism.

Amyloid triangles, squares, and loops of apolipoprotein C-III.

While a significant component of atherosclerotic plaques has been characterized as amyloid, the specific proteins remain to be fully identified. Probable amyloidogenic proteins are apolipoproteins (Apos), which are vital for the formation and function of lipoproteins. ApoCIII is an abundant protein implicated in atherosclerosis, and we show it forms a ribbonlike looped amyloid, strikingly similar to that previously reported for ApoAI and ApoCII. Triangles and squares with a width of ~50 nm were also observed, which may be a novel form of amyloid or related to previously reported amyloid rings.

Membrane remodeling by α-synuclein and effects on amyloid formation.

α-Synuclein (α-Syn), an intrinsically disordered protein, is associated with Parkinson's disease. Though molecular pathogenic mechanisms are ill-defined, mounting evidence connects its amyloid forming and membrane binding propensities to disease etiology. Contrary to recent data suggesting that membrane remodeling by α-syn involves anionic phospholipids and helical structure, we discovered that the protein deforms vesicles with no net surface charge (phosphatidylcholine, PC) into tubules (average diameter ∼20 nm). No discernible secondary structural changes were detected by circular dichroism spectroscopy upon the addition of vesicles. Notably, membrane remodeling inhibits α-syn amyloid formation affecting both lag and growth phases. Using five single tryptophan variants and time-resolved fluorescence anisotropy measurements, we determined that α-syn influences bilayer structure with surprisingly weak interaction and no site specificity (partition constant, Kp ∼ 300 M(-1)). Vesicle deformation by α-syn under a variety of different lipid/protein conditions is characterized via transmission electron microscopy. As cellular membranes are enriched in PC lipids, these results support possible biological consequences for α-syn induced membrane remodeling related to both function and pathogenesis.

Single-molecule study of G-quadruplex disruption using dynamic force spectroscopy.

Guanine-rich sequences in nucleic acids can fold into G quadruplexes, in which four guanines on a single strand combine to form G-tetrad planes stabilized by metallic ions. Sequence motifs which are predicted to form a G quadruplex are found throughout the genome and are believed to regulate a variety of biological processes. Detailed knowledge of the kinetics of G-quadruplex folding and unfolding would provide critical insight into these processes. To probe its structural stability, we used optical tweezers to disrupt single molecules of a single-stranded DNA G4 quadruplex. Dynamic force spectroscopy was employed, in which the distribution of rupture forces was measured for different loading rates and used to infer the nature of the transition state barrier for unfolding of the structure. The distance and height of the energy barriers were extracted for two observed conformations. The energy barrier was found to be close to the folded conformation, resulting in a high disruption force despite the relatively low energy barrier height.

Measuring the folding landscape of a harmonically constrained biopolymer.

Pioneering studies have shown that the probability distribution of opening length for a DNA hairpin, recorded under constant force using an optical trap, can be used to reconstruct the energy landscape of the transition. However, measurements made under constant force are subject to some limitations. Under constant force a system with a sufficiently high energy barrier spends most of its time in the closed or open conformation, with relatively few statistics collected in the transition state region. We describe a measurement scheme in which the system is driven progressively through the transition by an optical trap and an algorithm is used to extract the energy landscape of the transition from the fluctuations recorded during this process. We illustrate this technique in simulations and demonstrate its effectiveness in experiments on a DNA hairpin. We find that the combination of this technique with the use of short DNA handles facilitates a high-resolution measurement of the hairpin's folding landscape with a very short measurement time.

Effect of handle length and microsphere size on transition kinetics in single-molecule experiments.

When subject to constant tension, a DNA or RNA hairpin will typically make abrupt transitions between the open and closed state. Although the transition kinetics are an intrinsic property of the molecule, the transition rates measured in single-molecule experiments can be influenced by the configuration of the measurement system. We investigate the transition kinetics for a DNA hairpin held under constant force by an optical trap as a function of microsphere size and double-stranded DNA handle length. We find the apparent transition lifetime cannot be expressed as a function of the drag coefficient of the microsphere alone or as a function of time scales relevant to the optical trap. The apparent transition lifetime is found to be a linear function of the factor ß(eff)·α(handle), where ß(eff) is the effective drag coefficient of the microsphere near the surface and α(handle) is the stiffness of the DNA tether. The results provide insight into the perturbation to the hairpin transition kinetics due to experimental configuration and guidance for designing single-molecule experiments which determine the intrinsic molecular kinetics.

Noise associated with nonconservative forces in optical traps.

It is known that for a particle held in an optical trap the interaction of thermal fluctuations with a nonconservative scattering force can cause a persistent nonequilibrium probability flux in the particle position. We investigate position fluctuations associated with this nonequilibrium flux analytically and through simulation. We introduce a model which reproduces the nonequilibrium effects, and in which the magnitude of additional position fluctuations can be calculated in closed form. The ratio of additional nonconservative fluctuations to direct thermal fluctuations scales inversely with the square root of trap power, and is small for typical experimental parameters. In a simulated biophysical experiment the nonconservative scattering force does not significantly increase the observed fluctuations in the length of a double-stranded DNA tether.