Session W40: Focus Session: Single Molecule Biological Physics - Nucleic acids and Proteins

Sequence-dependent sliding kinetics of p53

Theoretical work has long proposed that one-dimensional sliding along DNA while simultaneously reading its sequence can accelerate transcription factors' (TFs) search for their target sites. More recently, functional sliding has been shown to require TFs to possess at least two DNA-binding modes. The tumor suppressor p53 has been directly observed to slide on DNA, and structural and single-molecule studies have provided evidence for a two-mode model for the protein. If the model is in fact applicable to p53, then the requirement that TFs read while they slide implies that p53's mobility on DNA should be affected by non-cognate sites and thus that its diffusivity should be generally sequence-dependent. Here we confirm this prediction with single-molecule microscopy measurements of p53's local diffusivity on non-cognate DNA. We show how a two-mode model accurately predicts the variation in local diffusivity while a single-mode model does not. Our work provides evidence that p53's sliding is indeed functional and suggests that the timing and efficiency of its activating and repressing transcription can depend on its non-cognate binding properties and its ability to change between multiple modes of binding, in addition to the much better-studied effects of cognate-site binding.   

Single-molecule study of protein-DNA target search mechanisms for dimer-active protein complexes

Protein-DNA interactions are essential to cellular processes, many of which require proteins to recognize a specific DNA target-site. This search process is well-documented for monomeric proteins, but not as well understood for systems that require dimerization at the target site for activity. We present a single-molecule study of the target-search mechanism of Protelomerase TelK, a recombinase-like protein that is only active as a dimer. We observe that TelK undergoes 1D diffusion on non-target DNA as a monomer, as expected, but becomes immobile on DNA as a dimer or oligomer despite the absence of its target site. We further show that TelK condenses non-target DNA upon dimerization, forming a tightly bound nucleo-protein complex. Together with simulations, our results suggest a search model whereby monomers diffuse along DNA, and subsequently dimerize to form an active complex on target DNA. These results show that target-finding occurs faster than nonspecific dimerization at biologically relevant protein concentrations. This model may provide insights into the search mechanisms of proteins that are active as multimeric complexes for a more accurate and comprehensive model for the target-search process by sequence specific proteins.   

Single-Molecule Encoders for Tracking Motor Proteins on DNA

Devices such as inkjet printers and disk drives track position and velocity using optical encoders, which produce periodic signals precisely synchronized with linear or rotational motion. We have implemented this technique at the nanometer scale by labeling DNA with regularly spaced fluorescent dyes. The resulting molecular encoders can be used in several ways for high-resolution continuous tracking of individual motor proteins. These measurements do not require mechanical coupling to macroscopic instrumentation, are automatically calibrated by the underlying structure of DNA, and depend on signal periodicity rather than absolute level. I will describe the synthesis of single-molecule encoders, data from and modeling of experiments on a helicase and a DNA polymerase, and some ideas for future work.   

Tuning Cargo Travel via Single Motor Velocity

Active intracellular transport is crucial for cell function, and defects are linked to diseases including neurodegeneration. Single molecule biophysical studies have revealed a great deal about the function of individual motors in vitro. However, it remains challenging to use single molecule properties of molecular motors to explain the complex range of cargo motions observed in vivo, in particular how motors work together in small ensembles. Recent reports have highlighted the sensitivity of ensemble transport to the single motor properties of processivity and force production. Here we investigate the previously unexplored role of motor velocity, and report a combined experimental and theoretical demonstration that single motor velocity crucially controls the ensemble function of two-motor transport.   

Study of Large Multimeric Biomolecules by Single-Molecule Manipulation and Imaging

Single-molecule manipulation enables us to study the properties of long chain, multimeric biomolecules. Perlecan, a giant secreted heparin sulfate proteoglycan, is a major component of basement membrane, bone stroma and blood vessels. It is involved in processes such as cell adhesion, migration and modulation of apoptosis. The changes in its synthesis and function are closely associated with many diseases, including cancer. Von Willebrand factor is a large multimeric protein circulating in blood, and is crucial for initiation of blood coagulation. We use atomic force microscope to obtain force curves and images of these proteins. We characterized the mechanical property of perlecan as well as the domain conformational changes of von Willebrand factor. The results demonstrate that single-molecule manipulation can probe directly the dynamics of large biomolecules that are usually not accessible with other methods.   

Uncovering the microscopic mechanism of strand exchange during RecA mediated homologous recombination using all-atom molecular dynamics simulations

Homologous recombination (HR) is a key step during the repair process of double-stranded DNA (dsDNA) breakage. RecA is a protein that mediates HR in bacteria. RecA monomers polymerize on a single-stranded DNA (ssDNA) separated from the broken dsDNA to form a helical filament, thus allowing strand exchange to occur. Recent crystal structures depict each RecA monomer in contact with three contiguous nucleotides called DNA triplets. Surprisingly, the conformation of each triplet is similar to that of a triplet in B-form DNA. However, in the filament the neighboring triplets are separated by loops of the RecA proteins. Single molecule experiments demonstrated that strand exchange propagation occurs in 3 base-pair increments. However, the temporal resolution of the experiments was insufficient to determine the exact molecular mechanism of the triplet propagation. Using all-atom molecular dynamics simulations, we investigated the effect of both the RecA protein and the conformation of the bound ssDNA fragment on the stability of the duplex DNA intermediate formed during the strand-exchange process. Specifically, we report simulations of force-induced unzipping of duplex DNA in the presence and absence of the RecA filament that explored the effect of the triplet ladder conformation.   

Transduction of Glycan-Lectin Binding using Near Infrared Fluorescent Single Walled Carbon Nanotubes for Glycan Profiling

In this work, we demonstrate a sensor array employing recombinant lectins as glycan recognition sites tethered via Histidine tags to Ni2+ complexes that act as fluorescent quenchers for semi-conducting single walled carbon nanotubes embedded in a chitosan to measure binding kinetics of model glycans. Two higher-affined glycan-lectin pairs are explored: fucose (Fuc) to PA-IIL and N-acetylglucosamine (GlcNAc) to GafD. The dissociation constants (KD) for these pairs as free glycans (106 and 19 $\mu $M respectively) and streptavidin-tethered (142 and 50 $\mu $M respectively) were found. The absolute detection limit for the current platform was found to be 2 $\mu $g of glycosylated protein or 100 ng of free glycan to 20 $\mu $g of lectin. Glycan detection is demonstrated at the single nanotube level (GlcNAc to GafD). Over a population of 1000 nanotubes, 289 of the SWNT sensors had signals strong enough to yield kinetic information (KD of 250 $\pm $ 10 $\mu $M). We are also able to identify the locations of ``strong-transducers'' on the basis of dissociation constant (4 sensors with KD $<$ 10 $\mu $M) or overall signal modulation (8 sensors with $>$ 5{\%} quench response). The ability to pinpoint strong-binding, single sensors is promising to build a nanoarray of glycan-lectin transducers as a method to profile glycans without protein labeling or glycan liberation pretreatment steps.   

Comprehensive single molecule dynamics and functions of lysozyme upon linear and cross-linked substrate using a carbon nanotube circuit

The dynamic processivity of individual lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates using a single-walled carbon nanotube transistor. The substrate-driven, hinge bending motions of lysozyme induce dynamic electronic signals in the underlying transistor to allow long-term monitoring of the same molecule, all without the limitations of fluorophore quenching or bleaching. For both types of substrates, lysozyme exhibits slow, processive turnover at 20 Hz and also rapid, nonproductive motions at 300 Hz. However, the latter type of motion nearly vanishes with the linear substrate, which lacks cross-links. Specifically, the nonproductive binding fills 43{\%} of the enzyme's total activity when the substrate has cross-links, but only 7{\%} with the cross-links are absent. The continuous, uninterrupted processing indicates that lysozyme can catalytically hydrolyze glycosidic bonds all the way to the end of a linear substrate, and that the motion attributed to nonproductive binding may be the lysozyme sidestepping the peptide cross-links.   

A hierarchical coarse-grained (all-atom to all residue) approach to peptides (P1, P2) binding with a graphene sheet

Recently, Kim et al. [1] have found that peptides P1: HSSYWYAFNNKT and P2: EPLQLKM bind selectively to graphene surfaces and edges respectively which are critical in modulating both the mechanical as well as electronic transport properties of graphene. Such distinctions in binding sites (edge versus surface) observed in electron micrographs were verified by computer simulation by an all-atomic model that captures the pi-pi bonding. We propose a hierarchical approach that involves input from the all-atom Molecular Dynamics (MD) study (with atomistic detail) into a coarse-grained Monte Carlo simulation to extend this study further to a larger scale. The binding energy of a free amino acid with the graphene sheet from all-atom simulation is used in the interaction parameter for the coarse-grained approach. Peptide chain executes its stochastic motion with the Metropolis algorithm. We investigate a number of local and global physical quantities and find that peptide P1 is likely to bind more strongly to graphene sheet than P2 and that it is anchored by three residues $^{4}$Y$^{5}$W$^{6}$Y. [1] S.N. Kim et al J. Am. Chem. Soc. 133, 14480 (2011).