Bulletin of the American Physical Society
APS March Meeting 2010
Volume 55, Number 2
Monday–Friday, March 15–19, 2010; Portland, Oregon
Session A7: Single Chain Experiments: from Polymers to Biophysics |
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Sponsoring Units: DBP DPOLY Chair: Alexander Grosberg, New York University Room: Portland Ballroom 254 |
Monday, March 15, 2010 8:00AM - 8:36AM |
A7.00001: Molecular architecture governs the kinetics of single molecule unfolding under force Invited Speaker: Proteins are a paradigm of complexity due to the broad energy scales involved in holding their folded structure intact under thermal fluctuations. Moreover, a subset of all proteins is known to withstand stretching forces on the order of 100 pN on the timescale of seconds. The dynamic mechanism by which these proteins support stress on the molecular level remains largely unknown. With the advent of single molecule techniques using the atomic force microscope (AFM), we measure the kinetics of unfolding as a function of a constant force for the archetypal mechanically stable proteins: the degradation protein ubiquitin and the 27th immunoglobulin domain (I27) in muscle. Instead of filtering the data, we develop a maximum likelihood method to analyze all force-clamp unfolding dwell times in order to deduce the underlying kinetics. We find that the large pool of data for both proteins is best fit with stretched exponential distributions, whose exponent depends on the molecular architecture of the protein. Our analysis of previously published kinetic data on ubiquitin as a function of force [PNAS, Garcia-Manyes et. al., 2009] follows stretched exponential kinetics at all forces. Rescaling the data by the exponent shows that the characteristic timescale for the rupture of the molecules increases slower than exponentially with the force, challenging the Bell model. The observed complex kinetics may therefore be of evolutionary importance, as it increases the protein's mechanical resilience. We discuss competing microscopic mechanisms by which the complex kinetic profiles may arise. [Preview Abstract] |
Monday, March 15, 2010 8:36AM - 9:12AM |
A7.00002: DNA Sequencing Using an Engineered Protein Nanopore Invited Speaker: Inexpensive and fast sequencing of DNA is of paramount importance to medicine, the life sciences and to many other applications. Because of the nanometer diameter of DNA a nanometer-scale reader directly interfaced to macroscopic observables seems particularly attractive. We are working on a new single molecule technique based on a biological pore embedded in a lipid bilayer. When a voltage is applied across the bilayer an ion current is measured that flows through the nanometer opening of the pore. Poly-negatively charged single stranded DNA passes through the pore and reduces the ion current with the remaining ion current being indicative of the nucleotide type in the constriction of the pore. The protein pore that we introduced to the field, MspA, has a shape ideally suited to nanopore sequencing, has robustness comparable to solid state devices, is easily reproduced with sub-nanometer level precision and is engineerable using genetic mutations. I will present proof-of-principle data showing that this technique can lead to a direct very inexpensive and fast sequencing technology. The experimental electronic signatures of the DNA translocation process provide an ideal test bed for molecular dynamics simulations, which in turn allows developing intuition and prediction of nanoscale dynamics. [Preview Abstract] |
Monday, March 15, 2010 9:12AM - 9:48AM |
A7.00003: Electrostatic Focusing of DNA into Nanoscale Invited Speaker: Solid-state nanopores are sensors capable of analyzing individual unlabelled DNA molecules in solution. While the critical information obtained from nanopores (e.g., DNA sequence) is the signal collected during DNA translocation, the throughput of the method is determined by the rate at which molecules arrive and thread into the pores. Using a combination of experiment and theoretical modeling, we study the process of DNA capture into silicon nanopores of molecular dimensions. We find an increase in capture rate as the DNA length increases from 800 to 8,000 basepairs and a length-independent capture rate for longer molecules. We show that this change of behavior is the signature of a transition from a free energy barrier dominated regime to a diffusion limited regime as the molecular weight increases. We also show that capture rates can be increased dramatically by enhancing the electric field in the vicinity of the pore by setting up salt gradients across the pore. [Preview Abstract] |
Monday, March 15, 2010 9:48AM - 10:24AM |
A7.00004: Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins Invited Speaker: Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone. [Preview Abstract] |
Monday, March 15, 2010 10:24AM - 11:00AM |
A7.00005: How Bacteriophage Genomes Get Inside Cells Invited Speaker: Modern molecular biology was founded in part on the basis of experiments done in the context of bacterial viruses. There has been a resurgence of interest in these viruses as a result of the fact that they serve as powerful model systems for the attempt to build detailed quantitative models of a biological system and to test those models with systematic, quantitative experimentation. One of the central unanswered questions in the study of these viruses is the precise mechanism whereby the genomic DNA enters its host. To that end, we have carried out both in vitro and in vivo single-molecule experiments aimed at measuring the DNA translocation process in real time. In this talk, I will report on a series of single-molecule experiments which explore the mechanism and rate of ejection in vitro and how it depends upon factors such as dye concentration, salt, surface preparations, etc. These experiments suggest ejection mechanisms that are at odds with the standard picture of DNA translocation by bacterial viruses. The second set of experiments amount to a single-molecule version of the Hershey-Chase experiment which permits the observation of individual viruses infecting individual bacteria. [Preview Abstract] |
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