Session Y42: Focus Session: Single Molecule Studies of Nucleotides and Nanomachines

Length selective accumulation of oligonucleotides in thermal gradients

Central to most Origin-of-Life scenarios is the possibility for pre-biotic organic molecules to interact in order to form increasingly complex, catalytic molecular machinery ultimately capable of autonomous replication. While strong evidence for the spontaneous synthesis of single nucleotides [1] recently arose, concentrations required to allow these building blocks to polymerize [2] and gain functionality, still seem improbable for early earth conditions. Here, we demonstrate experimentally that temperature gradients across pores, as found in rocks near hydrothermal vents [3], are sufficient to accumulate nucleotides efficiently from dilute solutions. In particular we show that depending on the pores' dimensions, it can act as a length-selective molecular filter. We suggest that equivalent systems could have served as meeting points for long and complex molecules, too rare to find each other in a dilute primordial ocean. Furthermore, we discuss under which conditions this selection could have triggered the evolutionary adaptation of molecular replicators, and how polymerase chain reaction assays could nowadays benefit from the presented concept. References: 1. M. Powner et al., Nature 459 (2009), 2. G. Costanzo et al., ChemBioChem 13 (2012), 3. P. Baaske et al., PNAS (2007)   

Reconstructing kinetic pathways from single-molecule FRET experiments using Bayesian inference

Single-molecule FRET studies have enabled observation of conformational transitions in individual molecules, allowing targeted investigations into the mechanistic function of molecular machines. Like in many single-molecule platforms, sm-FRET studies yield observations of hundreds of noisy time series, which report on the same underlying conformational steps, but exhibit significant variations in photophysical properties and kinetic rates. Reconstruction of a consensus kinetic pathway from such noisy measurements is statistically challenging. Hidden Markov Models are widely used to identify states and estimate the associated kinetic rates. Existing techniques perform inference on one time series at a time, yielding variable parameter estimates that must now be `averaged' using ad-hoc experiment specific post-processing steps. Here, we propose a technique known as Empirical Bayes estimation, which performs simultaneous analysis on a collection of trajectories in an experiment. This results in a single estimate for a consensus kinetic model, as well as a significantly reduced estimation error. By comparing models with different constraints, we show how these methods may be used to test detailed mechanistic hypotheses in a statistically principled, adaptable manner.   

Single Molecule Measurements Using Correlation Force Spectroscopy

Thermal noise represents a fundamental limit in force measurements. We describe single molecule measurements using two AFM cantilevers that have lower thermal noise than single-cantilever measurements. We achieve this by measuring the correlated thermal motions of two closely spaced cantilevers. Because only correlated thermal noise is measured, there is lower noise. In addition, the use of two cantilevers produces both decreased hydrodynamic fluid damping and decreased van der Waals forces acting on an AFM probe, both of which are interferences in single molecule measurements. Analysis of the correlated motions reveals molecular damping, a parameter that is not sensed with conventional (pulling) AFM single molecule force spectroscopy. When a molecule is straddled between the two cantilevers, the correlation arises from the solvent coupling as well as stiffness and damping of the molecule. We will describe the technique of correlation force spectroscopy and measurements of the mechanical properties of single polymer chains such as dextran.   

A kinetic clutch governs uncoiling by type IB topoisomerases

Type IB topoisomerases (Top1B) are essential enzymes that relax excessive DNA supercoiling associated with replication and transcription and are important drug targets for cancer chemotherapy. The natural compound camptothecin (CPT) and the cancer chemotherapeutics derived from it, irinotecan and topotecan, are highly specific inhibitors of human nuclear Type IB topoisomerase (nTop1). We employed a magnetic-tweezers based single-molecule DNA supercoil relaxation assay to measure the torque dependence of human nuclear Top1 relaxation (nTop1) and inhibition by CPT. For comparison, we examined the human mitochondrial (Top1mt) topoisomerase and an N-terminal deletion mutant of nTop1 (Top68). Despite substantial sequence homology in their core domains, nTop1 and Top1mt exhibit dramatic differences in sensitivity to torque and CPT, with Top68 betraying intermediate characteristics. In particular, nTop1 displays nearly torque-independent religation probability, distinguishing it from other Top1B enzymes studied to date. Kinetic modeling reveals a hitherto unobserved torque-independent transition linking the DNA rotation and religation phases of the enzymatic cycle. The parameters of this transition determine the torque sensitivity of religation, and the efficiency of CPT binding. This ``kinetic clutch'' mechanism explains the molecular basis of CPT sensitivity and more generally provides a framework with which to interpret Top1B activity and inhibition.   

Stretch Moduli of Ribonucleotide Embedded Short DNAs

Understanding the mechanical properties of DNA is essential to comprehending the dynamics of many cellular functions. DNA deformations are involved in many mechanisms when genetic information needs to be stored and used. In addition, recent studies have found that Ribonucleotides (rNMPs) are among the most common non-standard nucleotides present in DNA. The presences of rNMPs in DNA might cause mutation, fragility or genotoxicity of chromosome but how they influence the structure and mechanical properties of DNA remains unclear. By means of Atomic Force Microscopy (AFM) based single molecule spectroscopy, we measure the stretch moduli of double stranded DNAs (dsDNA) with 30 base pairs and 5 equally embedded rNMPs. The dsDNAs are anchored on gold substrate via thiol chemistry, while the AFM tip is used to pick up and stretch the dsDNA from its free end through biotin-streptavidin bonding. Our preliminary results indicate that the inclusion of rNMPs in dsDNA might significantly change its stretch modulus, which might be important in some biological processes.   

The interplay between single-stranded binding proteins on RNA secondary structure

RNA-protein interactions are critical for Biology because of their regulatory effects on mRNA and protein levels. There are typically several specific protein binding sites on an RNA molecule. A protein can bind one of these sites only if the RNA folds into a structure that leaves the entire binding site free of base pairs. Therefore, a protein binding to an RNA excludes some of the originally permitted RNA structures, causing a change in the structural ensemble. Thus, the probability of another protein to bind the same RNA at a different site will change upon binding of the first protein. To discover such effects, we combine methods of RNA secondary structure prediction with models of protein-RNA interaction. We focus on an RNA molecule with two protein binding sites. The ensemble of secondary structures of random RNA sequences is considered, and numerical calculations show the existence of a semi-long-range interaction between the protein binding sites mediated by the thermodynamics of the RNA structures. A brief analytic argument for this correlation is given, and a phase transition to a high-temperature phase, possibly related to the molten-glass phase transition of secondary RNA structures, is discussed.   

Mechanostability of Proteins and Virus Capsids

Molecular dynamics of proteins within coarse grained models have become a useful tool in studies of large scale systems. The talk will discuss two applications of such modeling. The first is a theoretical survey of proteins' resistance to constant speed stretching as performed for a set of 17134 simple and 318 multidomain proteins. The survey has uncovered new potent force clamps. They involve formation of cysteine slipknots or dragging of a cystine plug through the cystine ring and lead to characteristic forces that are significantly larger than the common shear-based clamp such as observed in titin. The second application involves studies of nanoindentation processes in virus capsids and elucidates their molecular aspects by showing deviations in behavior compared to the continuum shell model. Across the 35 capsids studied, both the collapse force and the elastic stiffness are observed to vary by a factor of 20. The changes in mechanical properties do not correlate simply with virus size or symmetry. There is a strong connection to the mean coordination number $< z >$, defined as the mean number of interactions to neighboring amino acids. The Young's modulus for thin shell capsids rises roughly quadratically with $< z >$ - 6, where 6 is the minimum coordination for elastic stability in three dimensions. \\[4pt] [1] M. Sikora, J. I. Sulkowska, and M. Cieplak, Mechanical strength of 17134 model proteins and cysteine slipknots. PLoS Computational Biology, 5:e1000547 (2009).\\[0pt] [2] M. Sikora nd M. Cieplak, Mechanical stability of multidomain proteins and novel mechanical clamps. Proteins. Struct. Fun. Bioinf. 79:1786-1799 (2011).\\[0pt] [3] M. Sikora and M. Cieplak, Formation of cystine slipknots in dimeric proteins. Phys. Rev. Lett. 109 208101 (2012).\\[0pt] [4] M. Cieplak and M. O. Robbins, Nanoindentation of virus capsids in a molecular model. J. Chem. Phys. 132:015101 (2010).\\[0pt] [5] M. Cieplak and M. O. Robbins, Nanoindentation of 35 virus capsids in a molecular model: Relating mechanical properties to structure (submitted).   

Nanomechanical Response of \textit{Pseudomonas aeruginosa} PAO1 Bacterial Cells to Cationic Antimicrobial Peptides

We have used an atomic force microscopy (AFM)-based creep deformation technique to study changes to the viscoelastic properties of individual Gram-negative \textit{Pseudomonas aeruginosa} PAO1 cells as a function of time of exposure to two cationic peptides: polymyxin B (PMB), a cyclic antimicrobial peptide, and the structurally-related compound, polymyxin B nonapeptide (PMBN). The measurements provide a direct measure of the mechanical integrity of the bacterial cell envelope, and the results can be understood in terms of simple viscoelastic models of arrangements of springs and dashpots, which can be ascribed to different components within the bacterial cell. Time-resolved creep deformation experiments reveal abrupt changes to the viscoelastic properties of \textit{P. aeruginosa} bacterial cells after exposure to both PMB and PMBN, with quantitatively different changes for the two cationic peptides. These measurements provide new insights into the kinetics and mechanism of action of antimicrobial peptides on bacterial cells.   

Ion Discrimination by Nanoscale Design

Proteins that form membrane-spanning channels excel at discriminating between molecules on the basis of subtle structural and chemical differences. For example, some channels distinguish between water and ions; others between Na+ (sodium) and K+ (potassium) despite identical charges and only sub-Angstrom differences in size. If we could understand these structure/function relationships, we could potentially harness biological design principles in robust nanoscale devices that mimic biological function for efficient separations. Using ab initio molecular simulations, we have interrogated the link between channel structure and selective transport, both in cellular channels and polymer membranes. Our results emphasize the surprisingly important role of the environment that surrounds ion-binding sites, as well as the coordination chemistry of the binding site for raising or lowering the free energy barrier to transport in both systems.   

Extracting Models in Single Molecule Experiments

Single molecule experiments can now monitor the journey of a protein from its assembly near a ribosome to its proteolytic demise. Ideally all single molecule data should be self-explanatory. However data originating from single molecule experiments is particularly challenging to interpret on account of fluctuations and noise at such small scales. Realistically, basic understanding comes from models carefully extracted from the noisy data. Statistical mechanics, and maximum entropy in particular, provide a powerful framework for accomplishing this task in a principled fashion. Here I will discuss our work in extracting conformational memory from single molecule force spectroscopy experiments on large biomolecules. One clear advantage of this method is that we let the data tend towards the correct model, we do not fit the data. I will show that the dynamical model of the single molecule dynamics which emerges from this analysis is often more textured and complex than could otherwise come from fitting the data to a pre-conceived model.