Open source platform for the execution and analysis of mechanical refolding experiments

Abstract Motivation: Single-molecule force spectroscopy has facilitated the experimental investigation of biomolecular force-coupled kinetics, from which the kinetics at zero force can be extrapolated via explicit theoretical models. The atomic force microscope (AFM) in particular is routinely used to study protein unfolding kinetics, but only rarely protein folding kinetics. The discrepancy arises because mechanical protein refolding studies are more technically challenging. Results: We developed software that can drive and analyse mechanical refolding experiments when used with the commercial AFM setup 'Picoforce AFM', Bruker (previously Digital Instruments). We expect the software to be easily adaptable to other AFM setups. We also developed an improved method for the statistical characterization of protein folding kinetics, and implemented it into an AFM-independent software module. Availability: Software and documentation are available at http://code.google.com/p/refolding under Apache License 2.0. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online

Characterization of the conformational space of the murine prion protein using single-molecule force spectroscopy techniques

The conversion of the cellular prion protein (PrPC) to its infectious counterpart (PrPSc) is the initial step of prion diseases. These neurodegenerative disorders are characterized by different incubation times, sympthoms and disease phenotypes. Structural heterogenity of PrP aggregates is responsible for this biological diversity. Understanding the structural rearrangements of PrP at the monomeric and oligomeric level is essential to gain insights into its aggregation processes. However traditional \u201cin-bulk\u201d techniques can only provide ensemble-averaged information for monomer and oligomer structures. We applied single-molecule force spectroscopy to characterize the heterogeneous structural ensemble of the murine PrP at the monomeric and at the oligomeric level. By stretching chimeric protein construct carrying one MoPrP molecule we found that the protein folds with a two state mechanism. Less frequently the protein can adopt more extended conformations that encompass also the N-terminal domain. These structures might be involved in subsequent aggregation processes. We also developed an assay to characterize the oligomerization processes using multiple PrP constructs. By analyzing the extension of these constructs under tension we characterized the structure between different PrP moieties, under different conditions. We found that reciprocal PrP orientation affects the length and mechanical resistance of these structures but their events frequency. Comparing the structures observed from monomers, dimers, trimers and tetramers we found that their frequency of events and their average length increased by increasing the number of PrP moieties. Remarkably, decreasing pH to more acidic values resulted in a higher frequency of events that involved structures between PrP moieties only in multimeric constructs. Instead, increasing the ionic strength significantly diminished their frequency, indicating how solution conditions can strongly alter the conformational transitions. These results provide a new scenario on PrP misfolding and aggregation processes, characterizing their early aggregation steps under different reaction conditions

Open source single molecule force spectroscopy

Single molecule force spectroscopy (SMFS) experiments provide an experimental benchmark for testing simulated and theoretical predictions of protein unfolding behavior. Despite it use since 1997, the labs currently engaged in SMFS use in-house software and procedures for critical tasks such as cantilever calibration and Monte Carlo unfolding simulation. Besides wasting developer time producing and maintaining redundant implementations, the lack of transparency makes it more difficult to share data and techniques between labs, which slows progress. In some cases it can also lead to ambiguity as to which of several similar approaches, correction factors, etc. were used in a particular paper.In this thesis, I introduce an SMFS sofware suite for cantilever calibration (calibcant), experiment control (unfold-protein), analysis (Hooke), and postprocessing (sawsim) in the context of velocity clamp unfolding of I27 octomers in buffers with varying concentrations of CaCl2. All of the tools are licensed under open source licenses, which allows SMFS researchers to centralize future development. Where possible, care has been taken to keep these packages operating system (OS) agnostic. The experiment logic in unfold-protein and calibcant is still nominally OS agnostic, but those packages depend on more fundamental packages that control the physical hardware in use. At the bottom of the physical-interface stack are the Comedi drivers from the Linux kernel. Users running other operating systems should be able to swap in analogous low level physical-interface packages if Linux is not an option.Ph.D., Physics -- Drexel University, 201