Open source platform for the execution and analysis of mechanical refolding experiments
ABSTRACT 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.
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.
Software and documentation are available at http://code.google.com/p/refolding under Apache License 2.0.
- SourceAvailable from: Daniel J Müller[Show abstract] [Hide abstract]
ABSTRACT: Membrane proteins comprise 30% of the proteome of higher organisms. They mediate energy conversion, signal transduction, solute transport and secretion. Their native environment is a bilayer in a physiological buffer solution, hence their structure and function are preferably assessed in this environment. The surface structure of single membrane proteins can be determined in buffer solutions by atomic force microscopy (AFM) at a lateral resolution of less than 1 nm and a vertical resolution of 0.1-0.2 nm. Moreover, single proteins can be directly addressed, stuck to the AFM stylus and subsequently unfolded, revealing the molecular interactions of the protein studied. The examples discussed here illustrate the power of AFM in the structural analysis of membrane proteins in a native environment.Nature Protocol 02/2007; 2(9):2191-7. DOI:10.1038/nprot.2007.309 · 8.36 Impact Factor
- Angewandte Chemie International Edition 05/2011; 50(19):4394-7. DOI:10.1002/anie.201006714 · 11.34 Impact Factor
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ABSTRACT: Membranes confining cells and cellular compartments are essential for life. Membrane proteins are molecular machines that equip cell membranes with highly sophisticated functionality. Examples of such functions are signaling, ion pumping, energy conversion, molecular transport, specific ligand binding, cell adhesion and protein trafficking. However, it is not well understood how most membrane proteins work and how the living cell regulates their function. We review how atomic force microscopy (AFM) can be applied for structural and functional investigations of native membrane proteins. High-resolution time-lapse AFM imaging records membrane proteins at work, their oligomeric state and their dynamic assembly. The AFM stylus resembles a multifunctional toolbox that allows the measurement of several chemical and physical parameters at the nanoscale. In the single-molecule force spectroscopy (SMFS) mode, AFM quantifies and localizes interactions in membrane proteins that stabilize their folding and modulate their functional state. Dynamic SMFS discloses fascinating insights into the free energy landscape of membrane proteins. Single-cell force spectroscopy quantifies the interactions of live cells with their environment to single-receptor resolution. In the future, technological progress in AFM-based approaches will enable us to study the physical nature of biological interactions in more detail and decipher how cells control basic processes.Reports on Progress in Physics 08/2011; 74(8). DOI:10.1088/0034-4885/74/8/086601 · 15.63 Impact Factor