Biomaterial Database

Tracking unfolding and refolding reactions of single proteins using atomic force microscopy methods. 2013

Paul J Bujalowski, and Andres F Oberhauser

Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, TX 77555, USA.

During the last two decades single-molecule manipulation techniques such as atomic force microscopy (AFM) has risen to prominence through their unique capacity to provide fundamental information on the structure and function of biomolecules. Here we describe the use of single-molecule AFM to track protein unfolding and refolding pathways, enzymatic catalysis and the effects of osmolytes and chaperones on protein stability and folding. We will outline the principles of operation for two different AFM pulling techniques: length clamp and force-clamp and discuss prominent applications. We provide protocols for the construction of polyproteins which are amenable for AFM experiments, the preparation of different coverslips, choice and calibration of AFM cantilevers. We also discuss the selection criteria for AFM recordings, the calibration of AFM cantilevers, protein sample preparations and analysis of the obtained data.

UI	MeSH Term	Description	Entries
D008958	Models, Molecular	Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures.	Molecular Models,Model, Molecular,Molecular Model
D009994	Osmolar Concentration	The concentration of osmotically active particles in solution expressed in terms of osmoles of solute per liter of solution. Osmolality is expressed in terms of osmoles of solute per kilogram of solvent.	Ionic Strength,Osmolality,Osmolarity,Concentration, Osmolar,Concentrations, Osmolar,Ionic Strengths,Osmolalities,Osmolar Concentrations,Osmolarities,Strength, Ionic,Strengths, Ionic
D002021	Buffers	A chemical system that functions to control the levels of specific ions in solution. When the level of hydrogen ion in solution is controlled the system is called a pH buffer.	Buffer
D002138	Calibration	Determination, by measurement or comparison with a standard, of the correct value of each scale reading on a meter or other measuring instrument; or determination of the settings of a control device that correspond to particular values of voltage, current, frequency or other output.	Calibrations
D003198	Computer Simulation	Computer-based representation of physical systems and phenomena such as chemical processes.	Computational Modeling,Computational Modelling,Computer Models,In silico Modeling,In silico Models,In silico Simulation,Models, Computer,Computerized Models,Computer Model,Computer Simulations,Computerized Model,In silico Model,Model, Computer,Model, Computerized,Model, In silico,Modeling, Computational,Modeling, In silico,Modelling, Computational,Simulation, Computer,Simulation, In silico,Simulations, Computer
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D055595	Mechanical Phenomena	The properties and processes of materials that affect their behavior under force.	Mechanical Concepts,Mechanical Processes,Mechanical Phenomenon,Mechanical Process,Concept, Mechanical,Concepts, Mechanical,Mechanical Concept,Phenomena, Mechanical,Phenomenon, Mechanical,Process, Mechanical,Processes, Mechanical
D055767	Immobilized Proteins	Proteins that are chemically bound to a substrate material which renders their location fixed. The immobilization of proteins allows their use in chemical reactions without being diluted by solvent.	Proteins, Immobilized
D058767	Protein Unfolding	Conformational transitions of the shape of a protein to various unfolded states.	Unfolding, Protein
D058849	Protein Refolding	Conformational transitions of a protein from unfolded states (after PROTEIN UNFOLDING) to a more folded state.	Refolding, Protein