Relatively little is known about the folding and stability of membrane proteins. Conventional thermal or chemical unfolding techniques probe the average behavior of large numbers of molecules and thus cannot resolve co-existing minor and major unfolding pathways and intermediates. Here, I applied single-molecule force measurements based on an atomic force microscope (AFM) to characterize the stability of the membrane protein bacteriorhodopsin (BR). In these mechanical unfolding experiments, an external pulling force played the role of the denaturant and lead to unfolding of the three-dimensional structure of individual proteins. It was found that single BRs unfold step-wise in a well-defined sequence of stable intermediates and in different unfolding pathways. Although single [alpha]-helices were sufficiently stable to unfold in individual steps they also exhibited certain probabilities to unfold in pairs. These observations support the "two-stage" and the "helical-hairpin" model of membrane protein folding. Dynamic force measurements showed that [alpha]-helices and helical hairpins are relatively rigid structures, which are stabilized by narrow energy barriers and have stabilities between 100-10?000 seconds. These forced unfolding experiments were complemented with the development of new force measurement techniques. It is demonstrated that hydrodynamic effects need to be considered to obtain more complete kinetic pictures of single molecules. In addition, two force spectroscopy approaches to measure the complex visco-elastic response of single molecules are presented and applied to BR. These experiments revealed that the unfolding patterns of single proteins are dominated by purely elastic polypeptide extension and determined the dissipative interactions associated with the unfolding of single [alpha]-helices. In addition, it was found that kinks result in a reduced unfolding cooperativity of [alpha]-helices.

Relatively little is known about the folding and stability of membrane proteins. Conventional thermal or chemical unfolding techniques probe the average behavior of large numbers of molecules and thus cannot resolve co-existing minor and major unfolding pathways and intermediates. Here, I applied single-molecule force measurements based on an atomic force microscope (AFM) to characterize the stability of the membrane protein bacteriorhodopsin (BR). In these mechanical unfolding experiments, an external pulling force played the role of the denaturant and lead to unfolding of the three-dimensional structure of individual proteins. It was found that single BRs unfold step-wise in a well-defined sequence of stable intermediates and in different unfolding pathways. Although single [alpha]-helices were sufficiently stable to unfold in individual steps they also exhibited certain probabilities to unfold in pairs. These observations support the "two-stage" and the "helical-hairpin" model of membrane protein folding. Dynamic force measurements showed that [alpha]-helices and helical hairpins are relatively rigid structures, which are stabilized by narrow energy barriers and have stabilities between 100-10?000 seconds. These forced unfolding experiments were complemented with the development of new force measurement techniques. It is demonstrated that hydrodynamic effects need to be considered to obtain more complete kinetic pictures of single molecules. In addition, two force spectroscopy approaches to measure the complex visco-elastic response of single molecules are presented and applied to BR. These experiments revealed that the unfolding patterns of single proteins are dominated by purely elastic polypeptide extension and determined the dissipative interactions associated with the unfolding of single [alpha]-helices. In addition, it was found that kinks result in a reduced unfolding cooperativity of [alpha]-helices.