Atomic force microscopy kymographs provide robust access to conformational transitions in membrane proteins

Abstract

Membrane proteins play critical roles in many biochemical processes, ranging from the efflux of pharmaceutical agents used in the treatment of diseases to the translocation of polypeptides in secretion pathways. High-resolution structural methods have shed light on the conformational landscape of many of these proteins, but understanding is limited by the static nature the data they provide. Atomic force microscopy (AFM) is a powerful single molecule technique that provides structural information of active membrane proteins in real time and in physiologically relevant conditions. By disabling the slow axis scan during AFM imaging, one can acquire kymographs, which are graphical representations of spatial position over time. Here, we demonstrate the utility of rigorous kymograph analysis at the 100-ms time scale for two different membrane proteins in supported lipid bilayers: (1) proton-driven protein translocation factor SecDF, and (2) multi-drug resistance associated efflux pump P-glycoprotein (Pgp). We find that changing specific components of the systems, such as domain-specific deletions in SecDF or introduction of non-hydrolysable nucleotides in Pgp, reduces the measured conformational dynamics in predictable ways. We further probed the limitations of kymograph analysis, which have not been extensively studied. We simulated various experimental challenges associated with the method, including protein orientation, instrument noise, and drift. Despite the complex behavior of kymographs that result from these conditions, we show that robust information can be obtained including state transitions and dwell times. Lateral drift of 75% of the protein dimension leads to only a 12% probability of erroneous transition detection. Average dwell time error achieves a maximum value of 6% for identified states. Our results indicate that AFM kymographs are a useful method for probing real-time conformational dynamics of active membrane proteins.