Resolving structure,dynamics and molecular organization of ion channels by ´Molecular recognition force microscopy (MRfM)´

"Molecular Recognition Force Microscopy" (MRFM) is a new atomic force microscopy (AFM) technique detecting the specific interaction (molecular recognition) of a ligand molecule on the AFM tip with receptors on a probe surface at single molecule resolution. Immobilization of bio-molecules on surfaces is thereby of key importance in order to resolve their structure, dynamics and molecular organization. The investigated molecule is thus to be presented on the probe surface in native conditions, so that the ligand molecule on the AFM tip can bind in unconstrained fashion. A major breakthrough was the development of a tethering protocol for antibodies to AFM tips via a flexible poly(ethylenglykol)800 chain (PEG800 ) which established the AFM as a bio-spezific detection technique with single molecule resolution. It was a significant task of this project to thouroughly test and validate the bio-conjugation chemistry used for the AFM tip. Various heterobifunctional PEGs were synthesized by conjugating N-hydroxy-succinimide- (NHS), 2- pyridyl-dithio- (PDP), maleimid- (Mal), vinylsulfonic- (VS), nitrilo triacetate - (NTA) or vitamin H (biotin) as functional groups, thus enabling covalent binding to amines or thiols and reversible but strong binding to hexahistidine tags or avidin, respectevily. The biotinylated PEG 800 -chain itself represents a new class of a heterobifunctional crosslinker because it has already a specific sensor (biotin) attached to its end. This simple measuring principle was used as model of MRFM for systematic investigations of the physical and chemical properties of differently amino-functionalized AFM tips. The amine density and the adsorptive behavior of these protocols was examined. A new standard for the quantification of thiols using 4,4`-DTDP was worked out and compared with an improved Ellman assay. These thiol determinations are essential as quality control for the coupling of thiol active sensor molecules. Different strategies were also developed for the probe fixation in bio-specific detection. As a most biological application, calcium-channels from skeletal muscle were adsorbed to mica to resolve topography and the interaction force of a polyclonal antibody, and to localize antibody binding epitopes. A monolayer, consisting of phospholipids containing a dithio group on the hydrophobic end for the covalent attachment to gold and a cholin or biotin group on the hydrophilic end, was deposited for the specific binding of biotinilated or hexa histidine tagged proteins, respectively. Different studies using labeled proteins showed the potential of this new concept for applications in AFM and surface plasmon resonance spectroscopy (SPR). This design appears suited as a general tool for the investigation of densely packed two-dimensional protein layers in various bio-sensing application. In conclusion, a broad range of strategies for bio-conjugation was developed and the great potential of AFM for high resolution structure investigations and for the detection of bio-specific interaction of biological components in native environment was shown.