Targeting structural flexibility in low density lipoprotein by integrating cryo-electron microscopy and high-speed atomic force microscopy.

Low-density lipoprotein (LDL) plays a crucial role in cholesterol metabolism. Responsible for cholesterol transport from the liver to the organs, LDL accumulation in the arteries is a primary cause of cardiovascular diseases, such as atherosclerosis. This work focuses on the fundamental question of the LDL molecular structure, as well as the topology and molecular motions of apolipoprotein B-100 (apo B-100), which is addressed by single-particle cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy (HS-AFM). Our results suggest a revised model of the LDL core organization with respect to the cholesterol ester (CE) arrangement. In addition, a high-density region close to the flattened poles could be identified, likely enriched in free cholesterol. The most remarkable new details are two protrusions on the LDL surface, attributed to the protein apo B-100. HS-AFM adds the dimension of time and reveals for the first time a highly dynamic direct description of LDL, where we could follow large domain fluctuations of the protrusions in real time. To tackle the inherent flexibility and heterogeneity of LDL, the cryo-EM maps are further assessed by 3D variability analysis. Our study gives a detailed explanation how to approach the intrinsic flexibility of a complex system comprising lipids and protein.

Single molecule distribution of RhD binding epitopes on ultraflat erythrocyte ghosts.

The Rh blood group system plays a key role in transfusion and organ transplant medicine. The complex transmembrane Rh polypeptides RhD and RhCE carry numerous antigens, including the extremely immunogenic D antigen. The Rh polypeptides form multimolecular Rh complexes with certain transmembrane and skeletal proteins, with so far only incompletely understood physiological functions. Determination of the energy landscape of individual Rh binding epitopes towards their specific interaction partners as well as their localization across the red blood cell (RBC) membrane requires single molecule approaches including large area high resolution recognition imaging. Atomic force microscopy based molecular recognition force spectroscopy in combination with single molecule recognition imaging fulfills these requirements. For unbiased single molecule results, nano-mechanical influences due to cell elasticity have to be eliminated. This is realized by generation of ultra flat erythrocyte ghosts on a solid support. We developed a protocol for the preparation of complete ultraflat erythrocyte ghosts and determined the molecular binding behaviour of different anti-D antibodies towards their binding epitopes on RhD positive and negative erythrocytes. Performing optimized topography and recognition imaging at 16 Mpixel resolution allowed localisation of individual RhD molecules at the single molecule level across an entire RBC. A map of Rh antigens across integer ultraflat RBC ghosts was generated with nanometer resolution. Here we show a homogeneous distribution on rim and dimple regions with comparable receptor densities. Furthermore, differences in the energy landscape between specific monoclonal antibodies were determined at the single molecule level.

Atomic Force Microscopy Imaging in Turbid Liquids: A Promising Tool in Nanomedicine.

Tracking of biological and physiological processes on the nanoscale is a central part of the growing field of nanomedicine. Although atomic force microscopy (AFM) is one of the most appropriate techniques in this area, investigations in non-transparent fluids such as human blood are not possible with conventional AFMs due to limitations caused by the optical readout. Here, we show a promising approach based on self-sensing cantilevers (SSC) as a replacement for optical readout in biological AFM imaging. Piezo-resistors, in the form of a Wheatstone bridge, are embedded into the cantilever, whereas two of them are placed at the bending edge. This enables the deflection of the cantilever to be precisely recorded by measuring the changes in resistance. Furthermore, the conventional acoustic or magnetic vibration excitation in intermittent contact mode can be replaced by a thermal excitation using a heating loop. We show further developments of existing approaches enabling stable measurements in turbid liquids. Different readout and excitation methods are compared under various environmental conditions, ranging from dry state to human blood. To demonstrate the applicability of our laser-free bio-AFM for nanomedical research, we have selected the hemostatic process of blood coagulation as well as ultra-flat red blood cells in different turbid fluids. Furthermore, the effects on noise and scanning speed of different media are compared. The technical realization is shown (1) on a conventional optical beam deflection (OBD)-based AFM, where we replaced the optical part by a new SSC nose cone, and (2) on an all-electric AFM, which we adapted for measurements in turbid liquids.