AFM characterization of tilt and intrinsic flexibility of Rhodobacter sphaeroides light harvesting complex 2 (LH2)

Atomic force microscopy (AFM) has developed into a powerful tool to investigate membrane protein surfaces in a close-to-native environment. Here we report on the surface topography of Rhodobacter sphaeroides light harvesting complex 2 (LH2) reconstituted into two-dimensional crystals. These photosynthetic trans-membrane proteins formed cylindrical oligomeric complexes, which inserted tilted into the lipid membrane. This peculiar packing of an integral membrane protein allowed us to determine oligomerization and tilt of the LH2 complexes, but also protrusion height and intrinsic flexibility of their individual subunits. Furthermore the surface contouring reliability and limits of the atomic force microscopy could be studied. The two-dimensional crystals examined had sizes of up to 5 mum and, as revealed by a 10 Angstrom cryo electron microscopy projection map, p22(1)2(1) crystal symmetry. The unit cell had dimensions of a = b = 150 Angstrom and gamma = 90degrees, and housed four nonameric complexes, two pointing up and two pointing down. AFM topographs of these 2D crystals had a lateral resolution of 10 Angstrom. Further, the high vertical resolution of similar to1 Angstrom, allowed the protrusion height of the cylindrical LH2 complexes over the membrane to be determined. This was maximally 13.1 Angstrom on one side and 3.8 Angstrom on the other. Interestingly, the protrusion height varied across the LH2 complexes, showing the complexes to be inserted with a 6.2degrees tilt with respect to the membrane plane. A detailed analysis of the individual subunits showed the intrinsic flexibility of the membrane protruding peptide stretches to be equal and independent of their protrusion height. Furthermore, our analysis of membrane proteins within this peculiar packing confirmed the high vertical resolution of the atomic force microscopy on biological samples, and led us to conclude that the image acquisition function was equally accurate for contouring protrusions with heights up to similar to15 Angstrom. (C) 2003 Elsevier Science Ltd. All rights reserved.