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Albany 2015:Book of Abstracts

Albany 2015
Conversation 19
June 9-13 2015
©Adenine Press (2012)

Visualization of αC regions in fibrin network formation by high-resolution AFM

Fibrinogen is one of the key proteins in thrombosis. It has been intensively studied with transmission electron microscopy and x-ray diffraction. But until now, a complete 3D structure of the molecule has not been resolved yet because of the lack of the structure of the two highly flexible αC regions. This study was aimed at determining whether the αC regions can be visualized in high-resolution atomic force microscopy.

Atomic force microscopy with super high resolution was used to image single molecules of fibrinogen and fibrin primary associates. The key approach was to use a graphite surface modified with the monolayer of amphiphilic carbohydrate-glycine molecules (Klinov et al., 2007) and unique supersharp cantilevers with 1 nm tip diameter (Klinov et al., 1998).

Fibrinogen αC regions were visualized and measured along with the complete domain structure of the protein. In 80% of molecules at pH 7.4 the D domain regions had one or two protrusions of average height 0.4 ± 0.1 nm and length 21 ± 6 nm, which is considerably less that would be expected of an unfolded polypeptide chain. The fibrinogen overall shape presented a mixture of straight and bent molecules. The bend angle is almost the same as in (Yermolenko et al., 2011). This gives a reason to believe that the two conformations are an integral feature of the protein in the solution and not just on a certain substrate surface.

Images of growing fibrin fibers with clearly visible αC regions have been obtained. Interacting alpha;C regions were clearly visible in protofibrils and large fibers reinforcing the notion of their involvement in long-range interactions between fibrin molecules. For example, αC regions intertwined near a branchpoint and looked like a zipper.

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Fig. (A) Height mode AFM images of single fibrinogen molecules on GM-modified HOPG. (B) A long protofibril with fifteen molecules and one structure mismatch marked by an arrow.

References
    Klinov D. V., Lagutina I. V., Prokhorov V. V., Neretina T., Khil P. P., Lebedev Y. B., Cherny D. I., Demin V. V., Sverdlov E. D. (1998) High resolution mapping DNAs by R-loop atomic force microscopy. NAR 26, 4603-10.

    Klinov D., Dwir B., Kapon E., Borovok N., Molotsky T., Kotlyar A. (2007) High-resolution atomic force microscopy of duplex and triplex DNA molecules. Nanotechnology 18, 225102.

    Yermolenko I. S., Lishko V. K., Ugarova T. P., Magonov S. N. (2011) High-resolution visualization of fibrinogen molecules and fibrin fibers with atomic force microscopy. Biomacromolecules 12, 370-9.


Anna D. Protopopova
Nikolay A. Barinov
Dmitry V. Klinov

Scientific Research Institute of Physical-Chemical Medicine
Moscow, Russia

Ph: (007) 499-2464843
Fx: (007) 499-2464409
ann.protopopova@ripcm.org.ru