Time-Dependent Conformational Changes in Fibrinogen Measured by Atomic Force Microscopy

• Published in: Langmuir Vol. 20; no. 20; pp. 8846 - 8852
• Main Authors: Agnihotri, Aashiish, Siedlecki, Christopher A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 28.09.2004
• ISSN: 0743-7463; 1520-5827
• DOI: 10.1021/la049239+

Tapping-mode atomic force microscopy was used to study the time-dependent changes in the structure of fibrinogen under aqueous conditions following adsorption on two model surfaces:  hydrophobic graphite and hydrophilic mica. Fibrinogen was observed in the characteristic trinodular form, and the dimensions of the adsorbed molecules were consistent with previously reported values for these surfaces. On the basis of the differences in the relative heights of the D and the E domains, four orientation states were observed for fibrinogen adsorbed on both the surfaces. On graphite, the initial asymmetric orientation states disappeared with spreading over time. Some small lateral movements of the adsorbed proteins were observed on mica during repeated scanning, whereas no such movement was observed on graphite, indicating strong adhesion of fibrinogen to a hydrophobic surface. Spreading kinetics of fibrinogen on the two surfaces was determined by measuring the heights of the D and E domains over a time period of ∼2 h. On graphite, the heights of both the D and E domains decreased with time to a lower plateau value of 1.0 nm. On mica, the heights of both the D and E domains showed an increase, rising to an upper plateau value of ∼2.1 nm. The spreading of the D and E domains on graphite was analyzed using an ‘exponential-decay-of-height' model. A spreading rate constant of ∼4.7 × 10-4 s-1 was observed for the whole fibrinogen molecule adsorbed on graphite, corresponding to a free energy of unfolding of ∼37 kT. Extrapolation of the exponential curve in the model to t = 0 yielded values of 2.3 and 2.2 nm for the heights of the D and the E domains at the time of contact with the hydropbobic graphite substrate, significantly less than their free solution diameters. A two-step spreading model is proposed to explain this observation.