Fibrinogen Hydrodynamic Properties from NMR-Diffusion Studies

Abstract

Fibrinogen is a blood plasma protein with crystallographically determined rod-like structure with globular domains, but it is unclear if this conformation is kept in blood. We studied fibrinogen in solution using NMR diffusometry. Pulsed-field gradient NMR self-diffusion measurements were made on a Bruker AVANCE-III-600 NMR-spectrometer equipped with a z-gradient inverse probehead (TBI, 5-mm-tube). Diffusion experiments were performed using a stimulated-echo sequence incorporating bipolar gradient pulses and a longitudinal eddy current delay (BPP-LED). The water signal was suppressed by pre-saturation. Data processing was performed using Topspin 2.1 software. The self-diffusion coefficient (SDC) was measured within T=278-315K for bovine fibrinogen (97% pure) at 0.3-43 mg/ml in a physiological buffer. By applying the Stokes-Einstein equation, we calculated an effective hydrodynamic radius, RH, of the fibrinogen molecule, corresponding to the radius of a sphere having the same mobility. At 37C in a highly dilute solution RH=6.7 nm. However, a concentration dependence of the fibrinogen SDC revealed a dramatic deviation from the spherical particle-like behavior. The SDC-[fibrinogen] plot was fitted well with a stretched exponential, characteristic of the sub-diffusion or hindered diffusion observed in crowded molecular systems with direct intermolecular interactions. Accordingly, the activation energy of the protein diffusive motion increased from 20 kJ/mol at 1.4 mg/ml (close to the activation energy of water) to 32 kJ/mol at 36 mg/ml, suggesting a marked fibrinogen self-interaction at higher concentrations, likely mediated by the αC regions. The mean value of RH=6.7 nm fits with a model for hydrodynamic motion of an elongated cylinder 45 nm in length with the cylinder radius of ∼1.70 nm, close to the known crystallographic dimensions. The results suggest that fibrinogen molecules in solution maintain a rod-like shape and undergo direct intermolecular interactions/collisions during diffusive motion. (Supported by the Program of Competitive Growth of Kazan Federal University)