Viscoelastic Sensing of Conformational Changes in Plasminogen Induced upon Binding of Low Molecular Weight Compounds

Abstract

Plasminogen is a precursor to the fibrinolytic enzyme plasmin and is known to undergo large conformational changes when subjected to low molecular lysine analogues such as tranexamic acid (TA) or ε-amino-n-caproic acid (EACA). Here, we demonstrate how well-controlled surface immobilization of biotinylated plasminogen allows for monitoring of the interaction between TA and EACA with plasminogen. The interaction was studied by the quartz crystal microbalance with dissipation monitoring (QCM-D) technique as well as by surface plasmon resonance (SPR) based sensing. QCM-D measures changes in acoustically coupled mass (by detection of changes in the resonance frequency of the crystal, Δf) and is sensitive to changes in mass adsorbed on the sensor surface including how liquid medium is associated with this material. Through the dissipation factor (i.e., changes in the energy dissipation of the crystal oscillation, ΔD), QCM-D is also sensitive to the viscoelastic properties of material adsorbed to the sensor surface. Upon binding of TA or EACA, changes in the plasminogen structure were recorded as distinct, although small, ΔD responses which were used to determine affinity constants. By comparing native and truncated plasminogen, we conclude that the observed dissipation shifts were caused by conformational changes in the proteins leading to changes in the viscoelastic properties of the protein layer on the surface. These results demonstrate a novel application of the QCM-D technique, paving the way for a whole new approach to screening of this target for novel lead structures.

A self-assembled DNA nanostructure-amplified quartz crystal microbalance with dissipation biosensing platform for nucleic acids

“…DNA, proteins, or cells). 5,6 The total frequency variation data, the sum of the mass contribution (DF mass ), viscous contribution (DF visc ) and charge contribution (DF charge ), 3,7 , are a signal for probing events on the chip surface. To analyze trace levels of an interesting nucleic acid sequence, several classical signal amplification strategies for piezoelectric sensors, such as using liposomes, 8 enzymes, 9 gold nanoparticles, 10 or quantum dots 11 as the mass contribution amplifiers, have been employed for enhancing detection sensitivity.…”

A Survey of the 2010 Quartz Crystal Microbalance Literature

“…Conformational changes in the HIV‐1 envelope glycoprotein, gp120 after ligand binding was followed by QCM and compared well with other analytical methods (Lee et al ., ). Plasminogen also undergoes conformational change upon interaction with lysine analogues such as tranexamic acid or ε‐amino‐ n ‐caproic acid and this interaction was investigated with the protein immobilised via a biotin tag (Nileback et al ., ). QCM could be used to detect conformational changes in the protein via changes in the viscoelastic properties of the protein surface.…”

How to Study Protein-protein Interactions

“…This enables further elucidation of changes in the structure of deposited film of material including burst of membrane vesicles on the sensor surface as well as conformational changes of attached proteins. 62,63 QCM senses mass directly, therefore no labelling of studied material is needed. The sensor surface can be functionalized with capture molecules for specific detection of the selected analyte.…”

“…Sensors can be prepared for interaction studies of ions, small molecules or proteins. [63][64][65] QCM can be set for detection of viruses and artificial particles or even for binding of cells from suspension. 61,66,67 Interactions of proteins from complex samples such as culture media or sera can also be measured accurately as the optical properties of samples have no effect on the measurement enabling studies in biologically relevant environments.…”