The Study of HIV-1 Vpr-Membrane and Vpr-hVDAC-1 Interactions by Graphene Field-Effect Transistor Biosensors
Journal
ACS Applied Materials & Interfaces
Journal Volume
3
Journal Issue
9
Pages
6351-6357
Date Issued
2020
Author(s)
Abstract
The viral protein R (Vpr) of human immunodeficiency virus 1 (HIV-1) is involved in many cellular processes during the viral life cycle; however, its associated mechanisms remain unclear. Here, we designed an Escherichia coli expression construct to achieve a milligram yield of recombinant Vpr. In addition, we fabricated a graphene field-effect transistor (G-FET) biosensor, with the modification of a supported lipid bilayer (SLB), to study the interaction between Vpr and its interaction partners. The Dirac point of the SLB/G-FET was observed to shift in response to the binding of Vpr to the SLB. By fitting the normalized shift of the Dirac point as a function of Vpr concentration to the Langmuir adsorption isotherm equation, we could extract the dissociation constant (Kd) to quantify the Vpr binding affinity. When the 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG) membrane was used as the SLB, the dissociation constant was determined to be 9.6 ± 2.1 μM. In contrast, only a slight shift of the Dirac point was observed in response to the addition of Vpr when the 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane was used as the SLB. Taking advantage of the much weaker binding of Vpr to the DOPC membrane, we prepared a human voltage-dependent anion channel isoform 1 (hVDAC-1)-embedded DOPC membrane as the SLB for the G-FET and used it to determine the dissociation constant to be 5.1 ± 0.9 μM. In summary, using the clinically relevant Vpr protein as an example, we demonstrated that an SLB/G-FET biosensor is a suitable tool for studying the interaction between a membrane-associated protein and its interaction partners. ? 2020 American Chemical Society.
Subjects
G-FET; HIV-1; lipid composition; membrane protein; VDAC; Vpr
Other Subjects
Binding energy; Biosensors; Diseases; Dissociation; Escherichia coli; Field effect transistors; Graphene; Life cycle; Lipid bilayers; Proteins; Viruses; 1 ,2-dioleoyl-sn-glycero-3-phosphocholine; Expression constructs; Graphene field-effect transistors; Human immunodeficiency virus-1; Langmuir adsorption isotherm equation; Membrane-associated proteins; Supported lipid bilayer (SLB); Voltage-dependent anion channels; Graphene transistors
Type
journal article