Abstract
Monitoring individual proteins in solution while simultaneously obtaining tertiary and quaternary structural information is challenging. In this study, translocation of the vascular endothelial growth factor (VEGF) protein through a solid-state nanopore (ssNP) produces distinct ion-current blockade amplitude levels and durations likely corresponding to monomer, dimer, and higher oligomeric states. Upon changing from a non-reducing to a reducing condition, ion-current blockage events from the monomeric state dominate, consistent with the expected reduction of the two inter-chain VEGF disulfide bonds. Cleavage by plasmin and application of either a positive or a negative NP bias results in nanopore signals corresponding either to the VEGF receptor recognition domain or to the heparin binding domain, accordingly. Interestingly, multi-level analysis of VEGF events reveals how individual domains affect their translocation pattern. Our study shows that careful characterization of ssNP results elucidates real-time structural information about the protein, thereby complementing classical techniques for structural analysis of proteins in solution with the added advantage of quantitative single-molecule resolution of native proteins.
Highlights
IntroductionVEGF used in this study was a recombinant human protein purchased from Biological Industries
The NP translocation results obtained at pH 7.6, above the pI of VEGF, were expected and matched the observed oligomeric states of VEGF on SDS-PAGE under reducing and non-reducing conditions
Our results, which were obtained at pH 7.2, below the pI of VEGF, were surprising and emphasized how slight pH change can affect the structure of the protein in solution
Summary
VEGF used in this study was a recombinant human protein purchased from Biological Industries. The purity of the sample was >98%. The stock powder protein was reconstituted with MilliQ water (Millipore), 10% glycerol and 1 mM of dithiothreitol (DTT) to the stock concentration of 20 μM and stored at −80 °C until use. Upon usage, this stock solution was mixed with the nanopore buffer to obtain the final concentration without any further additives. The protein concentration inside the nanopore chamber ranged from 0.5–100 n
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