title: EPR assessment of protein sites for incorporation of Gd(III) MRI contrast labels.
abstract: We have engineered apolipoprotein A-I (apoA-I), a major protein constituent of high-density lipoprotein (HDL), to contain DOTA-chelated Gd(III) as an MRI contrast agent for the purpose of imaging reconstituted HDL (rHDL) biodistribution, metabolism and regulation in vivo. This protein contrast agent was obtained by attaching the thiol-reactive Gd[MTS-ADO3A] label at Cys residues replaced at four distinct positions (52, 55, 76 and 80) in apoA-I. MRI of infused mice previously showed that the Gd-labeled apoA-I migrates to both the liver and the kidney, the organs responsible for HDL catabolism; however, the contrast properties of apoA-I are superior when the ADO3A moiety is located at position 55, compared with the protein labeled at positions 52, 76 or 80. It is shown here that continuous wave X-band (9 GHz) electron paramagnetic resonance (EPR) spectroscopy is capable of detecting differences in the Gd(III) signal when comparing the labeled protein in the lipid-free with the rHDL state. Furthermore, the values of NMR relaxivity obtained for labeled variants in both the lipid-free and rHDL states correlate to the product of the X-band Gd(III) spectral width and the collision frequency between a nitroxide spin label and a polar relaxation agent. Consistent with its superior relaxivity measured by NMR, the rHDL-associated apoA-I containing the Gd[MTS-ADO3A] probe attached to position 55 displays favorable dynamic and water accessibility properties as determined by X-band EPR. While room temperature EPR requires >1 m m Gd(III)-labeled and only >10 µ m nitroxide-labeled protein to resolve the spectrum, the volume requirement is exceptionally low (~5 µl). Thus, X-band EPR provides a practical assessment for the suitability of imaging candidates containing the site-directed ADO3A contrast probe.
authors.affiliation: Department of Experimental Medical Science, Lund University, Lund, Sweden.
authors.forename: Jens O
authors.initials: JO
authors.lastname: Lagerstedt
authors.forename: Jitka
authors.initials: J
authors.lastname: Petrlova
authors.forename: Silvia
authors.initials: S
authors.lastname: Hilt
authors.forename: Antonin
authors.initials: A
authors.lastname: Marek
authors.forename: Youngran
authors.initials: Y
authors.lastname: Chung
authors.forename: Renuka
authors.initials: R
authors.lastname: Sriram
authors.forename: Madhu S
authors.initials: MS
authors.lastname: Budamagunta
authors.forename: Jean F
authors.initials: JF
authors.lastname: Desreux
authors.forename: David
authors.initials: D
authors.lastname: Thonon
authors.forename: Thomas
authors.initials: T
authors.lastname: Jue
authors.forename: Alex I
authors.initials: AI
authors.lastname: Smirnov
authors.forename: John C
authors.initials: JC
authors.lastname: Voss
mesh_terms: D001665:Binding Sites; D003287:Contrast Media / Q000032:analysis / Q000138:chemical synthesis*; D015195:Drug Design; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005682:Gadolinium / Q000737:chemistry*; D008279:Magnetic Resonance Imaging / Q000379:methods*; D053769:Nanocapsules / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
chemical_list: D003287:Contrast Media; D053769:Nanocapsules; D011506:Proteins; D005682:Gadolinium