Metal- and metallocycle-binding sites engineered into polyvalent virus-like scaffolds.
Metal-binding motifs appear on protein scaffolds throughout nature and are critical for a vast array of functions that span structure, electron transfer, and catalysis. In an effort to reproduce and exploit this activity in vitro, described herein are versatile bacteriophage Qbeta particles bearing metal-binding motifs polyvalently. The three motifs, His(6), His(6)-His(6), and Cys-His(6), were incorporated into the capsid via a coexpression methodology at ratios of 1.1:1, 1.1:1, and 2.3:1 for wild-type to modified coat protein. Size-exclusion chromatography yielded elution profiles identical to wild-type particles, while Ni-NTA affinity chromatography resulted in retention times that increase according to Qbeta-His(6) < Qbeta-Cys-His(6) < Qbeta-His(6)-His(6). In addition to interacting with metal-derivatized surfaces, Qbeta-Cys-His(6) and Qbeta-His(6)-His(6) bind heme as evidenced by the appearance of new absorbances at 416 and 418 nm, respectively, upon addition of hemin-Cl. The heme-bearing particles were also found to be electrochemically active as a surface-confined system. While both constructs yield similar E(1/2) values anaerobically and with carbon monoxide present, and both display similar pH dependences, a standard rate constant k degrees could only be measured for Qbeta-Cys-His(6) (83 s(-1)), as electron transfer for Qbeta-His(6)-His(6) was too rapid to estimate. Experiments with rotated-disk electrodes yielded significant activity of the constructs toward dioxygen reduction. The versatility of the particles is further underscored by their multivalent nature, permitting simultaneously a range of activities for applications demanding polyfunctionality.
Udit
Andrew K
AK
Department of Chemistry, Occidental College, 1600 Campus Road, Los Angeles, California 90041, USA. udit@oxy.edu
Hollingsworth
William
W
Choi
Kang
K
D017908:Allolevivirus; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D032701:Biomimetics; D002213:Capsid / Q000737:chemistry / Q000378:metabolism; D004563:Electrochemistry; D004566:Electrodes; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D008670:Metals; D014764:Viral Proteins