| Autor: |
Chen J, van Dongen MA, Merzel RL, Dougherty CA, Orr BG, Kanduluru AK; Department of Chemistry and Center for Drug Discovery, Purdue University , West Lafayette, Indiana 47907, United States., Low PS; Department of Chemistry and Center for Drug Discovery, Purdue University , West Lafayette, Indiana 47907, United States., Marsh EN; Department of Biological Chemistry, University of Michigan , Ann Arbor, Michigan 48109, United States., Banaszak Holl MM; Macromolecular Science and Engineering, University of Michigan , Ann Arbor, Michigan 48109, United States. |
| Jazyk: |
angličtina |
| Zdroj: |
Biomacromolecules [Biomacromolecules] 2016 Mar 14; Vol. 17 (3), pp. 922-7. Date of Electronic Publication: 2016 Feb 03. |
| DOI: |
10.1021/acs.biomac.5b01586 |
| Abstrakt: |
Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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