A monovalent streptavidin with a single femtomolar biotin binding site.
Streptavidin and avidin are used ubiquitously because of the remarkable affinity of their biotin binding, but they are tetramers, which disrupts many of their applications. Making either protein monomeric reduces affinity by at least 10(4)-fold because part of the binding site comes from a neighboring subunit. Here we engineered a streptavidin tetramer with only one functional biotin binding subunit that retained the affinity, off rate and thermostability of wild-type streptavidin. In denaturant, we mixed a streptavidin variant containing three mutations that block biotin binding with wild-type streptavidin in a 3:1 ratio. Then we generated monovalent streptavidin by refolding and nickel-affinity purification. Similarly, we purified defined tetramers with two or three biotin binding subunits. Labeling of site-specifically biotinylated neuroligin-1 with monovalent streptavidin allowed stable neuroligin-1 tracking without cross-linking, whereas wild-type streptavidin aggregated neuroligin-1 and disrupted presynaptic contacts. Monovalent streptavidin should find general application in biomolecule labeling, single-particle tracking and nanotechnology.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Mark
M
Howarth
Daniel J-F
DJ
Chinnapen
Kimberly
K
Gerrow
Pieter C
PC
Dorrestein
Melanie R
MR
Grandy
Neil L
NL
Kelleher
Alaa
A
El-Husseini
Alice Y
AY
Ting
D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D019921:Biotinylation; D015816:Cell Adhesion Molecules, Neuronal; D003432:Cross-Linking Reagents / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000648:ultrastructure; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D036103:Nanotechnology / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D019809:Streptavidin / Q000737:chemistry* / Q000378:metabolism; D013569:Synapses / Q000378:metabolism; D013696:Temperature
D015816:Cell Adhesion Molecules, Neuronal; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C093438:neuroligin 1; D001710:Biotin; D019809:Streptavidin