High-affinity chelator thiols for switchable and oriented immobilization of histidine-tagged proteins: a generic platform for protein chip technologies.
Protein micro-/nanoarrays are becoming increasingly important in systematic approaches for the exploration of protein-protein interactions and dynamic protein networks, so there is a high demand for specific, generic, stable, uniform, and locally addressable protein immobilization on solid supports. Here we present multivalent metal-chelating thiols that are suitable for stable binding of histidine-tagged proteins on biocompatible self-assembled monolayers (SAMs). The architectures and physicochemical properties of these SAMs have been probed by various surface-sensitive techniques such as contact angle goniometry, ellipsometry, and infrared reflection-absorption spectroscopy. The specific molecular organization of proteins and protein complexes was demonstrated by surface plasmon resonance, confocal laser scanning, and atomic force microscopy. In contrast to the mono-NTA/His6 tag interaction, which has major drawbacks because of its low affinity and fast dissociation, drastically improved stability of protein binding by these multivalent chelator surfaces was observed. The immobilized histidine-tagged proteins are uniformly oriented and retain their function. At the same time, proteins can be removed from the chip surface under mild conditions (switchability). This new platform for switchable and oriented immobilization should assist proteome-wide wide analyses of protein-protein interactions as well as structural and single-molecule studies.
Institute of Biochemistry, Biocenter, Johann Wolfgang Goethe-University, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany.
Ali
A
Tinazli
Jilin
J
Tang
Ramûnas
R
Valiokas
Srdjan
S
Picuric
Suman
S
Lata
Jacob
J
Piehler
Bo
B
Liedberg
Robert
R
Tampé
D015374:Biosensing Techniques; D002614:Chelating Agents / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D040081:Protein Array Analysis; D011506:Proteins / Q000737:chemistry*; D013055:Spectrophotometry, Infrared; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D020349:Surface Plasmon Resonance; D013499:Surface Properties
D002614:Chelating Agents; D011506:Proteins; D013438:Sulfhydryl Compounds; D006639:Histidine