title: Oriented covalent immobilization of antibodies for measurement of intermolecular binding forces between zipper-like contact surfaces of split inteins.
abstract: In order to measure the intermolecular binding forces between two halves (or partners) of naturally split protein splicing elements called inteins, a novel thiol-hydrazide linker was designed and used to orient immobilized antibodies specific for each partner. Activation of the surfaces was achieved in one step, allowing direct intermolecular force measurement of the binding of the two partners of the split intein (called protein trans-splicing). Through this binding process, a whole functional intein is formed resulting in subsequent splicing. Atomic force microscopy (AFM) was used to directly measure the split intein partner binding at 1 μm/s between native (wild-type) and mixed pairs of C- and N-terminal partners of naturally occurring split inteins from three cyanobacteria. Native and mixed pairs exhibit similar binding forces within the error of the measurement technique (~52 pN). Bioinformatic sequence analysis and computational structural analysis discovered a zipper-like contact between the two partners with electrostatic and nonpolar attraction between multiple aligned ion pairs and hydrophobic residues. Also, we tested the Jarzynski's equality and demonstrated, as expected, that nonequilibrium dissipative measurements obtained here gave larger energies of interaction as compared with those for equilibrium. Hence, AFM coupled with our immobilization strategy and computational studies provides a useful analytical tool for the direct measurement of intermolecular association of split inteins and could be extended to any interacting protein pair.
authors.affiliation: Howard P. Isermann Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
authors.forename: Mirco
authors.initials: M
authors.lastname: Sorci
authors.forename: Bareket
authors.initials: B
authors.lastname: Dassa
authors.forename: Hongwei
authors.initials: H
authors.lastname: Liu
authors.forename: Gaurav
authors.initials: G
authors.lastname: Anand
authors.forename: Amit K
authors.initials: AK
authors.lastname: Dutta
authors.forename: Shmuel
authors.initials: S
authors.lastname: Pietrokovski
authors.forename: Marlene
authors.initials: M
authors.lastname: Belfort
authors.forename: Georges
authors.initials: G
authors.lastname: Belfort
mesh_terms: D000595:Amino Acid Sequence; D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D047668:Inteins / Q000502:physiology*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D019154:Protein Splicing / Q000502:physiology*; D017433:Protein Structure, Secondary
chemical_list: D055910:Antibodies, Immobilized; D001426:Bacterial Proteins