De novo Design of Homodimerizing Protein by use of the Knob-Socket Model
Poster Number
21
Format
Poster Presentation
Faculty Mentor Name
Jerry Tsai
Faculty Mentor Department
Chemistry
Abstract/Artist Statement
The knob-socket model aids in the identification of packing in protein secondary, tertiary and quaternary structure. First, the intra-helical packing patterns are first identified as a regular grid of 3 residue cliques or sockets connected by the peptide bond, a hydrogen bond, and van der Waals interactions. For interactions between helices, the tetrahedral knob-socket motif involves the single residue knob from one helix that packs into a socket coming from a different helix. Because amino acid composition of sockets and knob-sockets has shown preferences, the knob-socket model can be used for the de novo design of an anti-parallel homodimerizing, alpha-helical protein named KSa1. By utilizing histidine-tagged affinity chromatography, KSa1 confirmed knob-socket model’s applicability in the the design of a helix. Since the protein designed was so short in length, affinity chromatography after cutting off the histidine- SUMO tag was not with our current resources. To facilitate purification and therefore further study, KSa2 was designed that effectively duplicates the helix, so that the packing interactions between 2 helices occur within a protein chain. Using the current plasmid for KSa1, a restriction enzyme will be used to cut the plasmid allowing for insertion of a small linker and second KSa1 helix sequence. By adding a linker and second helix, the expressed protein for KSa2 should interact with itself, allowing for observation of not only the helical structure, but also the knob-socket interactions. Assuming DNA sequencing for the transformed plasmids showed the appropriate insertion of the additional sequence, visual confirmation of the structure could be confirmed through circular dichroism and protein Nuclear Magnetic Resonance experiments.
Location
DeRosa University Center, Ballroom
Start Date
30-4-2016 1:30 AM
End Date
30-4-2016 3:30 PM
De novo Design of Homodimerizing Protein by use of the Knob-Socket Model
DeRosa University Center, Ballroom
The knob-socket model aids in the identification of packing in protein secondary, tertiary and quaternary structure. First, the intra-helical packing patterns are first identified as a regular grid of 3 residue cliques or sockets connected by the peptide bond, a hydrogen bond, and van der Waals interactions. For interactions between helices, the tetrahedral knob-socket motif involves the single residue knob from one helix that packs into a socket coming from a different helix. Because amino acid composition of sockets and knob-sockets has shown preferences, the knob-socket model can be used for the de novo design of an anti-parallel homodimerizing, alpha-helical protein named KSa1. By utilizing histidine-tagged affinity chromatography, KSa1 confirmed knob-socket model’s applicability in the the design of a helix. Since the protein designed was so short in length, affinity chromatography after cutting off the histidine- SUMO tag was not with our current resources. To facilitate purification and therefore further study, KSa2 was designed that effectively duplicates the helix, so that the packing interactions between 2 helices occur within a protein chain. Using the current plasmid for KSa1, a restriction enzyme will be used to cut the plasmid allowing for insertion of a small linker and second KSa1 helix sequence. By adding a linker and second helix, the expressed protein for KSa2 should interact with itself, allowing for observation of not only the helical structure, but also the knob-socket interactions. Assuming DNA sequencing for the transformed plasmids showed the appropriate insertion of the additional sequence, visual confirmation of the structure could be confirmed through circular dichroism and protein Nuclear Magnetic Resonance experiments.