title: Recombination of protein fragments: a promising approach toward engineering proteins with novel nanomechanical properties.
abstract: Combining single molecule atomic force microscopy (AFM) and protein engineering techniques, here we demonstrate that we can use recombination-based techniques to engineer novel elastomeric proteins by recombining protein fragments from structurally homologous parent proteins. Using I27 and I32 domains from the muscle protein titin as parent template proteins, we systematically shuffled the secondary structural elements of the two parent proteins and engineered 13 hybrid daughter proteins. Although I27 and I32 are highly homologous, and homology modeling predicted that the hybrid daughter proteins fold into structures that are similar to that of parent protein, we found that only eight of the 13 daughter proteins showed beta-sheet dominated structures that are similar to parent proteins, and the other five recombined proteins showed signatures of the formation of significant alpha-helical or random coil-like structure. Single molecule AFM revealed that six recombined daughter proteins are mechanically stable and exhibit mechanical properties that are different from the parent proteins. In contrast, another four of the hybrid proteins were found to be mechanically labile and unfold at forces that are lower than the approximately 20 pN, as we could not detect any unfolding force peaks. The last three hybrid proteins showed interesting duality in their mechanical unfolding behaviors. These results demonstrate the great potential of using recombination-based approaches to engineer novel elastomeric protein domains of diverse mechanical properties. Moreover, our results also revealed the challenges and complexity of developing a recombination-based approach into a laboratory-based directed evolution approach to engineer novel elastomeric proteins.
authors.affiliation: Department of Chemistry, The University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
authors.forename: M M
authors.initials: MM
authors.lastname: Balamurali
authors.forename: Deepak
authors.initials: D
authors.lastname: Sharma
authors.forename: Anderson
authors.initials: A
authors.lastname: Chang
authors.forename: Dingyue
authors.initials: D
authors.lastname: Khor
authors.forename: Ricky
authors.initials: R
authors.lastname: Chu
authors.forename: Hongbin
authors.initials: H
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D064211:Connectin; D032621:DNA Shuffling / Q000379:methods*; D004548:Elasticity; D006801:Humans; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics; D049329:Nanostructures / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D011995:Recombination, Genetic
chemical_list: D064211:Connectin; D009124:Muscle Proteins; D010455:Peptides; D011994:Recombinant Proteins; C577285:TTN protein, human; D011494:Protein Kinases