title: Proline substitution of dimer interface β-strand residues as a strategy for the design of functional monomeric proteins.
abstract: Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline's unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.
authors.affiliation: Departments of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, Texas; Sealy Center for Structural Biology and Molecular Biophysics, The University of Texas Medical Branch, Galveston, Texas.
authors.forename: Prem Raj B
authors.initials: PR
authors.lastname: Joseph
authors.forename: Krishna Mohan
authors.initials: KM
authors.lastname: Poluri
authors.forename: Pavani
authors.initials: P
authors.lastname: Gangavarapu
authors.forename: Lavanya
authors.initials: L
authors.lastname: Rajagopalan
authors.forename: Sandeep
authors.initials: S
authors.lastname: Raghuwanshi
authors.forename: Ricardo M
authors.initials: RM
authors.lastname: Richardson
authors.forename: Roberto P
authors.initials: RP
authors.lastname: Garofalo
authors.forename: Krishna
authors.initials: K
authors.lastname: Rajarathnam
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D006860:Hydrogen Bonding; D016209:Interleukin-8 / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D056004:Molecular Dynamics Simulation; D011392:Proline; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D058767:Protein Unfolding
chemical_list: D016209:Interleukin-8; D011392:Proline
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