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Author Contact: ocho@wesleyan.edu
Local Identifier: 1703"},{"label":"Author-supplied Keywords","value":["Protein design","Molecular dynamics rosetta free energy perturbation (MDR-FEP)","Alchemical fast-morph simulations","Single-point mutation","Sidechain rotamers","Structural optimization","EHEE_rd2_0005","Miniproteins","Conformational heterogeneity","Conformational exchange"]},{"label":"Degree","value":["Master of Arts"]},{"label":"Description","value":"

The presence of multiple conformations in designed proteins, particularly therapeutic miniproteins, can lead to adverse effects. However, the conformational heterogeneity of designed proteins is rarely characterized or redesigned. We have used EHEE_rd2_0005 as a model miniprotein to investigate how conformational heterogeneity can be manipulated, potentially providing general strategies for optimizing a wide range of protein designs. Unassigned peaks in the nuclear Overhauser effect (NOE) structure of EHEE_rd2_0005 were observed, likely due to peak broadening from an intermediate-rate conformational exchange between two W35 sidechain rotamers. This motion of the W35 sidechain can disrupt NMR peaks because of the aromatic sidechain’s strong electromagnetic ring current effects on surrounding nuclei. Our goal is to identify a single-point mutation favoring one W35 rotamer without significantly altering the secondary structure. By introducing mutations to the miniprotein and determining the relative free energy values of two conformational states using alchemical fast-morph simulations, we predict the impact of specific mutations on conformational preference. We also employed molecular dynamics Rosetta free energy perturbation (MDR-FEP) to refine rigid-backbone Rosetta calculations to better align with molecular dynamics simulations. Among six expressed mutants, two demonstrated more peaks in their 15N-1H heteronuclear single quantum coherence (HSQC) spectra than the wild-type spectrum. These additional peaks may account for some of the missing peaks hypothesized to be broadened by W35 rotamer interconversion, indicating the partial success of these two mutations.

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