Experimental Studies of Functionally Diverse and Controllable Computationally Designed Protein Systems

• Main Author: Eibling, Matthew J
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2017
• Subjects: Biochemistry; Chemistry; Computational chemistry
• ISBN: 9780355619751; 035561975X

Protein structure dictates function. While natural proteins exhibit great versatility, the intricacies of controlled structure, assembly, and recognition are subtle and challenging to engineer. Computational protein design is a powerful tool useful for creating uniquely functional systems. Presented herein are experimental studies for two computationally designed, functionally rich protein systems. The first example discusses a four-helix bundle protein designed to bind a single copy of an optically active Zn-porphyrin donor—phenyl bridged—naphthaldiimide acceptor (PZn-Ph-NDI) charge-separating chromophore in a well-structured local environment and study its electron transfer (ET) properties. PZn-Ph-NDI is covalently bound within the bundle core by a single histidine. Redshifts in PZn-Ph-NDI Soret and Q-bands measured by UV/Vis, a ratio shift in the double-peak PZn-Ph-NDI fluorescence, and a circular dichroism “Cotton Effect” strongly suggest that PZn-Ph-NDI is encapsulated in the designed pocket of the four-helix bundle core. Pump-probe studies revealed the protein undergoes a switch in the effective dielectric constant (ϵs) following photoinduced ET, from ϵs ≈ 8 to ϵs ≈ 3. Studies of chromophores in engineered proteins with well-structured interiors can facilitate elucidation of ET processes within protein environments. The second example discusses a designed protein useful for controlling association and separation of Au nanoparticles (AuNP) and Au nanorods (AuNR) with zinc-coordinating proteins. The protein was designed to present multiple Zn2+ coordination sites and cooperatively self-associate to form an antiparallel helical homodimer. When bound to the surface of AuNPs or end-grafted to AuNRs via cysteine, the protein provides a reversible molecular linkage. Association and changes in interparticle separation were monitored by redshifts in surface plasmon resonance (SPR) and by transmission electron microscopy (TEM). Titrations with Zn2+ revealed sigmoidal transitions indicative of cooperative assembly. Specifying the number of helical (heptad) repeat units conferred control over protein length and interparticle separation. Two different length proteins were designed via extension of the helical structure. TEM and extinction measurements revealed distributions of interparticle separations consistent with the expected protein structures. Particle association, interparticle separation, and SPR properties can be tuned using computationally designed proteins, where protein structure, folding, length, and response to molecular species such as Zn2+ can be engineered.