Publication Date


Document Type


First Advisor

Horn, James R.

Degree Name

Ph.D. (Doctor of Philosophy)

Legacy Department

Department of Chemistry and Biochemistry




The strept(avidin)-biotin interactions has been utilized across numerous biological applications, including cell imagining, diagnostics and affinity chromatography. These applications exploit the high affinity (K[sub d]≈10⁻¹⁴-10⁻¹⁶M) and specificity between strept(avidin) and its low-molecular weight target biotin. With such widespread use, there is considerable interest in engineering variants of strept(avidin) proteins with enhanced properties, such as tunable binding affinity, reduced size and altered valency. With regard to reduced size, engineered strept(avidin) monomers, which are one-fourth the size of wild-type strept(avidin), have exhibited a tendency to reform tetramers or form aggregates and have possessed reduced biotin affinity. In chapter 2, we aimed to overcome such shortcomings through modifications a dimeric avidin-like protein, called rhizavidin. To engineer a monomeric rhizavidin variant, a series of residues within the dimer interface were mutated to introduce charge repulsion and steric hindrance. In addition, to prevent possible aggregation upon monomerization, several nonpolar residues within the interface were mutated to polar residues. Following this approach, we successfully generated a functional monomeric rhizavidin variant (K[sub d] = 3 nM), by destabilizing the dimeric rhizavidin interface. In addition, insights were revealed regarding the role of oligomerization on rhizavidin/biotin binding affinity. There is interest in monomeric variants of the strept(avidin) family for cellular labeling and nanotechnology applications. However, all existing engineered monomer variants of the streptavidin family, including the rhizavidin monomer engineered in chapter 2, have reduced binding affinity and stability. An alternative approach to produce monomeric variants is the development of a single polypeptide dimeric monovalent rhizavidin protein, where only one functional biotin binding subunit exists. To produce monovalent rhizavidin, we first engineered a single-chain rhizavidin (scRhiz) fusion protein, by constructing a scaffold in which rhizavidin's two subunits were joined together as a single polypeptide chain. The scRhiz variant displayed similar functional and structural properties to wild-type rhizavidin. Consequently, the scRhiz variant provided the basis to engineer a single-chain monovalent form of rhizavidin (scRhiz-M), by incorporating site-specific mutations to generate a single dead subunit. Monovalent rhizavidin was shown to retain comparable binding affinity, off-rate kinetics and thermostability of wild-type rhizavidin. In addition, scRhiz-AP, a single polypeptide fusion with the enzyme alkaline phosphatase, was constructed to show the potential of scRhiz to be used as a bifunctional fusion protein in applications, such as immunoassays. Antibody engineering efforts have been utilized to produce antibody fragments that possess reduced size, such as Fab antigen fragments (Fabs), single-chain variable fragments (scFv) and single domain antibodies (VHHs). Development of these fragments for therapeutics and affinity reagents requires optimization of several properties including affinity, specificity, half-life, and clearance rate. A general method for improving the affinity of antibody fragments is in vitro affinity maturation. This method relies on display-based systems to screen libraries of unique antibody variants to isolate clones that possess improved affinity. An alternative approach to in vitro affinity maturation involves increasing the functional affinity (i.e., avidity). We previously generated a bivalent VHH antibody by simple incubation of biotinylated VHH with rhizavidin. Here, we continued this research to develop a bivalent Fab antibody fragment using in vivo biotinylation and the dimeric avidin protein, rhizavidin. To evaluate functional affinity of the Fab-Rhiz bivalent complex, surface plasmon resonance (SPR) experiments were conducted to compare the binding dissociation kinetics of the monovalent and bivalent complex. Rational design or combinatorial approaches are traditional protein engineering methods to regulate protein-ligand interactions. However, as these approaches require changing residues in the protein-ligand interface, some of these changes may have adverse effects that permanently alter the protein structure and function. An alternative approach to regulate protein-ligand interactions is the use of allosteric effectors, which regulate affinities by conformational selection. Here, we investigated the ability of conformationally-specific synthetic antibodies (sABs) to regulate the maltose binding protein (MBP) binding affinity for maltose. The binding energetics (ΔG°, ΔH° and -TΔS°) were examined using isothermal titration calorimetry (ITC) and a thermodynamic cycle was generated to describe the binding events in relation to maltose binding affinity and conformational effects of MBP/sAB interactions. Our results showed the closed-conformation sABs enhanced the affinity for maltose with K[sub d] values in the low nanomolar range. While, the sABs for the open-conformation, reduced the affinity for maltose substantially. In addition, the conformationally-selective sABS help dissect the energetic costs of MBP's open/close conformation transition. Finally, the ability to produce sABs against the maltose-binding competent state of MBP allow a route to "affinity mature" the maltose/MBP interaction through protein-protein interactions. These results demonstrate the usefulness of synthetic conformationally-selective sABs to serve as reagents that can regulate ligand binding affinity, reveal the thermodynamics of changes in protein conformation, and enhance binding affinity for protein-ligand interactions.


Advisors: James R. Horn.||Committee members: Gary Baker; Timonthy Hagen; Venumadhav Korampally; Victor Ryzhov.||Includes bibliographical references.||Includes illustrations.


xii, 118 pages




Northern Illinois University

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