Hydrophobic membrane proteins perform a variety of important functions in the cell, but their structures are notoriously difficult to solve. Thus, new strategies to obtain crystals of membrane proteins for structure determination are critical. We aim to develop a toolbox of peptide specific single-chain antibody fragment chaperones engineered for hyper-crystallizability. These peptide sequences can be introduced into various regions of membrane proteins without interfering with protein function. The resulting protein-chaperone complex is expected to form a crystal lattice mediated by chaperone interactions. We have developed candidate scFv chaperone proteins binding hexa-histidine (His6) and EYMPME (EE) tags with improved biophysical features influencing crystallization propensity, including peptide affinity, stability and solubility. The scFv libraries were generated using a novel ligation-free technique, MegAnneal, allowing us to rapidly generate large libraries based on 3D5 scFv. We identified two candidate chaperones, 3D5/His_683, specific for His6 and 3D5/EE_48, specific for EE tags. Variants exhibit high solubility (up to 16.6 mg/ml) and nanomolar peptide affinities; complexes of 3D5/EE_48 with EE-tagged proteins were isolated by gel filtration. We have developed design rules for EE peptide placement at terminal, inter-domain or internal loop regions of the target protein to balance peptide accessibility for chaperone binding while retaining rigid protein-chaperone complexes suitable for crystallization. The 3D5/ His_683 crystallized in four different conditions, utilizing multiple space groups. The 3D5/EE_48 scFv was crystallized (3.1 Å), revealing a ~52 Å channel in the crystal lattice, which may accommodate a small peptide-tagged target protein. Our evolution experiments altered scFv surface residues, resulting in use of different crystallization contacts. Analysis of these crystal contacts and those used by crystallized 14B7 scFv variants, led us to postulate that lattice formation is driven by strong crystal contacts. To test this hypothesis, we introduced amino acid changes expected to reduce the affinity of the 3D5/EE_48 energetically dominant crystal contacts. This approach to crystal contact engineering may allow semi-rational control over lattice networks preferred by scFv chaperones. Co-crystallization trials with model proteins are on-going. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries. / text

The glycine riboswitch is a structured RNA found upstream of genes in mRNA transcripts in many bacteria, functioning as a biofeedback gene regulator. Upon binding glycine, a complete RNA transcript including gene sequences is transcribed, effectively turning on gene expression. In an effort to understand the intricacies of its functioning, many mutants of the riboswitch were made and characterized during Ph. D. work, resulting in discovery of a P0 duplex/kink-turn motif involving a few nucleotides upstream of the established glycine riboswitch sequence which changed its ligand binding characteristics (Chapter 1). Previously, the two aptamers of the riboswitch were thought to cooperatively bind glycine, but with the inclusion of this leader sequence which forms a kink turn motif with the linker between the two aptamers, glycine binding in one aptamer no longer requires glycine binding in the other. Furthermore, the Kd from three species tested are now a similar, lower value of about 5 µM, indicating authenticity of this new consensus sequence. Glycine binding and interaptamer interaction both enhanced one another in trans aptamer assays. Another discovery from this was a shortened construct including all of aptamer II but only part of aptamer I in which a few specific nucleotides prevented glycine binding in aptamer II (Chapter 2). This may provide insight into the nature of interaptamer interactions in the full switch; addition of an oligonucleotide complimentary to these nucleotides restored glycine binding ability to aptamer II. With future development, this could also be a useful molecular biology tool, using two signals, glycine and an oligonucleotide, to allow gene expression. To precisely understand how any macromolecule functions, a 3D structure, obtainable by x-ray crystallography, is vital. A new technique to accomplish that for RNA, precedented in the protein world, is Fab chaperoned crystallography, which has advantages compared to RNA alone. A phage displayed library of Fabs with reduced codon diversity designed for RNA was created, the YSGR Min library (Chapter 3). Its Fabs had specificities and affinities equal to or greater than previous libraries which were originally created for phage displayed selection against proteins. Fab chaperoned RNA crystallography is currently in progress for the glycine riboswitch; the best resolution thus far is 5.3 … (Chapter 4). In addition to providing molecular insight into its gene regulation mechanism, a structure of the glycine riboswitch could be applied for use in structure based drug design of novel antibiotics targeting the riboswitch to disrupt important downstream carbon cycle genes in pathogenic bacteria.