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The BTB domain is a eukaryotic protein-protein interaction motif found in variety of proteins. This thesis describes an investigation into the general and specific properties of the sequence, structure and self-association properties of this domain. The work is divided by two complementary approaches. Chapter 2 describes compu-tational work in assembling a collection of BTB domain sequences from completely se-quenced eukaryotic genomes. This chapter describes analyses on this collection including the genomic distribution, domain architectures, identification of putative novel domains and predictions of interactions. Chapters 3, 4 and 5 are founded on experimental analyses on BTB domains from human BTB-ZF proteins. Chapter 3 describes the structure of the BTB domain from Leu-kemia/Lymphoma Related Factor (LRF). The structure closely resembles the previously determined structures of BTB domains. The structure showed a large number of sequence substitutions on the surface of the LRF BTB domain that is equivalent to the surface in-volved in an interaction between the BTB domain from B-Cell Lymphoma 6 (BCL6) and a peptide derived from the SMRT co-repressor (the SMRT-BBD). We show the LRF BTB domain does not interact with this peptide. Chapter 4 describes the structures of the BTB domains from FAZF and Miz-1. These proteins conserve most of the BTB fold but show some unexpected changes. The BTB domain from FAZF lacks domain swapping which is a novel feature. The BTB do-main from Miz-1 contains a naturally truncated N-terminus and a novel movement of 10 residues away from a conserved three-stranded β-sheet. We show these BTB domains are dimeric within a specific concentration range and that they do not interact with the SMRT-BBD. Chapter 5 describes the structure of the BTB domain from Kaiso. This structure showed interactions between Kaiso BTB domain dimers that extend through the crystal. We identified similar interactions between dimers in a number of other structures of other BTB domains which suggested a common mode of oligomerization.

The BTB domain is a eukaryotic protein-protein interaction motif found in variety of proteins. This thesis describes an investigation into the general and specific properties of the sequence, structure and self-association properties of this domain. The work is divided by two complementary approaches. Chapter 2 describes compu-tational work in assembling a collection of BTB domain sequences from completely se-quenced eukaryotic genomes. This chapter describes analyses on this collection including the genomic distribution, domain architectures, identification of putative novel domains and predictions of interactions. Chapters 3, 4 and 5 are founded on experimental analyses on BTB domains from human BTB-ZF proteins. Chapter 3 describes the structure of the BTB domain from Leu-kemia/Lymphoma Related Factor (LRF). The structure closely resembles the previously determined structures of BTB domains. The structure showed a large number of sequence substitutions on the surface of the LRF BTB domain that is equivalent to the surface in-volved in an interaction between the BTB domain from B-Cell Lymphoma 6 (BCL6) and a peptide derived from the SMRT co-repressor (the SMRT-BBD). We show the LRF BTB domain does not interact with this peptide. Chapter 4 describes the structures of the BTB domains from FAZF and Miz-1. These proteins conserve most of the BTB fold but show some unexpected changes. The BTB domain from FAZF lacks domain swapping which is a novel feature. The BTB do-main from Miz-1 contains a naturally truncated N-terminus and a novel movement of 10 residues away from a conserved three-stranded β-sheet. We show these BTB domains are dimeric within a specific concentration range and that they do not interact with the SMRT-BBD. Chapter 5 describes the structure of the BTB domain from Kaiso. This structure showed interactions between Kaiso BTB domain dimers that extend through the crystal. We identified similar interactions between dimers in a number of other structures of other BTB domains which suggested a common mode of oligomerization.

N-methyl D-aspartate (NMDA) receptors represent integral signal transducers for excitatory glutamate neurotransmission. While NMDA receptors are critical for synaptic plasticity, the molecular events underlying this process are not fully elucidated. The potential role of NP22, a novel neuronal protein, as a downstream mediator of NMDA receptor function is explored. NP22 protein expression in genetic and pharmacological models of NMDA receptor hypofunction is examined and no significant changes are reported. Characterization of the NP22 protein complex via tandem-affinity and FLAG-purification coupled with mass spectrometry was used and no novel protein interactions are reported. GFP-tagged NP22 colocalization with F-actin decreases in cell processes of transiently transfected HEK293 cells in response to elevated intracellular calcium, while similar colocalization reductions are not seen in stably transfected HEK293 under a comparable treatment regiment. Changes in intracellular calcium affecting NP22 biology can be useful in the ongoing characterization of this novel protein.

AGAMOUS-Like 15 (AGL15) encodes a MADS-domain transcription factor that is preferentially expressed in the plant embryo, and may function as a regulator in embryonic developmental programs. A number of direct downstream targets of AGL15 have been identified, and while some of these target genes are induced in response to AGL15, others are repressed. Additionally, direct target genes have been analyzed that exhibit strong association with AGL15 in vivo, yet in vitro, AGL15 binds only weakly. Taken together these data suggest that AGL15 may form heterodimers, or ternary complexes with other proteins, thus modulating the specificity and function of AGL15 in planta. Yeast two-hybrid screens were undertaken to identify novel proteins able to interact with AGL15, and a number of interesting and potentially biologically important AGL15-interacting partners are reported here. These include members of a histone deacetylase complex, a COLD SHOCK DOMAIN (CSD)-containing protein, a Khomology domain/CCCH type zinc finger containing protein, a bZIP transcription factor, a homeobox-leucine zipper protein, a LATERAL ORGAN BOUNDARIES (LOB) domain containing protein, and an Agenet domain containing protein. Interactions between AGL15 and other MADS domain factors that are expressed in embryonic tissue, including SEPALLATA 3 (SEP3) have also been indentified. The regions of AGL15 that mediate interactions with the aforementioned proteins were mapped, and the capacity of these proteins to interact with other plant MADS-domain proteins tested. It is reported herein that AGL15 interacts with members of the SWI-INDEPENDENT 3/HISTONE DEACETYLASE (SIN3/HDAC) complex, and that AGL15 target genes are also responsive to an AGL15 interacting protein that is also a member of this complex, SIN3 ASSOCIATED POLYPEPTIDE OF 18 KD (SAP18). AGL15 can repress transcription in vivo, and a region essential to this repressive function contains an LxLxL motif that is conserved among putative orthologs of AGL15. What is more, the aforementioned motif mediates the association of AGL15 with SAP18 in yeast two-hybrid assays, thus providing a possible mechanism for explaining how role AGL15 regulates gene expression via recruitment of a histone deacetylase complex.

The aim of this thesis is the study of protein resistant oligo(ethylene glycol) (OEG) self-assembled monolayers (SAMs) using in situ techniques, such as neutron reflectivity (NR), polarisation modulation infrared spectroscopy (PMIR) and small-angle x-ray scattering (SAXS). In order to elucidate the mechanisms that lead to the nonfouling properties of these SAMs, the SAM-water, protein-protein and protein-SAM interactions have been studied separately. NR measurements, focused on the solid-liquid interface between OEG SAMs and water, show clear evidence of an extended layer with reduced density water. The reduction in density is up to 10% compared to the bulk value, and extends up to 5 nm into the bulk. The effective area (density reduction x length) of this reduced density water layer did not significantly change when the temperature was reduced to 5°C. In a complementary study, the interaction of water with protein-resistant HS(CHV₂)₁₁(OCH₂CH₂)₃OMe monolayers was examined using in and ex situ PMIR. In particular, shifts in the position of the characteristic C-O-C stretching vibration were observed after the monolayers had been exposed to water. The shift in frequency increased when the SAM was observed in direct contact with a thin layer of water. It was found that the magnitude of the shift also depended on the surface coverage of the SAM. These results suggest a rather strong interaction of oligo(ethylene glycol) SAMs with water and indicate the penetration of water into the upper region of the monolayer. These findings indicate the presence of a tightly bound water layer at the SAM-water interface. Further NR studies of the interface between OEG SAMs and a highly concentrated protein solution revealed an oscillating protein density profile. A protein depleted region of about 4-5 nm close to the SAM was followed by a more densely populated region of 5-6 nm. These oscillations were then rapidly damped out until the bulk value was reached. The influence of temperature and salt concentration on the protein density profile was small, indicating a rather minor contribution of electrostatic interactions to the protein repulsive force. SAXS measurements of OEG coated gold colloids mixed with proteins in solution did also not show any pronounced salt concentration dependence of the colloid-protein interaction. The strong association of water with the SAM and the layer of tightly bound water, together with the lack of electrostatic repulsion, suggest that the adsorption of proteins is energetically hindered by the presence of a strongly bound hydration layer.

LNX1 belongs to a family of multi-PDZ domain containing RING-type E3 ligases. Several interactions have been mapped to its PDZ domains, but the role of each domain in LNX function has not yet been determined. To study individual PDZ domain function in the context of full length protein I generated point mutations in peptide binding sites of each of PDZ domain, and in a putative phosphoinositide binding site of LNX1 PDZ4. Peptide binding was successfully disrupted by an arginine or lysine to alanine mutation in the peptide binding cleft. A LNX1 PDZ4 mutant with lysine residues in a putative phosphoinositide binding site mutated to glutamate displayed decreased membrane localization. The impact of each PDZ mutation on cell morphology and substrate ubiquitination was also investigated. I identified a potential role for PDZ binding in auto-inhibition of RING function. Additionally, novel interactions between LNX1 and Frizzled family members were identified and characterized.

LNX1 belongs to a family of multi-PDZ domain containing RING-type E3 ligases. Several interactions have been mapped to its PDZ domains, but the role of each domain in LNX function has not yet been determined. To study individual PDZ domain function in the context of full length protein I generated point mutations in peptide binding sites of each of PDZ domain, and in a putative phosphoinositide binding site of LNX1 PDZ4. Peptide binding was successfully disrupted by an arginine or lysine to alanine mutation in the peptide binding cleft. A LNX1 PDZ4 mutant with lysine residues in a putative phosphoinositide binding site mutated to glutamate displayed decreased membrane localization. The impact of each PDZ mutation on cell morphology and substrate ubiquitination was also investigated. I identified a potential role for PDZ binding in auto-inhibition of RING function. Additionally, novel interactions between LNX1 and Frizzled family members were identified and characterized.

Membrane proteins are essential cellular components, responsible for a wide variety of biological functions. In order to better understand such aspects of cell activity, researchers have pursued detailed structural analysis of this class of proteins. Because of the complexities in isolating and studying membrane proteins in their native environment, detergents are often employed as a membrane mimetic media. This thesis examines several features of transmembrane (TM) protein structure and folding in detergents through which we are able to gain insights into membrane protein folding, as well as explore the suitability of detergents as membrane-mimetic environments. We first compare the helix-helix association of a series of model TM sequences in a native bilayer to the corresponding association in a detergent environment. We find that while various classes of helix-helix interaction motifs are preserved in detergents, alterations in detergent solvation may, in turn, lead to altered association affinity. We further explore this phenomenon through investigation of the consequences of the insertion of a strongly polar residue into a TM segment. In these studies we find a correlation between sequence-dependent alterations in detergent solvation and predicted in vivo folding. We also extend such analyses to a variety of detergents and native TM segments, finding that native secondary structure, as it occurs in the context of a full-length protein, is generally well preserved in a variety of detergents. Finally, we assess the determinants of membrane protein folding using two-transmembrane segment constructs, in the process optimizing expression, production and characterization techniques for a diverse range of transmembrane protein sequences. Overall this thesis finds that, detergents are capable of solubilizing membrane proteins in a form suitable for in-depth structural characterization that may not be feasible in other environments. Thus, as an approximation of a native membrane, detergents are able to preserve certain features of membrane proteins such as helix-helix association and native secondary structure.

Membrane proteins are essential cellular components, responsible for a wide variety of biological functions. In order to better understand such aspects of cell activity, researchers have pursued detailed structural analysis of this class of proteins. Because of the complexities in isolating and studying membrane proteins in their native environment, detergents are often employed as a membrane mimetic media. This thesis examines several features of transmembrane (TM) protein structure and folding in detergents through which we are able to gain insights into membrane protein folding, as well as explore the suitability of detergents as membrane-mimetic environments. We first compare the helix-helix association of a series of model TM sequences in a native bilayer to the corresponding association in a detergent environment. We find that while various classes of helix-helix interaction motifs are preserved in detergents, alterations in detergent solvation may, in turn, lead to altered association affinity. We further explore this phenomenon through investigation of the consequences of the insertion of a strongly polar residue into a TM segment. In these studies we find a correlation between sequence-dependent alterations in detergent solvation and predicted in vivo folding. We also extend such analyses to a variety of detergents and native TM segments, finding that native secondary structure, as it occurs in the context of a full-length protein, is generally well preserved in a variety of detergents. Finally, we assess the determinants of membrane protein folding using two-transmembrane segment constructs, in the process optimizing expression, production and characterization techniques for a diverse range of transmembrane protein sequences. Overall this thesis finds that, detergents are capable of solubilizing membrane proteins in a form suitable for in-depth structural characterization that may not be feasible in other environments. Thus, as an approximation of a native membrane, detergents are able to preserve certain features of membrane proteins such as helix-helix association and native secondary structure.