The study of ligand binding specificities of the lipid binding proteins : recombinant human a-tocopherol transport protein (a-ttp), supernatant protein factor (spf) and S. cerevisiae Sec 14p for vitamin e (rrr-a-tocopherol) and other hydrophobic ligands.

One of the various functions of proteins in biological systems is the transport of small molecules, for this purpose proteins have naturally evolved special mechanisms to allow both ligand binding and its subsequent release to a target site; a process fundamental to many biological processes. Transport of Vitamin E (a-tocopherol), a lipid soluble antioxidant, to membranes helps in the protection of polyunsaturated fatty acids against peroxidative damage. In this research, the ligand binding characteristics of several members of the CRALTRIO family of lipid binding proteins was examined; the recombinant human a-Tocopherol Transfer Protein (a-TIP), Supernatant Protein Factor (SPF)ffocopherol Associated Protein (TAP), Cellular Retinaldehyde Binding Protein (CRALBP) and the phosphatidylinositol transfer protein from S. cerevisiae Sec 14p. Recombinant Sec 14p was expressed and purified from E. coli for comparison of tocopherol binding to the two other recombinant proteins postulated to traffic a-tocopherol. Competitive binding assays using [3H]-a-tocopherol and Lipidex-l000 resin allowed determination of the dissociation constants ~) of the CRAL-TRIO proteins for a-tocopherol and - 20 hydrophobic ligands for evaluation of the possible biological relevance of the binding interactions observed. The KIs (nM) for RRR-a-tocopherol are: a-TIP: 25.0, Sec 14p: 373, CRALBP: 528 and SPFffAP: 615. This indicates that all proteins recognize tocopherol but not with the same affinity. Sec 14p bound its native ligand PI with a KI of381 whereas SPFffAP bound PI (216) and y-tocopherol (268) similarly in contrast to the preferential binding ofRRR-a-tocopherol by a-TIP. Efforts to adequately represent biologically active SPFff AP involved investigation of tocopherol binding for several different recombinant proteins derived from different constructs and in the presence of different potential modulators (Ca+2, Mg+2, GTP and GDP); none of these conditions enhanced or inhibited a-tocopherol binding to SPF. This work suggests that only aTTP serves as the physiological mediator of a-tocopherol, yet structural homology between proteins allows common recognition of similar ligand features. In addition, several photo-affmity analogs of a-tocopherol were evaluated for their potential utility in further elucidation of a-TTP function or identification of novel tocopherol binding proteins.

The Arabidopsis NPR1 Protein Is a Receptor for the Plant Defense Hormone Salicylic Acid

Systemic Acquired Resistance (SAR) is a type of plant systemic resistance occurring against a broad spectrum of pathogens. It can be activated in response to pathogen infection in the model plant Arabidopsis thaliana and many agriculturally important crops. Upon SAR activation, the infected plant undergoes transcriptional reprogramming, marked by the induction of a battery of defense genes, including Pathogenesis-related (PR) genes. Activation of the PR-1 gene serves as a molecular marker for the deployment of SAR. The accumulation of a defense hormone, salicylic acid (SA) is crucial for the infected plant to mount SAR. Increased cellular levels of SA lead to the downstream activation of the PR-1 gene, triggered by the combined action of the Non-expressor of Pathogenesis-related Gene 1 (NPR1) protein and the TGA II-clade transcription factor (namely TGA2). Despite the importance of SA, its receptor has remained elusive for decades. In this study, we demonstrated that in Arabidopsis the NPR1 protein is a receptor for SA. SA physically binds to the C-terminal transactivation domain of NPR1. The two cysteines (Cys521 and Cys529), which are important for NPR1’s coactivator function, within this transactivation domain are critical for the binding of SA to NPR1. The interaction between SA and NPR1 requires a transition metal, copper, as a cofactor. Our results also suggested a conformational change in NPR1 upon SA binding, releasing the C-terminal transactivation domain from the N-terminal autoinhibitory BTB/POZ domain. These results advance our understanding of the plant immune function, specifically related to the molecular mechanisms underlying SAR. The discovery of NPR1 as a SA receptor enables future chemical screening for small molecules that activate plant immune responses through their interaction with NPR1 or NPR1-like proteins in commercially important plants. This will help in identifying the next generation of non-biocidal pesticides.

Elliptical orbital studies of H21 and H2 molecules /|nS. K. Gupta. -- 260 St. Catharines, Ont. : [s. n.],

Calculations are performed on the \S <:Jd ground states of d ' + the H and HC) molecules using a basis set of non-integral ~ ~ I elliptical orbitals. Different variational wavefunctions constructed i- for H~ involved one parameter to three par~~eter variation. In order to l"'educe the ntunber of parameters in most commonly 0- used basis orbitals set, the importance of the term (,+~) Y\ over the term ;u 'Where n is a variational pararneter and the value of cr may be given by boundary condition or cusp condition is outlined in Chapters II and III. It is found that the two parameter -+ small and moderate internuclear separations. c;. In order to find out the importance of the term (I +~ ) Y\ over ;U for the two electron problem, the variational energy is computed for the H~ molecule from unrestricted two parameter closed shell wavefunctions including the term U+ft)