Potassium channel-interacting protein 1 (KChIP1), a Ca²⁺ sensor protein, regulates the function of A-type Kv4 potassium channels and increases their cell surface expression. Myristoylation at the N-terminus of KChIP1 has been suggested to facilitate membrane-binding, but was not sufficient for stable membrane assaociation. The aim of the present study is to investigate whether EF-hand motifs of KChIP1 are crucial for membranal targeting in addition to the N-terminal myristoyl group, and how the membrane association of KChIP1 is influenced by lipid compositions. According to hydropathy profile, EF-hands 3 and 4 of KChIP1 showed highly hydrophobicity. After deleting EF-hands 3 and 4, the altered microenvironment of Trp residue and decreased hydrophobicity were found in truncated KChIP1, but it still maintained £\-helix structure. Furthermore, truncated KChIP1 exhibited lower lipid-binding ability, affecting intracellular membrane localization and was almost diminished underlying increasing membrane permeability by digitonin in cells, suggesting that intact EF-hands 3 and 4 may be related to the anchorage of KChIP1 on cellular membrane. KChIP1, but not mutant, specifically bound with phosphatidylserine by lipid binding assay and the FTIR spectra showed the change of £\-helix structure by binding lipid large unilamellar vesicles was dependent on phosphatidylserine. Either phosphatidylserine or potassium channels enhanced KChIP1 to form tetramer for targeting to phospholipids by using chemical cross-linking assay. Taken together, our data highly suggest that intact of EF-hands 3 and 4 should structurally and functionally involve in fulfilling the physiological activity of KChIP1.

The simultaneous separation of anionic and cationic proteins has been achieved by addition of high concentration of poly(diallyldimethylammonium chloride) (PDDAC) in capillary electrophoresis. A capillary was filled with PDDAC so that it would act as ion-pair reagents in the separation of anionic proteins. On the other hand, the PDDAC can also be used as coating additives for the analysis of cationic proteins. Increasing the concentration of PDDAC in the separation buffer had the ability to improve the separation efficiency, change the electrophoretic mobility, and alter the separation selectivity; however, this was not true in the case of analyzing proteins by using the PDDAC larger than 1.6%. By both using a buffer containing 1.6% PDDAC and applying pH-stepwise techniques, 13 proteins with a wide range of pI (4.7¡V11.1) and molecular masses (6.5¡V198.0 kDa) could be separated within 30 min in a single run. In addition to this separation, we observed notonly more peaks from alph-chymotrypsinogen A and aprotinin but also the bovine serum albumin (BSA) dimer and trimer. The second part describes a method for enrichment and separation of acidic and basic proteins using the centrifugal ultrafiltration followed by polyelectrolyte-filled capillary electrophoresis. In order to improve stacking and separation efficiencies of proteins, the separation buffer containing 1.6% poly(diallyldimethylammonium chloride) was added with gold nanoparticles (AuNPs), poly(ethylene oxide), cetyltrimethyl ammonium bromide, and poly(vinyl alcohol). As a result, the use of AuNPs as additives exhibited better efficiency in separation, stacking and analysis time. Even for large-volume samples (110 nL), the separation efficiencies of acidic and basic proteins remained greater than 104 and 105 plates/m, respectively. To further enhance detection sensitivity, protein samples were enriched using the centrifugal ultrafiltration, followed by our proposed stacking method. As a result, the detection sensitivity was improved up to 314-fold as compared with normal hydrodynamic injection. Additionally, the limits of detection at a signal-to-noise of 3 for most proteins are down to nanomolar range. We have validated the application of our method by means of analyses of 50 nM lysozyme in saliva samples. The proposed method was also successfully applied to the analyses of egg-white proteins, which have large differences in molecular weight and pI.

The all-atom protein backbone reconstruction problem (PBRP) is to rebuild the 3D coordinates of all atoms on the backbone which includes N, C, and O atoms. In the previous work, we find that the prediction accuracy of the 3D positions of the O atoms is not so good, compared with the other two atoms N and C. Thus, our goal is to refine the positions of the O atoms after the initial prediction of N, C, and O atoms on the backbone has been done by the previous work. Based on the AMBER force field, we modify the energy function to a simplified one with the statistical data on the bond lengths and bond angles of the 21 distinct amino acids (including the nonstandard one). Then, we propose a two-phase refinement method (TPRM) to find the position of each O atom independently that optimizes the modified energy function. We perform our method on two test sets of proteins. The experimental results show that the reconstruction accuracy of our method is better than the previous ones. The solution of our method is also more stable than most of the previous work. Besides, our method runs much faster than the famous prediction tool, SABBAC.

No description available.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 148-170).

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 64-72).

Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 151-157). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--California Institute of Technology, 1994. UM #94-06,216. / Advisor names found in the Acknowledgements pages of the thesis. Title from home page. Viewed 01/15/2010. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 110-115). Available also in a digital version from Dissertation Abstracts.

Thesis (M.S. in Bio.)--School of Biology, Georgia Institute of Technology, 2004. Directed by Harish Radhakrishna. / Includes bibliographical references (leaves 55-65).