Inorganic cations play decisive roles in many cellular and physiological processes and are essential components of plant nutrition. Therefore, the uptake of cations and their redistribution must be precisely controlled. Vacuolar antiporters are important elements in mediating the intracellular sequestration of these cations. CAXs (for CAtion eXchanger) are members of a multigene family and appear to predominately reside on vacuoles. Defining CAX regulation and substrate specificity have been aided by utilizing yeast as an experimental tool. Studies in plants suggest CAXs regulate apoplastic Ca2+ levels in order to optimize cell wall expansion, photosynthesis, transpiration and plant productivity. CAX studies provide the basis for making designer transporters that have been used to develop nutrient enhanced crops and plants for remediating toxic soils. In my second study, I have characterized and defined autoinhibitory domain of Arabidopsis CAX3. Several CAX transporters, including CAX1, appear to contain an approximately 40 amino acid N-terminal regulatory regions (NRR) that modulates transport through N-terminal autoinhibition. Deletion of the NRR from several CAXs (sCAX) enhances function in plant and yeast expression assays; however, to date, there are no functional assays for CAX3. In this report, we create a series of truncations in the CAX3 NRR and demonstrate activation of CAX3 in both yeast and plants by truncating a large portion of the NRR. Experiments on endomembrane-enriched vesicles isolated from yeast expressing activated CAX3 demonstrate that the gene encodes Ca2+/H+ exchange with properties distinct from CAX1. These studies demonstrate shared and unique aspects of CAX1 and CAX3 transport and regulation. My third study is to express and purify CAX proteins for X-ray crystallographic analysis. In this study, I initiated crystallization of vacuolar membrane localized CAX protein from eukaryotes. Membrane proteins continue to be challenging targets for structural biology because of their hydrophobic nature. We have demonstrated here that eukaryotic Ca2+/H+ exchanger can be successfully expressed in E. coli based expression system. Collectively, our findings suggest that CAX protein can be successfully expressed, detergent solublized and purified from E. coli with a yield sufficient for functional and structural studies.

No description available.

This thesis describes the development of a novel approach for prediction of the three-dimensional structure of transmembrane regions of membrane proteins directly from amino acid sequence and basic transmembrane region topology. The development rationale employed involved a knowledge-based approach. Based on determined membrane protein structures, 20x20 association matrices were generated to summarise the distance associations between amino acid side chains on different alpha helical transmembrane regions of membrane proteins. Using these association matrices, combined with a knowledge-based scale for propensity for residue orientation in transmembrane segments (kPROT) (Pilpel et al., 1999), the software predicts the optimal orientations and associations of transmembrane regions and generates a 3D structural model of a gi ven membrane protein, based on the amino acid sequence composition of its transmembrane regions. During the development, several structural and biostatistical analyses of determined membrane protein structures were undertaken with the aim of ensuring a consistent and reliable association matrix upon which to base the predictions. Evaluation of the model structures obtained for the protein sequences of a dataset of 17 membrane proteins of detennined structure based on cross-validated leave-one-out testing revealed generally high accuracy of prediction, with over 80% of associations between transmembrane regions being correctly predicted. These results provide a promising basis for future development and refinement of the algorithm, and to this end, work is underway using evolutionary computing approaches. As it stands, the approach gives scope for significant immediate benefit to researchers as a valuable starting point in the prediction of structure for membrane proteins of hitherto unknown structure.

Due to the inherent problems with their structural elucidation in the laboratory, the computational prediction of membrane protein structure is an essential step toward understanding the function of these leading targets for drug discovery. In this work, the development of a genetic algorithm technique is described that is able to generate predictive 3D structures of membrane proteins in an ab initio fashion that possess high stability and similarity to the native structure. This is accomplished through optimisation of the distances between TM regions and the end-on rotation of each TM helix. The starting point for the genetic algorithm is from the model of general TM region arrangement predicted using the TMRelate program. From these approximate starting coordinates, the TMBuilder program is used to generate the helical backbone 3D coordinates. The amino acid side chains are constructed using the MaxSprout algorithm. The genetic algorithm is designed to represent a TM protein structure by encoding each alpha carbon atom starting position, the starting atom of the initial residue of each helix, and operates by manipulating these starting positions. To evaluate each predicted structure, the SwissPDBViewer software (incorporating the GROMOS force field software) is employed to calculate the free potential energy. For the first time, a GA has been successfully applied to the problem of predicting membrane protein structure. Comparison between newly predicted structures (tests) and the native structure (control) indicate that the developed GA approach represents an efficient and fast method for refinement of predicted TM protein structures. Further enhancement of the performance of the GA allows the TMGA system to generate predictive structures with comparable energetic stability and reasonable structural similarity to the native structure.

Membrane proteins roughly constitute 30% of open reading frames in a genome and form 70% of current drug targets. They are classified as integral, peripheral membrane proteins and polypeptide toxins. α-helices and β -strands are the principal secondary structures observed in integral membrane proteins. This thesis presents the results of studies on analysis and correlation of sequence and structure of helices constituting integral helical membrane proteins. The aim of this work is to understand the helix stabilization, distortion as well as packing in terms of amino acid sequences and the correlated structures they adopt. To this end, analyses of datasets of X-ray crystal structures of integral helical membrane proteins and their comparison with a dataset of representative folds of globular proteins was carried out. Initial analysis was carried out using a non-redundant dataset of 75 membrane proteins to understand sequence and structural preferences for stabilization of helix termini. The subsequent analysis of helix distortions in membrane proteins was carried out using an updated dataset of 90 membrane proteins. Chapter 1 of the thesis reviews experimental as well as theoretical studies that have provided insights into understanding the structure of helical membrane proteins. Chapter 2 details the methods used during the course of the present investigations. These include the protocol used for creation of the non-redundant database of membrane and globular proteins. Various statistical methods used to test significance of the position-wise representation of amino acids in helical regions and the differences in globular and membrane protein datasets have been listed. Based on the tests of significance, a methodology to identify differences in propensity values that are statistically significant among two datasets has been devised. Programs used for secondary structure identification of membrane proteins namely Structure Identification (STRIDE) and Assignment of Secondary Structure in Proteins (ASSP) as well as those used for characterization of helical geometry (Helanal-Plus) have also been enlisted. In Chapter 3, datasets of 865 α-helices in 75 membrane proteins and 2680 α- helices from 626 representative folds in globular proteins defined by the STRIDE program have been analyzed to study the sequence determinants at fifteen positions within and around the α-helix. The amino acid propensities have been studied for positions that are important for the process of helix initiation, propagation, stabilization and termination. Each of the 15 positions has unique sequence characteristics reflecting their role and contribution towards the stability of the α-helix. A comparison of the sequence preferences in membrane and globular proteins revealed common residue preferences in both these datasets confirming the importance of these positions and the strict residue preferences therein. However, short/medium length α-helices that initiated/terminated within the membrane showed distinct amino acid preferences at the N-terminus (Ncap, N1, N2) as well as the C-terminus ( Ccap, Ct) when compared to α-helices belonging to membrane and globular proteins. The sequence preferences in membrane proteins were governed by the helix initiating and terminating property of the amino acids as well as the external environment of the helix. Results from our analysis also conformed well with experimentally tested amino acid preferences in a position-specific amino acid preference library of the rat neurotensin receptor (Schlinkmann et al (2012) Proc Natl Acad Sci USA 109(25):1890-5) as well as crystal structures of GPCR proteins. In the light of the environment dependent amino acid preferences found at α- helix termini, a survey was carried out to find various helix capping motifs adopted at both termini of α-helices in globular and membrane proteins to stabilize these helix termini. The results from these findings have been reported in Chapter 4. A sequence dependent structural preference is found for capping motifs at helix termini embedded inside and protruding outside the membrane. The N-terminus of α-helices was capped by hydrogen bonds involving free main chain amide groups of the first helical turn as donors and amino acid side chains as acceptors, as against the C-terminus which showed position-dependent characteristic backbone conformations to cap the helix. Overall helix termini inside the membrane did not show a very high number of capping motifs; instead these termini were stabilized by helix- helix interactions contributed by the neighboring helices of the helical bundle. In Chapter 5, we examine transmembrane helical (TMH) regions to identify as well as characterize the various types of helix perturbations in membrane proteins using ASSP and Helanal-Plus. A survey of literature shows that the term ‘helix kink’ has been used rather loosely when in fact helical regions show significant amounts of variation and transitions in helical parameters. Hence a systematic analysis of TMH regions was undertaken to quantify different types of helix perturbations, based on geometric parameters such as helical twist, rise per residue and local bending angle. Results from this analysis indicated that helices are not only kinked but undergo transitions to form interspersed stretches of 310 helices and π-bulges within the bilayer. These interspersed 310 and π-helices showed unique sequence preferences within and around their helical body, and also assisted in main- taining the helical structure within the bilayer. We found that Proline not only kinked the helical regions in a characteristic manner but also caused a tightening or unwinding in a helical region to form 310 and π-helix fragments respectively. The helix distortions also resulted in backbone hydrogen bonds to be missed which were stabilized by hydrogen bonds from neighboring residues mediated by their side chain atoms. Furthermore, a packing analysis showed that helical regions with distortions were able to establish inter-helical interactions with more number of transmembrane segments in the helical bundle. The study on helix perturbations presented in the previous chapter, brought to light a previously unreported 19 amino acid π-helix fragment interspersed between α-helices in the functionally important transmembrane helix 2 (TM2) belonging to Mitochondrial cytochrome-c-oxidase (1v55). Chapter 6 describes a case study of the structurally similar but functionally different members within the Heme-Copper- Superoxidases (HCO) superfamily that were considered for a comparative analysis of TM2. An analysis of 7 family members revealed that the π-helix shortens, fragments in two shorter π-helices or was even absent in some family members. The long π-helix significantly decreased the total twist and rise of the entire helical fragment thus accommodating more hydrophobic amino acids within the bilayer to avoid hydrophobic mismatch with the bilayer. The increased radius of the TM2 helical fragment also assisted in helix packing interactions by increasing the number of residues involved in helix-helix interactions and hydrogen bonds. Chapter 7 documents the conclusions from the different analyses presented in each of the above chapters. Overall, it is found that membrane proteins optimize the biophysical and chemical constraints of the external environment to strategically place select amino acids at helix termini to ‘start’ and ‘stop’ α-helices. The stabilization of these helix termini is a consequence of sequence dependent structural preferences to form helix capping motifs. The studies on helix transitions and distortions highlight that membrane proteins are not only packed as α-helices but also accomodate 310- and π-helical fragments. These transitions and distortions help in harboring more hydrophobic amino acids and aiding inter-helical interactions important for maintaining the fold of the membrane protein. Appendix A describes a comparison of α-helix assignments in globular and membrane proteins by two algorithms, one based on Cα trace (ASSP) and the other using a combination of hydrogen bond pattern along with backbone torsion angles φ and ψ (STRIDE).