|dc.description.abstract||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).||en_US