Characterization of proline - containing Alpha-Helices & modelling studies on bacteriorhodopsin

Abstract

Membrane proteins have a wide range of vital functions and serve as enzymes, transporters, receptors, pores, etc. To understand the diverse functions of membrane proteins and fully explain their modes of action, it is necessary to have detailed knowledge of their three dimensional structures. Although the amino acid sequences of many membrane proteins are now known from recombinant DNA technologies, their three dimensional conformations have begun to emerge only in recent years. The integral membrane protein bacteriorhodopsin (BR) is one such extensively studied example. It is found in the purple membrane of Halobacterium halobium and contains seven transmembrane helices. BR also contains a prosthetic group, retinal, attached to Lys 216 of helix G through a Schiff base linkage. Upon absorption of light, retinal undergoes an all trans to 13 cis isomerization, resulting in proton transport from the cytoplasmic side to the extracellular side against a gradient. Extensive studies have been carried out on BR to understand the mechanism of proton transport. A number of residues have been identified as structurally and/or functionally important. To gain insight into the detailed three dimensional structure of BR, computer modelling was undertaken as part of this thesis work. Recently, the resting state structure of BR at a resolution of 3.5 Å parallel to the membrane surface and 10.0 Å in the vertical direction has been solved by electron cryo microscopy (Henderson et al., 1990, J. Mol. Biol., 213, 899). The modelling studies described here were continued for the photocycle intermediates. Among the seven helices of BR, three contain a proline residue in the middle. FTIR studies show that at least one of these proline residues undergoes structural changes during the photocycle. Since little structural information is available on proline containing helices, it was essential to characterize such helices to model BR. Therefore, high resolution crystal structures from the Protein Data Bank were analyzed. The geometry of proline containing helices was obtained from this analysis. Subsequently, energy minimization and molecular dynamics simulations were carried out on model peptides containing a centrally placed proline residue. Because many transport proteins contain such helices, these results are important and immediately applicable to modelling membrane proteins. The low resolution BR structure provides a good starting point for studying proton transport mechanisms, which remain incompletely understood. Computer modelling was used to obtain intermediate BR structures representing different stages of the photocycle. Energy minimizations of the resting state and intermediate structures were carried out, followed by analyses of interactions and roles of important amino acid residues, compared with experimental findings. The thesis is presented in five chapters:

Chapter 1 - Introduction This chapter introduces:

structural features of membrane proteins, the protein folding problem, secondary and tertiary structure prediction algorithms, differences between globular and membrane proteins, experimental techniques (spectroscopy, X ray diffraction, electron microscopy) and their limitations, membrane protein topology and transmembrane segment predictions (hydrophobicity and hydrophobic moment plots), the roles of ionizable and polar residues within bilayers, the special structural role of proline in transport proteins, an overview of the structure and function of bacteriorhodopsin.

The chapter provides the background necessary for the studies on proline containing helices and BR described later.

Chapter 2 - Proline Containing Helices High resolution crystal structures were analyzed to study the geometry of helices containing a central proline. Analysis of internal backbone parameters revealed:

consistent patterns of deviation from ideal helical geometry, the presence of a helix kink caused by proline, definitions of kink angle (between helical axes before and after proline) and a wobble angle (orientation of helix II relative to helix I).

Algorithms were developed to compute these parameters. Additional analyses examined whether such helices occur on the surface or in the interior of proteins. Energy minimization studies using flexible geometry showed that proline occurring mid helix is energetically favorable, contrary to earlier rigid geometry studies. A standard set of internal parameters was established for use in model building.

Chapter 3 - Modelling and Minimization of Bacteriorhodopsin This chapter describes:

the structure and photocycle of BR, previously proposed models for proton pumping, experimental studies identifying key residues.

Initial modelling aimed to construct a continuous hydrogen bonded chain from cytoplasmic to extracellular side. The structure obtained showed good agreement with the recently reported electron cryo microscopy model. Modelling was extended to photocycle intermediates. Energy minimizations of the resting and intermediate states were performed, followed by analysis of:

bend related parameters of proline containing helices, intra and inter helical hydrogen bonds, residues surrounding retinal, structural changes relevant to proton transfer.

Chapter 4 - Molecular Dynamics Studies MD simulations were performed on model peptides, including a helix F model of BR containing:

a central proline, aromatic residues Trp 182, Tyr 185, and Trp 189.

Simulations (100 ps after 25 ps equilibration) revealed:

backbone fluctuations consistent with crystal structure and energy minimization observations, helix oscillations between straight and highly bent conformations, correlated motion among bend related parameters, proline induced changes in side chain orientations, limited side chain flexibility in proline containing helices.

Energy minimization of MD structures identified two distinct minima for proline (up and down conformations), separated by small barriers permitting frequent interconversion.

Chapter 5 - Summary and Conclusions The chapter summarizes significant applications of characterizing proline containing helices and compares BR results with recent studies. It concludes with a discussion of future research directions in modelling proline containing helices and bacteriorhodopsin.