Mechanisms of Primary Energy Transduction in Biology

Mårten Wikström, Mårten Wikström

• Éditeur: Royal Society of Chemistry
• Année: 2017
• ISBN:
  9781788013635
• Édition: 1
• Sujet: Biowissenschaften
• Sous-sujet: Biophysik

This book describes the events of primary energy transduction in life processes. Life as we know it depends on pumping protons across membranes. New tools to study the protein complexes involved has led to recent intensified progress in the field.
Primary Energy Transduction in Biology focusses on recent structural results and new biophysical insights. These have been made possible by recent advances in high-resolution protein structures, in physical techniques to study reactions in real time, and in computational methods to study and refine both structures and their dynamics. Written and edited by leading experts, chapters discuss the latest key questions in cell respiration, photosynthesis, bioenergetics, proton transfer, electron transfer and membrane transport.
Biochemists, biophysicists and chemical biologists will find this book an essential resource for a complete understanding of the molecular machines of bioenergetics.

CHAPTER 1

Making Maquette Models of Bioenergetic Structures

CHRISTOPHER C. MOSER,* NATHAN M. ENNIST, JOSHUA A. MANCINI AND P. L. DUTTON

University of Pennsylvania, Johnson Research Foundation, Department of Biochemistry and Biophysics, Philadelphia, PA, USA

*Email: [email protected]

1.1 Unravelling Protein Complexity

Extensive work developing X-ray crystal structures of core natural bioenergetic proteins exposed the arrangement of hundreds to thousands of amino acids that surround and bind a range of different cofactors that support bioenergetic light absorption and respiratory and photosynthetic electron and proton transfer.^1–12 Such atomic resolution offers the promise of a detailed understanding of how these naturally evolved systems function to direct electron and proton transfer to support life, or how they may dysfunction in disease.

Indeed, such structures have provided a good appreciation of how electrons tunnel from one redox centre to another across the intervening electrically insulating protein medium to form chains of redox centres that connect catalytic centres of bond breaking and making. A survey of dozens of individual bioenergetic electron-transfer reactions, both productive and unproductive, using both natural and unnatural redox centres, has shown that a relatively simple formula depending on only three parameters captures the quantum mechanics adequately to provide estimates of electron-tunnelling rates within an order of magnitude.^13,14 By constructing a matrix of electron-tunnelling rates between all redox centres in a protein, we can model the progress of electron transfer to reveal the relative weaknesses and strengths in natural electron-transfer chain design.^15–17

However, factors other than electron-tunnelling distance can dominate performance, especially at the catalytic centres terminating electron-transfer chains.¹⁸ Here, the complexity of natural bioenergetic protein structures obscures which engineering elements are critical for function and which elements may be incidental remnants of an evolutionary legacy. While mutation of individual residues can verify their functional importance, mutation imperfectly clarifies the residue's role. This is because natural selection has an inherent tendency to create systems in which component parts become progressively interdependent^19,20—the protein-level equivalent to Muller's genetic ratchet.^21,22 Previously independent amino acid residues came to depend upon one another for continued function. Thus mutation of a residue with only indirect support of catalytic function may devastate function and seem to be of central importance. In addition, any one amino acid in natural proteins tends to support many functions, a molecular expression of Darwin's principle of multiple utility;²³ for example, a particular amino acid may support not only chemical activity, but also protein folding, stability and dynamics.^24,25

The need to unravel this complexity inspired design and construction of simplified maquettes of natural proteins aiming for intentional simplicity, robustness and independent function. De novo designed proteins use first principles of protein folding and are untouched by natural selection. These constructs can be exploited to reverse-engineer natural protein function and rigorously test hypotheses. The basic functional principles resolved in this way may then be used to construct novel protein systems that introduce new functions or surpass natural proteins in robustness and performance.

De novo design and construction of cofactor binding protein maquettes exposes the default properties of cofactors in a protein matrix independent of natural selection and gain insight into how redox proteins may have operated early in the evolution of life.^26,27 Such designs help us to isolate and understand the means by which protein can be engineered to manipulate cofactor properties towards desired function.²⁸ Furthermore, by independently recapitulating critical properties of natural bioenergetic proteins in designed constructs, we gain confidence in protein engineering principles—the maquette approach is the redox-protein counterpart of Feynman's principle “What I cannot create, I do not understand”.²⁹ This chapter surveys the present state of cofactor functionalized bioenergetic maquette design.

1.2 Robust, Modular, Helix-bundle Scaffold for Electron-transfer Architecture

De novo protein design is highly diverse, including architectures quite unlike natural proteins. For example, some early maquette designs sought to guide protein assembly through a template assisted synthetic protein (TASP) strategy whereby separate helical peptides were site specifically attached to a synthetic cyclic peptide template.^30–32 In this chapter, we will focus on designs that can be expressed in a cell; such designs can benefit from integration with natural cellular biochemistry and offer the possibility of conferring new functions to the organism.

De novo protein designs commonly exploit the fundamental principle of binary patterning of amino acid heptad repeats to promote spontaneous helical bundle association.^33,34 Hydrogen bonding between alpha-helical amide backbone oxygens and nitrogens folds two helical turns for every seven amino acids. Selecting a sequence in which residues on one face of the helix are non-polar, while the remaining are polar, drives helical self-association to bury hydrophobic residues in the bundle core. The structurally resolved two-helix coiled-coil protein tropomyosin³⁵ was used as an early model, in which heptad positions were labelled sequentially from a through g, with the a and d positions associated with core hydrophobic residues. By shifting the hydrophobic : hydrophilic balance from two to three non-polar residues per heptad at positions a, d and e, four-helix bundles form spontaneously.

For example, the heptad repeat LQQLLQX where L is Leu, Q is Gln and X is E (Glu) for one sequential pair of helices and K (Lys) for the other pair, forms a fully antiparallel helical bundle arrangement common in natural helical coiled-coil proteins. High-affinity wrapping of the helices in a left handed coiling is driven by the “knob-into-hole” interactions first predicted by Francis Crick, in which amino acid side chains (knobs) pack into spaces between the side chains on adjacent helices (holes).³⁶ Connecting the four helices into a single chain with Gly rich loops facilitates independent adjustment of the sequences of each helix, assisting in site-specific binding of redox cofactors and permitting surface residue salt-bridges between helices that stabilize the structure. For example, in the sequence just described, negatively charged E residues in helices one and two salt-bridge with positively charged K residues in helices three and four when helices are threaded in a counter-clockwise pattern, as shown in Figure 1.1a. It is not uncommon to generate helical bundle maquettes that resist thermal unfolding even at 100 °C.^25,37,38

Figure 1.1 Binary patterning of helical heptad repeats drives hydrophobic self-association into a four-helix bundle. (a) seven amino acids completing two turns of an alpha-helix are labelled a through g. In four-helix bundle maquettes, amino acids at positions a, d and e are non-polar, and the other positions are polar. Helices one through four are connected by flexible loops. (b) Binary patterning leads to spontaneous bundle association forming long non-polar cores, as seen by a maquette crystal structure. Cofactors are anchored at desired positions in these cores. (c) As an example of negative design, this helical threading brings charged groups of similar sign together and is electrostatically disfavoured compared to (a) where nearby charges are of opposi...