Computation Design of Bioenergetic Membrane Proteins

Abstract

The de novo design of bioenergetic proteins provides insight into the underlying principles of protein folding and assembly, permits the study of electron transport in minimal biomimetic systems, and produces new components for synthetic biology. Key respiratory and photosynthetic complexes, and unrelated transmembrane cytochromes, share a four-helix bundle architecture at their core that positions two b-type hemes for transmembrane electron transport. This fundamental scaffold is an ideal basis for creating man-made bioenergetic membrane proteins. This thesis describes two main approaches to producing such an artificial protein through (i) the introduction of two b-type heme binding sites into an existing minimal membrane protein (REAMP), and (ii) the conversion of a water-soluble diheme four-helix bundle (4D2) to a membrane protein by re-designing the protein surface to be lipid-soluble. Approach (i) produced a protein capable of membrane-localised recombinant expression and rudimentary heme binding, though suffered from non-specific aggregation and minimal purification yields. Approach (ii) produced transmembrane 4D2s (TM4D2s) with considerably improved biocompatibility, stability, secondary structure content, purification yields and heme binding capability. The final design round, where helix-helix interactions mediated by knobs-into-holes (KIH) packing were preserved, was successful, and yielded CytbX, a highly-stable artificial integral membrane protein capable of efficient membrane insertion and heme binding in vivo. The embedded hemes of CytbX are accessible to small diffusive redox partners, enabling CytbX to take part in simple electron transfer reactions with both natural and designed proteins. To the best of our knowledge, CytbX represents the first example of a de novo membrane protein capable of full in vivo assembly with endogenous cofactors. The findings described in this thesis therefore have far-reaching implications for the design of novel biocompatible transmembrane cytochromes with broad applications in synthetic biology and bioengineering.

Date of Award	21 Mar 2023
Original language	English
Awarding Institution	University of Bristol
Supervisor	Paul Curnow (Supervisor) & J L R Anderson (Supervisor)