Combining different experiment and instrument approaches to reconstruct how a protein moves and function
Creators: name: Ng, Wenfa
Publisher: name: figshare
Year: 2016
Subjects: subject: Biochemistry
subject: 60199 Biochemistry and Cell Biology not elsewhere classified
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: 60112 Structural Biology (incl. Macromolecular Modelling)

description: Understanding the function of proteins, enzymes and biomolecules in general often relies on cellular assays where, for example, an introduced substrate is converted into a product, whose quantity can be measured. While knowing the substrate range and product distribution of an enzyme offers important insights, there remains a gigantic black box covering the how and why of enzyme function. To answer these questions, a field of biology known as structural biology uses sophisticated imaging tools such as X-ray diffraction, and increasingly, cryo electron microscopy to obtain snapshots of enzymes in action, such as binding and cleaving a substrate. The enduring difficulty of structural biology, however, remains that of obtaining diffraction quality crystals of target proteins (particularly in complex with specific ligands) able to yield atomic resolution (2-3 Angstrom) structures after mapping atomic position onto electron density data. But the enduring challenge in understanding structure function relationship of proteins is the paucity of structural insights given only single or a few snapshots are usually caught in high resolution, where the overarching theme is that of enzymes dancing in multiple steps choreograph executing a functional move. To this end, structural biologists have increasingly turn to long duration molecular dynamics simulation to elucidate possible structural movements in, for example, an enzyme binding and locking in to a substrate. Such knowledge are important to understanding the progressive series of changes to the binding cleft as well as its effect on conformations of other parts of the macromolecular complex such as a distant ATP hydrolysis pocket providing further energy to power downstream structural changes. Structural biology was traditionally viewed as a distinct branch of biochemistry interested in the biophysics of enzyme function, but advent of facile molecular biology tools for heterologous expression of different domains of a specific protein target in knockout animal models have given the field fresh tools to understand how interactions between different domains of a macromolecular complex provide the defining function, for example, of a sterol transporter. A relatively simple blood draw would allow the cholesterol levels in blood of the recombinant knockout animal to be analysed; thereby, answering questions, in confidence, on hypothesized reasons of domain-domain interactions. With the structure in hand and knowledge of its amino acids sequence, bioinformatics sequence analysis and homology modelling is another area where structural biology aims to retrospectively understand the evolutionary trajectory of an enzyme viewed today, while seeking directions where the protein could be tuned by directed evolution enabled protein engineering for gaining new functions in a slightly altered structural motif. Combining various molecular biology tools, animal functional assays, computational biology’s dynamic view of proteins together with X-ray diffraction structures of the complex in question, a recent study demonstrates the wealth of understanding that emanates from a holistic investigation of the structure and function of human sterol transporter domain G5 and G8 of the ATP binding cassette (ABC) transporter superfamily in a paper entitled “Crystal structure of the human sterol transporter ABCG5/ABCG8” Link.