Combining different approaches to recreate the movement and function of proteins Wenfa Ng figshare 04 Dec 2016.pdf (223.48 kB)
Combining different experiment and instrument approaches to reconstruct how a protein moves and function
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.