Reversible dioxygen binding in solvent-free liquid myoglobin

Abstract

The ensemble of forces that stabilize protein structure and facilitate biological function are intimately linked with the ubiquitous aqueous environment of living systems. As a consequence, biomolecular activity is highly sensitive to the interplay of solvent-protein interactions, and deviation from the native conditions, for example by exposure to increased thermal energy or severe dehydration, results in denaturation and subsequent loss of function. Although certain enzymes can be extracted into non-aqueous solvents without significant loss of activity, there are no known examples of solvent-less (molten) liquids of functional metalloproteins. Here we describe the synthesis and properties of room-temperature solvent-free myoglobin liquids with near-native structure and reversible dioxygen binding ability equivalent to the haem protein under physiological conditions. The realization of room-temperature solvent-free myoglobin liquids with retained function presents novel challenges to existing theories on the role of solvent molecules in structural biology, and should offer new opportunities in protein-based nanoscience and bionanotechnology.

Heme Binding in Gas-Phase Holo-Myoglobin Cations: Distal Becomes Proximal?

“…However, recently it has been shown that gradual removal of water from myoglobin solution and replacing it with polymer surfactants largely preserves the native fold of hMb and its ligand-binding function [6]. This result gave a reason to rethink the role of water in myoglobin activity, and reinstated interest in studying details of the hMb structure in water-free environment.…”

“…To be fair, the surfactant replacing technique left approximately four H 2 O molecules bound on the surface of each Mb molecule, including the water molecule in the distal heme pocket [6]. In contrast to water-surfactant replacement, electrospray ionization (ESI) produces completely water-free gas-phase hMb ions even without extensive heating.…”

Enzyme activity in liquid lipase melts as a step towards solvent-free biology at 150 °C

“…Significantly, the room-temperature myoglobin biofluids contained significant levels of dynamic secondary and tertiary structure 30 , and remarkably, bound gaseous ligands such as carbon monoxide, sulphur dioxide, and oxygen reversibly in the complete absence of a solvent 31 . Although the persistent structure, function and dynamics exhibited by the solvent-free liquid myoglobin were remarkable, reversible gaseous oxygen binding could be considered a relatively primitive and robust biological process, which would occur provided that the haem prosthetic group was still present in the protein.…”

Strategies for cell membrane functionalization

“…Conformational rearrangement of the amphiphilic polymer surfactant promotes protein stability and aqueous solubility (due to the hydrophilic PEG segment), 97,98 and mediates membrane tethering for around one week in culture (via the hydrophobic tail). 13 Myoglobin conjugates retained their oxygen-binding capacity 99 and were delivered to hMSCs to provide an in situ oxygen reservoir to enhance the production of matrix fibres at the Armstrong and Perriman Strategies for cell membrane functionalization 1101 centre of engineered cartilage constructs. 13 Importantly, both cell priming and protein lipidation necessitate careful modification of the protein surface, as aggressive bioconjugation strategies can lead to denaturation and subsequent loss of biological function.…”