Effect of N-terminal Residues on the Structural Stability of Recombinant Horse L-chain Apoferritin in an Acidic Environment

Yoshizawa, Keiko; Mishima, Yumiko; Park, Sam-Yong; Heddle, Jonathan G.; Tame, J.R.H.; Iwahori, Keisuke; Kobayashi, Mime; Yamashita, Ichiro

doi:10.1093/jb/mvm187

Cited by 29 publications

(35 citation statements)

References 32 publications

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“…The apoferritin structures reported here are essentially identical to the previously reported structures of HSAF and recombinant horse L apoferritin (27)(28)(29)(30). The protomer forms a bundle of four long helices; a fifth short helix at the molecular C terminus caps the bundle at one end.…”

Section: Resultssupporting

confidence: 82%

A Unitary Anesthetic Binding Site at High Resolution

Vedula¹,

Brannigan

Economou

et al. 2009

Journal of Biological Chemistry

156

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Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABA A receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA A receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA A receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.Most general anesthetics alter the activity of ligand-gated ion channels, and electrophysiology, photolabeling, and transgenic animal experiments imply that this effect contributes to the mechanism of anesthesia (1-9). Although the molecular mechanism for this effect is not yet clear, photolabeling studies indicate that anesthetics bind within the transmembrane regions of Cys-loop ligand-gated ion channels such as the nicotinic acetylcholine and the ␥-aminobutyric acid (GABA) 2 type A receptors (2, 9 -11). Practical difficulties associated with overexpression, purification, and crystallization of ion channels have thus far stymied investigation of the structural and energetic bases underlying anesthetic recognition. However, general anesthetics also bind specifically to sites in soluble proteins, including firefly luciferase, human serum albumin (HSA), and horse spleen apoferritin (HSAF) (12)(13)(14), and x-ray crystal structures have been determined for complexes of these proteins with several general anesthetics (14 -16). In particular, HSAF is an attractive model for studying anesthetic-protein interactions because it has the highest affinity for anesthetics of any protein studied to date, has a unique anesthetic binding site, and is a multimer of 4-helix bundles, much like the putative anesthetic binding regions in ligand-gated channels. In addition, apoferritin is commercially available and crystallizes readily. Most importantly, however, the affinity of HSAF f...

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Section: Resultssupporting

confidence: 82%

A Unitary Anesthetic Binding Site at High Resolution

Vedula¹,

Brannigan

Economou

et al. 2009

Journal of Biological Chemistry

156

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“…23 Like many cage proteins, ferritins spontaneously assemble and are stable, requiring relatively harsh conditions for disassembly. 24,25 For capturing or releasing active proteins which may oen be quite fragile, this represents a challenge. Some archaeal ferritins offer a potential solution as they show reversible assembly in response to relatively mild changes in salt concentration (salt-dependency).…”

Section: Ferritinmentioning

confidence: 99%

Enzyme encapsulation by protein cages

et al. 2020

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“…Finally, ice-nucleating proteins expressed by Pseudomonas exhibit a repetition unit containing threonine amino acids with hydroxyl functional groups that are able to template ice. Aggregates involving only a few of these proteins are water soluble and induce ice nucleation up to −7 • C. Larger aggregates nucleate ice up to −2 • C but require the intact outer cell membrane to be stable (Polen et al, 2016;Zachariassen and Kristiansen, 2000).…”

Section: Introductionmentioning

confidence: 99%

Protein aggregates nucleate ice: the example of apoferritin

et al. 2020

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Abstract. Biological material has gained increasing attention recently as a source of ice-nucleating particles that may account for cloud glaciation at moderate supercooling. While the ice-nucleation (IN) ability of some bacteria can be related to membrane-bound proteins with epitaxial fit to ice, little is known about the IN-active entities present in biological material in general. To elucidate the potential of proteins and viruses to contribute to the IN activity of biological material, we performed bulk freezing experiments with the newly developed drop freezing assay DRoplet Ice Nuclei Counter Zurich (DRINCZ), which allows the simultaneous cooling of 96 sample aliquots in a chilled ethanol bath. We performed a screening of common proteins, namely the iron storage protein ferritin and its iron-free counterpart apoferritin, the milk protein casein, the egg protein ovalbumin, two hydrophobins, and a yeast ice-binding protein, all of which revealed IN activity with active site densities > 0.1 mg−1 at −10 ∘C. The tobacco mosaic virus, a plant virus based on helically assembled proteins, also proved to be IN active with active site densities increasing from 100 mg−1 at −14 ∘C to 10 000 mg−1 at −20 ∘C. Among the screened proteins, the IN activity of horse spleen ferritin and apoferritin, which form cages of 24 co-assembled protein subunits, proved to be outstanding with active site densities > 10 mg−1 at −5 ∘C. Investigation of the pH dependence and heat resistance of the apoferritin sample confirmed the proteinaceous nature of its IN-active entities but excluded the correctly folded cage monomer as the IN-active species. A dilution series of apoferritin in water revealed two distinct freezing ranges, an upper one from −4 to −11 ∘C and a lower one from −11 to −21 ∘C. Dynamic light scattering measurements related the upper freezing range to ice-nucleating sites residing on aggregates and the lower freezing range to sites located on misfolded cage monomers or oligomers. The sites proved to persist during several freeze–thaw cycles performed with the same sample aliquots. Based on these results, IN activity seems to be a common feature of diverse proteins, irrespective of their function, but arising only rarely, most probably through defective folding or aggregation to structures that are IN active.

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Effect of N-terminal Residues on the Structural Stability of Recombinant Horse L-chain Apoferritin in an Acidic Environment

Cited by 29 publications

References 32 publications

A Unitary Anesthetic Binding Site at High Resolution

A Unitary Anesthetic Binding Site at High Resolution

Enzyme encapsulation by protein cages

Protein aggregates nucleate ice: the example of apoferritin

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