Metalloprotein Analysis
Ion Beam Analysis of Metals in Proteins
Geoffrey W. Grime
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Geoffrey W. Grime et al. Journal of the American Chemical Society 2020 142 (1), 185-197. DOI: 10.1021/jacs.9b09186
Why measure metals in proteins?
☐ Proteins are basic components of biochemical processes including cell function, tissue building, disease processes and metal transport (e.g. haemoglobin).
☐ Proteins are linear chains of amino acids folded into complex 3D shapes. Shape determines function.
☐ Many proteins contain small numbers of metal atoms. X-ray crystallography is routinely used to determine the position of each non-hydrogen atom in a protein, including metals. However, this relies on fitting the electron density, which does not allow the unambiguous identification of the metal.
☐ Micro particle induced X-ray emission (µPIXE) can identify and quantify unknown metal atoms in proteins.
The scale of the problem
☐ Over one third of all proteins with known 3-D structure are identified as containing metal atoms or binding sites.
☐ Elements with round white symbols have been identified in a protein structure.
Our work indicates that up to 30% of metal assignments may be incorrect, possibly leading to erroneous biological conclusions being drawn from the structures.
Analysis of metals in proteins using PIXE
☐ Absolute measurement of metal concentration is not easy
☐ Metal concentrations are low
☐ Proteins must be dissolved in buffer solutions so amount of protein may not be well known
The scale of the problem
Elspeth’s insight in 1993:
Most proteins have known numbers of the amino acids methionine and cysteine in their sequence with each contain one sulphur atom. Use the known number of S atoms as an internal standard!
c is measured concentration
A is atomic weight
N is the number of detected metal atoms of interest per molecule
Z, S refer to the metal atom of interest and sulphur
☐ Using ratios eliminates many sources of error affecting absolute measurement.
☐ Other elements may be used as the reference (e.g. P in nucleic acids, Se following seleno-methionine substitution.)
Methionine molecule
Human Insulin: The importance of Zinc (Zn)
Diabetics who inject insulin may suffer from amyloidosis, an inflammation at the injection site.
- Caused by insulin aggregating to form fibrils and spherulites that compromise its action.
- PIXE showed that zinc is expelled on aggregation of insulin.
- PIXE was crucial for the study and this finding is helping develop treatments for amyloidosis.
Malaria vaccines: do our antibodies help each other?
Need to understand mechanism of synergy of antibodies against malaria to improve vaccine design. Calcium appears vital for maximum response.
☐ CyRPA is a protein of the malaria parasite.
☐ A panel of antibodies that bind to CyRPA and strongly inhibit parasite growth in vitro (Cy.00x) were tested and their 3-D structures found.
☐ PIXE used to identify and quantify metals and confirmed that calcium was vital to the binding.
The presence of iron in PhoX raises the possibility that microbial phosphate acquisition is limited by iron bio-availability.
P. fluorescens PhoX alkaline phosphatase
Uptake of phosphate by micro-organisms is mediated by phosphatases such as PhoX
- PhoX contains an active-site cofactor with 5 metal binding sites
PIXE indicated 3 Ca ions and 2 Fe ions.
No other candidate metals (Mg, Mn, Co, Ni, Zn) were detected.
The presence of iron in PhoX raises the possibility that microbial phosphate acquisition is limited by iron bio-availability.
