Cross-validation of distance measurements in motile proteins reveals certain inconsistencies

The researchers from PD Dr. Gregor Hagelueken's group at the Institute of Structural Biology of the UKB used so-called PELDOR spectroscopy to study the movement of so-called substrate-binding proteins. These proteins grab their substrate and transport it to a specific location in the cell. To observe this precisely, the researchers attached tiny magnets - the researchers call them "spin labels" - to the proteins and measured distances that are only about a billionth of a meter long.

They then transmitted their results to the research group of Prof. Dr. Thorben Cordes at LMU Munich. There, comparative measurements were carried out using so-called FRET spectroscopy. Tiny dye molecules were used instead of spin labels.

"Although both methods are used very frequently, no one has yet systematically investigated whether the results are really comparable," says Hagelueken.

Although it turned out that the measurement results were comparable in most cases, the researchers encountered inconsistencies in two cases.

We then thoroughly investigated what caused the differences and found what we were looking for. In one case, it turned out that the dye molecules stuck to the protein and thus falsified the measurement."

Martin Peter, Bonn post-doctoral researcher

In the second case, the addition of a type of antifreeze, which was necessary because of the low measurement temperature of below -220 degrees Celsius, led to unexpected deviations.

"We were able to show that despite the high accuracy of the methods, re-measuring with another nano ruler is always a good idea," Hagelueken says.

Source:
Journal reference:

Peter, M.F., et al. (2022) Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET. Nature Communications. doi.org/10.1038/s41467-022-31945-6.

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