Journal
ANALYTICAL CHEMISTRY
Volume 92, Issue 3, Pages 2620-2627Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.9b04490
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Funding
- Young Investigator Grant from the Swedish Research Council [621-2011-4395]
- Olle Engkvist Byggmastare Foundation [2012/428]
- Royal Swedish Academy of Sciences [FOAl2 V-111]
- AForsk Foundation [11-360]
- Foundation of Helge Ax:son Johnson
- Foundation of Wilhelm and Martina Lundgren [2016-1403]
- European Union [726396]
- European Commission [FP7-PEOPLE-2013-ITN-607793]
- Swedish Research Council [2014-5908]
- European Research Council (ERC) [726396] Funding Source: European Research Council (ERC)
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Changes in the tertiary conformation of adsorbed biomolecules can induce detectable shifts (Delta theta(r)) in the surface plasmon resonance (SPR) angle. Here it is shown how to calculate the corresponding shifts in the adsorbate's center of mass (Delta z(avg)) along the sensing surface normal from the measured Delta theta(r). The novel developed model was used for determining the mean distance between the cytochrome (CYT) and flavodehydrogenase (DH) domains of the enzyme cellobiose dehydrogenase (CDH) isolated from the fungi Neurospora crassa, Corynascus thermophilus, and Myriococcum thermophilum as a function of pH, [Ca2+], and substrate concentration. SPR confirmed the results from earlier electrochemical and SAXS studies stating that the closed conformation, where the two domains are in close vicinity, is stabilized by a lower pH and an increased [Ca2+]. Interestingly, an increasing substrate concentration in the absence of any electron acceptors stabilizes the open conformation as the electrostatic repulsion due to the reaped electrons pushes the DH and CYT domains apart. The accuracy of distance determination was limited mostly by the random fluctuations between replicate measurements, and it was possible to detect movements <1 nm of the domains with respect to each other. The results agreed with calculations using already established models treating conformational changes as contraction or expansion of the thickness of the adsorbate layer (t(protein)). Although the models yielded equivalent results, in this case, the Delta z(avg)-based method also works in situations, where the adsorbate's mass is not evenly distributed within the layer.
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