Spectroscopic investigation of the structure and function of the copper ATPase CopB of Enterococcus hirae


Spectroscopic investigation of the structure and function of the copper ATPase CopB of Enterococcus hirae

Groß, M.; Solioz, M.; Fahmy, K.

The Cu+-ATPase CopB of Enterococcus hirae is a bacterial P-type ATPase involved in resistance to high levels of environmental copper by expelling excess copper. The membrane protein CopB was purified from an over-expressing strain and solubilized in dodecyl-maltoside. By UV circular dichroism the secondary structure is predicted to contain 40-50 % alpha-helices and 10-15% beta-sheets in agreement with estimates based on homology with the Ca ATPase SERCA1. We present CD-spectroscopic data on thermal unfolding of the protein to address the influence of the binding of the ATP analogs ATPgS and the fluorescent analog mant-ATP on the protein stability. Such analogs are used to mimic functional states of the ATPase but undergo different interactions with the binding site that are not well characterized. We propose a competition-based assay for nucleotide binding using CD-spectroscopy to deduce the occupancy of the nucleotide-binding site by non-fluorescent nucleotides. Alternatively, the change of intrinsic fluorescence of mant-ATP upon binding to the ATPase is exploited in these assays. Finally, we show how the simultaneous measurement of protein CD and nucleotide fluorescence in thermal denaturation experiments may help to determine the stability of several functional conformational states of CopB.

Keywords: circular dichroism; nucleotide binding

  • Contribution to proceedings
    8th European Biophysics Congress, 23.-27.08.2011, Budapest, Hungary
    European Biophysics Journal with Biophysics Letters 40 (2011), 168

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Publ.-Id: 15699