Equilibrium unfolding experiments provide access to protein thermodynamic stability revealing basic aspects of protein structure-function relationships. A limitation of these experiments stands on the availability of large amounts of protein samples. Here we present the use of the NanoDrop for monitoring guanidinium chloride-induced unfolding by Soret absorbance of monomeric heme proteins. Unfolding experiments using 2 μl of reactant are validated by fluorescence and circular dichroism spectroscopy and supported with five heme proteins including neuroglobin, cytochrome b5, and cyanoglobin. This work guarantees 2 orders of magnitude reduction in protein expense. Promising low-cost protein unfolding experiments following other chromophores and high-throughput screenings are discussed.
Low-cost equilibrium unfolding of heme proteins using 2 μl samples / E., Guca; C., Roumestand; Vallone, Beatrice; C. A., Royer; M., Dellarole. - In: ANALYTICAL BIOCHEMISTRY. - ISSN 0003-2697. - STAMPA. - 443:1(2013), pp. 13-15. [10.1016/j.ab.2013.08.006]
Low-cost equilibrium unfolding of heme proteins using 2 μl samples
VALLONE, Beatrice;
2013
Abstract
Equilibrium unfolding experiments provide access to protein thermodynamic stability revealing basic aspects of protein structure-function relationships. A limitation of these experiments stands on the availability of large amounts of protein samples. Here we present the use of the NanoDrop for monitoring guanidinium chloride-induced unfolding by Soret absorbance of monomeric heme proteins. Unfolding experiments using 2 μl of reactant are validated by fluorescence and circular dichroism spectroscopy and supported with five heme proteins including neuroglobin, cytochrome b5, and cyanoglobin. This work guarantees 2 orders of magnitude reduction in protein expense. Promising low-cost protein unfolding experiments following other chromophores and high-throughput screenings are discussed.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.