title: Low-cost equilibrium unfolding of heme proteins using 2 μl samples.
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.
authors: Centre de Biochimie Structurale, INSERM U1054, CNRS UMR 5048, 34090 Montpellier, France.
authors: Ewelina
authors: E
authors: Guca
authors: Christian
authors: C
authors: Roumestand
authors: Beatrice
authors: B
authors: Vallone
authors: Catherine A
authors: CA
authors: Royer
authors: Mariano
authors: M
authors: Dellarole
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002942:Circular Dichroism / Q000191:economics; D015786:Cytochromes b5 / Q000737:chemistry*; D005914:Globins / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D007700:Kinetics; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D000078787:Neuroglobin; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence / Q000191:economics; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D054793:Truncated Hemoglobins / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; C102539:GlbN protein, bacteria; D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D054793:Truncated Hemoglobins; D006418:Heme; D005914:Globins; D015786:Cytochromes b5; D019791:Guanidine
keywords: Chemical denaturation; Heme proteins; NanoDrop; Protein stability