1. Reversible two-step unfolding of heme–human serum albumin: a 1H-NMR relaxometric and circular dichroism study
- Author
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Paolo Ascenzi, Giampiero De Sanctis, Gabriella Fanali, Magda Gioia, Massimo Coletta, Mauro Fasano, Fanali, Gabriella, DE SANCTIS, Giampiero, Gioia, Magda, Coletta, Massimo, Ascenzi, Paolo, Fasano, Mauro, Fanali, G, DE SANCTIS, G, Gioia, M, Coletta, M, and Fasano, M.
- Subjects
Models, Molecular ,Guanidinium chloride ,absorption spectroscopy ,Protein Denaturation ,Protein Folding ,Circular dichroism ,Magnetic Resonance Spectroscopy ,Heme binding ,denaturation ,Protein Conformation ,Heme ,proton nuclear magnetic resonance ,Myristic Acid ,Biochemistry ,Inorganic Chemistry ,chemistry.chemical_compound ,Models ,Fatty acid binding ,medicine ,Humans ,Denaturation (biochemistry) ,human ,protein structure ,Settore BIO/10 ,relaxation time ,Guanidine ,Serum Albumin ,Binding Sites ,guanidine ,heme ,human serum albumin ,myristic acid ,saturated fatty acid ,article ,binding site ,circular dichroism ,priority journal ,protein conformation ,protein folding ,protein stability ,signal transduction ,Circular Dichroism ,Protons ,Chemistry ,Molecular ,Human serum albumin ,Folding (chemistry) ,Crystallography ,medicine.drug - Abstract
Human serum albumin (HSA) participates in heme scavenging, the bound heme turning out to be a reactivity center and a powerful spectroscopic probe. Here, the reversible unfolding of heme–HSA has been investigated by 1H-NMR relaxometry, circular dichroism, and absorption spectroscopy. In the presence of 6 equiv of myristate (thus fully saturating all available fatty acid binding sites in serum heme–albumin), 1.0 M guanidinium chloride induces some unfolding of heme–HSA, leading to the formation of a folding intermediate; this species is characterized by increased relaxivity and enhanced dichroism signal in the Soret region, suggesting a more compact heme pocket conformation. Heme binds to the folding intermediate with K d = (1.2 ± 0.1) × 10−6 M. In the absence of myristate, the conformation of the folding intermediate state is destabilized and heme binding is weakened [K d = (3.4 ± 0.1) × 10−5 M]. Further addition of guanidinium chloride (up to 5 M) brings about the usual denaturation process. In conclusion, myristate protects HSA from unfolding, stabilizing a folding intermediate state in equilibrium with the native and the fully unfolded protein, envisaging a two-step unfolding pathway for heme–HSA in the presence of myristate.
- Published
- 2008