title: FRET between a donor and an acceptor covalently bound to human serum albumin in native and non-native states.
abstract: Fluctuation in the inter-domain distance of a protein, human serum albumin (HSA), in the native, molten globule and denatured states is studied by Förster resonance energy transfer (FRET). For this purpose, a donor (CPM) and an acceptor (Alexa Fluor 488) are covalently attached to HSA. Unfolding of the protein is induced by pH changes as well as by the addition of 6 M GdnHCl and addition of 1.5 M of a room temperature ionic liquid (RTIL, [pmim][Br]). The efficiency of FRET (εFRET) and hence donor (D) - acceptor (A) distances of protein molecules in the native and non-native states are determined using FRET. In the native state (N), there is only one value of εFRET and D-A distance. In the non-native states (molten globule and unfolded) there are multiple values of εFRET and D-A distances. This suggests the presence of multiple conformers in equilibrium in the non-native states. When the protein is unfolded (on addition of GdnHCl or RTIL), separation between the two domains (I and II) increases and as a result εFRET decreases. In the presence of both GdnHCl and RTIL, the protein undergoes compaction (to form N'). However, in spite of the decrease in the overall radius, the D-A distance in the compact state (N') is found to be larger than that in the native state (N) of the protein. In contrast, two acid induced molten globule states of HSA (formed at pH 2 and 4) exhibit high εFRET indicating short D-A distances. In summary, we show that under chemical denaturation HSA undergoes stepwise unfolding and different domains unfold independently.
authors.affiliation: Department of Physical Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700032, India. pckb@iacs.res.in.
authors.forename: Rajdeep
authors.initials: R
authors.lastname: Chowdhury
authors.forename: Shyamtanu
authors.initials: S
authors.lastname: Chattoraj
authors.forename: Supratik Sen
authors.initials: SS
authors.lastname: Mojumdar
authors.forename: Kankan
authors.initials: K
authors.lastname: Bhattacharyya
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D052578:Ionic Liquids / Q000737:chemistry; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature
chemical_list: D052578:Ionic Liquids; D012709:Serum Albumin; D019791:Guanidine