Molecular mechanisms of the anomalous thermal aggregation of green fluorescent protein

Multi-State Proteins: Approach Allowing Experimental Determination of the Formation Order of Structure Elements in the Green Fluorescent Protein

“…The presence of the first stage, when the unfolding of the polypeptide chain of GFP - cycle3 does not occur but the mutual packing of the secondary structure elements is destroyed, is supported by NMR studies [22]. It was demonstrated that because of the presence of water molecules within the GFP-can at an early stage of melting of this protein its structure is “fractured” and this “fracture” differs from the usual unfolding of the polypeptide chain.…”

“…After studying literature data and conducting preliminary experiments [6], [21], [22], we understood that because of the strong aggregation of GFP - cycle3 upon folding it was possible to measure reliably only constants of unfolding rates of this protein. At the same time even if we succeed to get constants of unfolding rates at all stages of GFP - cycle3 unfolding with optical methods, this would not allow understanding the sequence of unfolding of different states of this protein.…”

“…Naturally it is not quite clear what conformation GFP - cycle3 acquires consequently. The experimental results show only that the changes occur in the total protein without unfolded regions (explanations are in [22]). The fact that beta-strands 1–3 (shown in red color in Fig.…”

Beta-Barrel Scaffold of Fluorescent Proteins

“…Virtually all recent work on the processes of FP unfolding–refolding has been performed with the cycle 3 (Enoki et al, 2006, 2004; Fukuda et al, 2000; Huang et al, 2007, 2008; Melnik et al, 2011a) or sfGFP (Andrews et al, 2009, 2007; Stepanenko et al, 2012b) proteins being subjected to different denaturing effects, including the chemical denaturants GdnHCl (Andrews et al, 2007; Fukuda et al, 2000; Huang et al, 2007) and GTC (Stepanenko et al, 2012b), changes in the pH of the solution (Enoki et al, 2006, 2004), and changes in the ionic strength (Hsu et al, 2010). …”

“…Since FPs have a unique chromophore and a single tryptophan residue, Trp57, the fluorescence characteristics of which are sensitive to the structure of the protein, and nonradiative energy transfer from Trp to the chromophore in the neutral state exists in the native state of the protein, fluorescent methods have been used in practically all papers to monitor changes in the structure of the protein under different treatment conditions (Andrews et al, 2007; Enoki et al, 2006, 2004; Fukuda et al, 2000; Huang et al, 2008; Melnik et al, 2011a; Orte et al, 2008; Stepanenko et al, 2012b). In addition, to characterize the processes of unfolding-refolding, CD (Enoki et al, 2004; Huang et al, 2007), SAX (Enoki et al, 2006), single-molecule fluorescence (Orte et al, 2008), and single-molecule mechanical unfolding (Bornschlogl and Rief, 2011; Dietz et al, 2006; Dietz and Rief, 2004; Mickler et al, 2007) as well as theoretical approaches (Andrews et al, 2008; Reddy et al, 2012) were used.…”