An NMR view of the unfolding process of rusticyanin: Structural elements that maintain the architecture of a β-barrel metalloprotein

摘要: The dynamics of the unfolded state in proteins and its comparison with that folded molecule can provide clues as to structural elements essential maintaining three-dimensional conformation a protein (Fersht Daggett 2002; Ferguson Fersht 2003; Vendruscolo et al. 2003). In turn, substantial thermodynamic information on folding/unfolding process also be obtained from these kinds studies (Dinner 2000; Gianni this respect, NMR is one most widely used thorough techniques. unique tool allows characterization mobility features each single amino acid (Dyson Wright Bruschweiler Jimenez 2003b; Schwalbe 2003). Metalloproteins, general, copper proteins, particular, are stabilized large degree by prosthetic group (i.e., metal ion). principle, stabilizing effect occurs independently role ion function macromolecule. Blue (BCPs) relatively small, soluble electron transfer (Malmstrom Leckner 1998; Gray Randall Vila Fernandez 2001). They all possess β-barrel structure arranged so-called Greek key topology, shown Figure 1 ▶. redox potential higher BCPs than aqueous solution, i.e., reduced form more stable oxidized species. highly rigid. fact, values generalized order parameter, S2, data, range 0.83 0.93 (Kalverda 1999; Thompson Bertini 2001a; 2003a). This rigidity due network hydrogen bonds extending between β-strands which turn necessary fulfill their (electron transport). active site particularly strongly bound sulfur atom cysteine ligand imidazol nitrogen atoms two histidine residues (HisN HisC, respectively, according proximity sequence N- C-terminal ends). an axial methionine, weakly metal, completes pseudotetrahedral coordination (Randall 2000) (see Fig. ▶). geometry, remains essentially unaltered upon oxidation or reduction ion, imposed scaffold. not preferred for copper(II) thus, found strained (entatic/rack mechanism) 1994). characteristic permits low re-organization energy, required efficient (Gray Crane Traditionally, has been related high 1964, concept was reviewed since BCP Az (Winkler 1997; Wittung-Stafshede Marks 2004). Recently, elegant theoretical study factorized determinants relative potentials blue sites (Li study, investigators conclude entatic/rack mechanism major BCPs. These findings lead us consider stability and, folding governing potentials) BCPs. Figure 1. (A) Stereo view image rusticyanin. Secondary indicated. (B) Topology Rc. located Rc (1cur file; Botuyan 1996 ... Another important issue address metalloproteins whether they take up prior folding. Binding (in whatever state) presumably too slow biologically relevant (Pozdnyakova Consequently, must bind state, fact known do (Leckner 1997a,b; Pozdnyakova However, cell toxic (O’Halloran Culotta 2000). As result, form. Thus, it demonstrate binding copper(I) protein. We have studied dynamic means NMR. among possesses highest family, 680 mV (Nunzi other words, ion. extremely at acidic pH (<2.5) (Cox Boxer 1978; Ingledew Cobley 1980). existence hydrophobic core could explain, least partially, singularities (Botuyan 1996; Walter Donaire Nevertheless, correlation properties well understood still matter debate (Olsson Li To date, played either. Hence, represents interesting case identify critical make especially mediators. In we describe first, titration apo, Cu(I), Cu(II)Rc guanidinium chloride (GdmCl); second, direct evidence both states) completely state; third, solvent exchange incipient early step unfolding process. former experiments help interpret scaffold way folds. latter secondary involved starting We recently described formation aggregated species GdmCl, using several biochemical spectroscopic methods (Alcaraz Here, aggregation again evidenced. present results allowed regions resistant responsible stability.