Study of a Major Intermediate in the Oxidative Folding of Leech Carboxypeptidase Inhibitor: Contribution of the Fourth Disulfide Bond

Folding of small disulfide-rich proteins: clarifying the puzzle

“…Therefore, the productive precursor of native BPTI is the disulfide-secure des intermediate and the rate-limiting step of the folding process is either a reshuffling to form des or the escape from the dead-end metastable des and des species. In the oxidative folding of LCI, the des and des species, lacking the native disulfide bond that connects the α-helix to the β-sheet and stabilizes the β-sheet core, respectively, also seem to behave as metastable, disulfide-insecure intermediates with a high content of a native-like structure [46]. Lysozyme (129 residues; four disulfide bonds) is a more complex protein that comprises two folding domains, called the α-and β-domains.…”

Protease Inhibitors as Models for the Study of Oxidative Folding

“…The stability of the N 0 structure makes unfolding reactions energetically expensive so that the rate of this rearrangement is slow. In addition, the formation of N* and N SH-SH from N 0 is not a direct reaction but implies the creation of a transient population of two intermediates containing nonnative disulfide bonds, [5][6][7][8][9][10][11][12][13][14] and , which correspond to the productive nonnative intermediates in Creighton's scheme. Once N SH-SH is formed, as described above, the flexibility and accessibility of the free Cys14 and Cys34 thiols allow rapid oxidation to form the native protein.…”

“…In principle, the oxidation of the two free cysteines in any of these species would result in the formation of a native and functional LDTI. However, when the intermediates were trapped and isolated and their folding was re-initiated at neutral pH, it became clear that neither des nor des [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] can form directly native LDTI but rather reshuffle their disulfides to form des , which turns out to be the only productive intermediate in the folding pathway. The convergence of all 2S intermediates into des is due to both the relative conformational properties of these species and the different reactivity of their free cysteines.…”

“…AROLAS AND VENTURA [5][6][7][8][9][10][11][12][13][14], and , among which only the last one contains two native disulfide bonds; that is, it may be termed des . Although the formation of the Cys5-Cys55 disulfide bond in des would lead to native BPTI, this step finds a high energy barrier for its completion due to the highly folded structure of this intermediate.…”

Flexibility of the “rigid” classics or rugged bottom of the folding funnels of myoglobin, lysozyme, RNase A, chymotrypsin, cytochrome c, and carboxypeptidase A1

“…For each protein, structural alignments were conducted using the MultProt (http://bioinfo3d.cs.tau.ac.il/MultiProt/) 64 for pairs of structures characterized by the largest difference according to the PDBFlex analysis. Analyzed structures include PDB entries 105mA (blue ribbon) and 2eb8A (green ribbon) 65 for myoglobin, 1lkrB (blue ribbon) and 1a2yC (green ribbon) for lysozyme, 1f0vB (blue ribbon) 62 and 3fkzA (green ribbon) 66 for RNase A, 1oxgA (blue ribbon) 67 and 1ex3A (green ribbon) 68 for chymotrypsin, 1u75B (blue ribbon) 69 and 3nbsC (green ribbon) 63 for cytochrome c, and 2abzB (blue ribbon) 70 and 1hdqA (green ribbon) 71 for carboxypeptidase A1. Structures presented in this plot were generated using a molecular graphics program VMD.…”