Protein Interactions Leading to Conformational Changes Monitored by Limited Proteolysis:  Apo Form and Fragments of Horse Cytochromec

Abstract

Proteolysis experiments have been used to monitor the conformational transitions from an unfolded to a folded state occurring when the apo form of horse cytochrome c (cyt c) binds the heme moiety or when two fragments of cyt c form a native-like 1:1 complex. Proteinase K was used as a proteolytic probe, in view of the fact that the broad substrate specificity of this protease allows digestion at many sites along a polypeptide chain. The rather unfolded apo form of cyt c binds heme with a concomitant conformational transition to a folded species characterized by an enhanced content of helical secondary structure. While the holoprotein is fully resistant to proteolytic digestion and the apoprotein is digested to small peptides, the noncovalent complex of the apoprotein and heme exhibits an intermediate resistance to proteolysis, in agreement with the fact that the more folded structure of the complex makes the protein substrate more resistant to proteolysis. The noncovalent native-like complex of the two fragments 1-56 and 57-104 of cyt c, covering the entire polypeptide chain of 104 residues of the protein, is rather resistant to proteolysis, while the individual fragments are easily digested. Fragment 57-104 is fast degraded to several peptides, while fragment 1-56 is slowly degraded stepwise from its C-terminal end, leading initially mostly to fragments 1-48 and 1-40 and, at later stages of proteolysis, fragments 1-38, 1-35, 1-33, and 1-31. Thus, proteolysis data indicate that the heme containing fragment 1-56 has a rather compact core and a C-terminal flexible tail. Upon prolonged incubation of the complex of fragments 1-56 and 57-104 (nicked cyt c) with proteinase K, a chain segment is removed from the nicked protein, leading to a gapped protein complex of fragments of 1-48 and 57-104 and, on further digestion, fragments 1-40 and 57-104. Of interest, the chain segment being removed by proteolysis of the complex matches the omega-loop which is evolutionarily removed in cyt c of microbial origin. Overall, rates and/or resistance to proteolysis correlates well with the extent of folding of the protein substrates, as deduced from circular dichroism measurements. Thus, our results underscore the utility of proteolytic probes for analyzing conformational and dynamic features of proteins. Finally, a specific interest of the cyt c fragment system herewith investigated resides in the fact that the fragments are exactly the exon products of the cyt c gene.

Interactions of Cytoplasmic Dynein Light Chains Tctex-1 and LC8 with the Intermediate Chain IC74

“…Resistance to proteolysis has recently been shown to correlate with the extent of folding of apocytochrome c upon binding to heme (36). Interaction with other molecules often increases the stability of proteins, so that the unfolding-folding equilibrium is shifted toward the folded conformation.…”

Protein Interactions Within the Cytoplasmic Dynein Complex as Probed by Mass Spectrometry and Nmr

“…Limited tryptic digestion and mass spectrometry [2] localized binding of Tctex-1 to the vicinity of K104 and K105, and localized binding of LC8 to the region downstream of K130. Circular dichroism, fluorescence, sedimentation velocity and proteolysis studies indicate that N-IC74 has limited secondary and tertiary structure at near physiological solution conditions.…”

Evaluation of risedronate as an antibiofilm agent

“…Although nuclear magnetic resonance and X-ray crystallography are commonly employed to determine proteinligand binding sites and protein conformation changes induced by complex formation, limited proteolysis coupled to mass spectrometry has also been demonstrated to provide important information about solution-phase protein structural changes by determining intermediates in protein folding studies (Leite et al, 2000) and mapping interfaces in protein-protein (Spolaore et al, 2001) and protein-DNA (Cohen et al, 1995) complexes.…”