Enzymic Cleavage of Proteins

Keil, B.

doi:10.1007/978-1-4612-5832-2_24

Cited by 25 publications

(19 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4). Since thermolysin cleaves peptide bonds with poor specificity, even if with some preference at the N-terminus of hydrophobic and bulky amino acid residues (lie, Leu, Phe) [18,31], clearly the selective proteolysis herewith observed is dictated by the specific structural and dynamic features of the protein substrate dissolved in aqueous TFE. In previous studies, we have emphasized that the site of limited proteolysis in globular proteins are exposed and flexible loops and that, in particular, helices are not prone to proteolytic attack [13 17].…”

Section: Discussionmentioning

confidence: 89%

Limited proteolysis of cytochrome c in trifluoroethanol

et al. 1995

View full text Add to dashboard Cite

Horse heart cytochrome c is cleaved by thermolysin in 50% aqueous TFE (v/v) at neutral pH (25°C, 24 h) at the Gly56-Ile 57 peptide bond of the 104-residue chain of the protein.Additional, but anyway minor, fragmentation at the Gly4S-Phe 46 and MetS°-Ile s~ peptide bonds is also observed. On the other hand, in buffer only and in the absence of TFE, cytochrome c is digested by thermolysin to numerous small peptides. Considering the broad substrate specificity of the TFE-resistant thermolysin, clearly the conformational state of the protein substrate dictates the observed selective proteolysis. It is proposed that the highly helical secondary structure acquired by cytochrome c when dissolved in aqueous TFE hampers binding and adaptation of the protein substrate at the active site of the protease and that peptide bond fission occurs at flexible chain segments characterized by a low a-helix propensity. globular proteins are characterized by enhanced chain flexibility, since a correlation exists between sites of proteolysis and sites of high segmental mobility, these last determined crystallographically [13].Here we show that horse heart cytochrome c in the presence of aqueous TFE is cleaved by the TFE-resistant proteolytic enzyme thermolysin [18] to few fragments only, whereas in aqueous solution and in the absence of alcohol many small peptides are formed. The preferential proteolysis was interpreted on the basis of the conformational properties of cytochrome c dissolved in aqueous TFE, as deduced from circular dichroism (CD) and fluorescence emission measurements. Materials and methods

show abstract

Section: Discussionmentioning

confidence: 89%

Limited proteolysis of cytochrome c in trifluoroethanol

et al. 1995

View full text Add to dashboard Cite

show abstract

“…The peptide bond Asn 34-Leu 35 is the site of selective initial cleavage, followed by an additional, but slower, cut at Thr 45-Phe 46. This selective hydrolysis is quite striking, if one considers that thermolysin shows a broad substrate specificity with only some preferences for hydrophobic and bulky amino acid residues (Ile, Leu, Phe) (Heinrikson, 1977;Keil, 1982). Therefore, there are plenty of peptide bonds in the RNase polypeptide chain (see Fig.…”

Section: Limited Proteolysismentioning

confidence: 99%

“…For this study of limited proteolysis of a model protein in its TFE-state, the thermophilic enzyme thermolysin (Heinrikson, 1977;Holmes & Matthews, 1982) appeared to be a most suitable proteolytic probe, because of its noteworthy stability under relatively harsh solvent conditions, including aqueous organic solvents (Wayne & Fruton, 1983;Welinder, 1988;Kitaguchi & Klibanov, 1989) and broad substrate specificity (Heinrikson, 1977;Keil, 1982). Thus, it was anticipated that thermolysin would cleave the polypeptide chain of RNase in its TFE-state at sites characterized by the flexibility required for an efficient proteolysis and not by the specificity of the protease.…”

mentioning

confidence: 99%

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

et al. 1997

View full text Add to dashboard Cite

We have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE-resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124-residue chain of RNase in its TFE state (20-42"C, 6-24 h) at peptide bond Asn 34-Leu 35, followed by a slower cleavage at peptide bond Thr 45-Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1-34 and 35-124) (RNase Thl) or three (1-34, 35-45 and 46-124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Thl maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (-ATm 16 "C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Thl is closely similar to that of the intact protein was confirmed unambiguously by two-dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30-41, i.e., at residues nearby the cleaved Asn 34-Leu 35 peptide bond. RNase Thl retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 3 1-39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41-48 region forms a &strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Thl and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE-state that likely dictate the selective proteolysis phenomenon by thermolysin.Keywords: CD; limited proteolysis; NMR; ribonuclease A; thermolysin; trifluoroethanol Short-chain alcohols, such as methanol, ethanol, propanol, chloroethanol, and, in particular, trifluoroethanol, have been used frequently to induce a-helical conformations in otherwise randomly coiled or partially structured polypeptides (Toniolo et al., 1975;

show abstract

“…Thus, exposure and flexibility characteristics appear to dictate the exclusive peptidebond fission at Lys47 in the HM2 molecule. Nevertheless, the observed selective peptide-bond hydrolysis at Lys47 by trypsin can be dictated, in part, also by the substrate specificity of the protease (Walsh, 1972;Keil, 1982). This possibility should be considered, especially to explain the resistance to hydrolysis of the peptide bond Lys45-Pro46 (Keil, 1982).…”

Section: S Y T D C T E S G a N Y C L C V G S N V C G E G K N C Q L mentioning

confidence: 99%

Probing the Structure of Hirudin from Hirudinaria manillensis by Limited Proteolysis

Vindigni¹,

Filippis²,

Zanotti³

et al. 1994

European Journal of Biochemistry

View full text Add to dashboard Cite

Hirudin is the most potent and specific inhibitor of the blood-clotting enzyme thrombin so far known. Several hirudin variants were isolated mostly from Hirudo medicinalis and shown to be polypeptide chains of approximately 7 kDa with three internal disulfide bridges. In this study, limited proteolysis has been used to probe aspects of the structure and dynamics of a hirudin variant HM2 isolated from Hirudinaria manillensis. Proteolysis of the polypeptide chain of 64-amino-acid residues of hirudin HM2 by protease from Staphylococcus aureus V8, trypsin, thermolysin and subtilisin occurs at region 41 -49 of the chain. The N-terminal fragments 1-41 and 1-47 were isolated to homogeneity and shown to maintain inhibitory action on thrombin, though much lower than the intact protein. The results were interpreted on the basis of a proposed three-dimensional structure of hirudin HM2 deduced by protein modelling the known structure of hirudin variant HV1 from Hirudo medicinalis (75% sequence similarity between HM2 and HV1). Both proteolysis experiments and protein modelling provide evidence for the existence in hirudin HM2 of a Nterminal well-structured domain (core) and a C-terminal flexible polypeptide segment. Determination of the accessible surface area of the three-dimensional model of hirudin HM2 showed that the sites of preferential cleavages are at the surface of the polypeptide molecule.

show abstract

Enzymic Cleavage of Proteins

Cited by 25 publications

References 57 publications

Limited proteolysis of cytochrome c in trifluoroethanol

Limited proteolysis of cytochrome c in trifluoroethanol

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

Probing the Structure of Hirudin from Hirudinaria manillensis by Limited Proteolysis

Contact Info

Product

Resources

About