Conformational studies of alpha-globin in 1-propanol: propensity of the alcohol to limit the sites of proteolytic cleavage.

Abstract

Selective condensation of the unprotected fragments of a-globin-namely, a1..30 and a31.141-is catalyzed by Staphylococcus aureus V8 protease in the presence of 25% 1-propanol. The propensity of 1-propanol to induce the ahelical conformation and to generate a "native-like" topology for the polypeptide chain has been now investigated in an attempt to understand the molecular basis of this enzymecatalyzed stereospecific condensation. Removal of heme from the a-chain decreases the overall a-helical conformation of the protein considerably. A significant amount of the a-helical conformation is restored in the presence of25% 1-propanol and the digestion of a-globin by V8 protease becomes more selective concomitant with the increase in helicity. V8 protease digestion of a-globin at pH 6.0 and 40C occurs at Glu-30, Asp47, Glu-27, and Glu-23 in the absence of 1-propanol. In the presence of 25% 1-propanol, the digestion is selective to the peptide bond of Glu-30. This selectivity appears to be a characteristic feature of the native conformation of a-chain (polypeptide chain with bound heme). 1-Propanol induces the a-helical conformation into RNase S. peptide also. However, this increased helical conformation did not protect the RNase S peptide from V8 protease digestion at the Glu-9-Arg-10 peptide bond. RNase S peptide is in an a-helical conformation in RNase S, an interacting fragment-complementing system of S protein and S peptide. S peptide is resistant to V8 protease hydrolysis in this conformation. Thus, the resistance of a peptide bond in a segment of a protein to protease digestion appears to be a consequence of the secondary structure as well as the tertiary interactions of this segment with the rest of the molecule. The results suggest that the 1-propanol induces a-helical conformation into segments of a-globin as well as packing of these helices in a native-like topology.There has been considerable interest in recent years in the structure of proteins in organic solvents (1). The lowtemperature studies of enzymes, cryoenzymology, as well as low-temperature crystallography have necessitated the use of organic solvents in these protein structural studies (2). In addition, many enzyme-catalyzed reactions are also being investigated in organic media to enhance the industrial applications, specifically to take advantage of the high selectivity and specificity of the enzyme (3). With proteases, it has been possible to achieve "reverse proteolysis"-i.e., use proteases to catalyze the synthesis of peptide bonds by incorporating large amounts of organic solvents (i.e., like ethylene glycol, glycerol, etc.) in the reaction mixtures (4). The reformation of ribonuclease A from RNase S (5) and nuclease A from nuclease T (6) are the classical examples of this reverse proteolysis approach for semisynthesis of proteins (7).The protease-catalyzed reverse proteolysis for the semisynthesis of noncovalent analogues of proteins has been emerging as a useful procedure to prepare covalent variants of proteins (7). The presence...

Improvement of Staphylococcus aureus‐V8‐protease hydrolysis of bovine haemoglobin by its adsorption on to a solid phase in the presence of SDS: peptide mapping and obtention of two haemopoietic peptides

“…The location of the cleavage sites and of the peptides isolated from haemoglobin hydrolysate with V8 protease is shown in Figure 2. All the peptide bonds hydrolysed in human haemoglobin and reported in the literature [19,20] were observed. Moreover, 17 additional cleavages never reported before were observed in the presence of SDS: three on the α chain and 14 on the β chain.…”

“…Bovine haemoglobin hydrolyses by V8 protease were performed at pH 6 because, at this pH, human haemoglobin, which presents great sequence homology with bovine haemoglobin, could be cleaved at both Glu and Asp sites, even if the hydrolysis was incomplete [20]. As shown in Figure 1(a), the hydrolysis of native haemoglobin in solution proceeded very slowly and was incomplete.…”

“…In the native form, none of the Glu or Asp peptide bonds of the α and β chains is accessible to digestion by V8 protease. Partial hydrolyses of human haemoglobin were often observed under denaturing conditions [19] from isolated chains [20] or from tryptic fragments [21,22]. Generally, Asp bonds remained refractive to cleavage.…”

Limited Proteolysis of Lysozyme in Trifluoroethanol

“…acetonitrile, methanol, 2-propanol) (Welinder, 1988), and other protein denaturing agents (SDS, urea, guanidinium chloride) (Rivibre et al, 1991), has been studied previously with the aim of developing usefuI techniques for PAGE peptide mapping (Cleveland et al, 1977) and for producing protein fragments suitable for sequence analysis. Both organic solvents (Welinder, 1988;Houen and Sando, 1991;Iyer and Acharya, 1987;Acharya et al, 1992) and SDS (Cleveland et al, 1977) appear to control and restrict the degree of proteolysis of a protein substrate. However, the reduced catalytic activity of thermolysin in the presence of 50% (by vol.)…”

Product‐conformation‐driven ligation of peptides by V8 protease

“…Although the selective coupling of unprotected peptides through reverse proteolysis is one of the early protein semisynthetic reactions documented, delineation of molecular aspects that facilitate the reverse reactions of a protease continues to be a subject of considerable interest (Homandberg and Laskowski 1979; Wallace 1995; Nacharaju and Acharya 1999). Ligation of the discontinuity site of the fragment‐complementing systems catalyzed by protease in the presence of organic cosolvent is the best studied example of this class of protein semisynthetic reaction (Homandberg and Laskowski 1979; Komoriya et al 1980; Graf and Li 1981; Jullierat and Homandberg 1981; Homandberg et al 1982; Iyer and Acharya 1987; Kullman 1987; De Filippis et al 1990; Chang et al 1994; Davey et al 1995; Wallace 1995; Woods et al 1996; Cerovsky et al 1997; Nacharaju and Acharya 1999). In these systems, noncovalent interactions of two or more segments of a protein maintain a native‐like folding despite the discontinuity in the polypeptide chain and provide the proximity to the ligating ends of the peptides, which is crucial for the protease‐catalyzed splicing reaction.…”