Structural and functional importance of β- turn in proteins. Studies on proline-containing peptides.

We report here two sets of results on proline-containing linear peptides, one of which brings out the role of the β-turn conformation in the structure of nascent collagen while the other points to the functional importance of the β-turn in calcium-binding proteins. Based on the data on peptides containing the -Pro-Gly- sequence, we had proposed and experimentally verified that the β-turn conformation in these peptides is a structural requirement for the enzymic hydroxylation of the proline residues in the nascent (unhydroxylated) procollagen molecule. Our recent data, presented here, on the conformation of peptides containing both the -Pro-Gly- and -Gly-Pro- sequences reveal that while the β-turn in the substrate molecule is required at the catalytic site of prolyl hydroxylase, the polyproline-II structure is necessary for effective binding at the active site of the enzyme. Thus, peptides containing either the β-turn or the polyproline-II structure alone are found to act only as inhibitors while those with the polyproline-II followed by β-turn serve as substrates of the enzyme. In another study, we have synthesized the two linear peptides: Boc-Pro-D-Ala-Ala- NHCH3 and Boc-Pro-Gly-Ala-NHCH3 each of which adopts, in solution, a structure with two consecutive β-turns, as judged from circular dichroism, infrared and nuclear magnetic resonance data. Drastic spectral changes are seen in these peptides on binding to Ca2+. Both the peptides show a distinct specificity to Ca2+ over Mg2+, Na+ and Li+. A conformational change in the peptides occurs on Ca2+ binding which brings together the carbonyl groups to coordinate with the metal ion. These results imply a functional role for the β-turn in Ca2+ - binding proteins.

Comparison of the conformation and stability of the native dimeric, monomelic, tetrameric and the desensitized forms of the nucleotide pyrophosphatase from mung bean (Phaseolus aureus) seedlings.

A homogenous and crystalline form of nucleotide pyrophosphatase (EC 3.6.1.9) from Phaseolus aureus (mung bean) seedlings was used for the study of the regulation of enzyme activity by adenine nucleotides. The native dimeric form of the enzyme had a helical content of about 65% which was reduced to almost zero values by the addition of AMP. In addition to this change in the helical content, AMP converted the native dimer to a tetramer. Desensitization of AMP regulation, without an alteration of the molecular weight, was achieved either by reversible denaturation with 6 Μ urea or by passage through a column of Blue Sepharose but additionof phydroxymercuribenzoate desensitized the enzyme by dissociating the native dimer to a monomer. The changes in the quaternary structure and conformation of the enzyme consequent to AMP interaction or desensitization were monitored by measuring the helical content, EDTA inactivation and Zn2+ reactivation, stability towards heat denaturation, profiles of urea denaturation and susceptibility towards proteolytic digestion. Based on these results and our earlier work on this enzyme, we propose a model for the regulation of the mung bean nucleotide pyrophosphatase by association-dissociation and conformational changes. The model emphasizes that multiple mechanisms are operative in the desensitization of regulatory proteins.

Allosteric serine hydroxymethyltransferase from monkey liver: temperature induced conformational transitions.

The homogeneous serine hydroxymethyltransferase from monkey liver was optimally activate at 60°C and the Arrhenius plot for the enzyme was nonlinear with a break at 15°C. The monkey liver enzyme showed high thermal stability of 62°C, as monitored by circular dichroism at 222 nm, absorbance at 280 nm and enzyme activity. The enzyme exhibited a sharp co-operative thermal transition in the range of 50°-70° (Tm= 65°C), as monitored by circular dichroism. L-Serine protected the enzyme against both thermal inactivation and thermal disruption of the secondary structure. The homotropic interactions of tetrahydrofolate with the enzyme was abolished at high temperatures (at 70°C, the Hill coefficient value was 1.0). A plot of h values vs. assay temperature of tetrahydrofolate saturation experiments, showed the presence of an intermediate conformer with an h value of 1.7 in the temperature range of 45°-60°C. Inclusion of a heat denaturation step in the scheme employed for the purification of serine hydroxymethyltransferase resulted in the loss of cooperative interactions with tetrahydrofolate. The temperature effects on the serine hydroxylmethyltransferase, reported for the first time, lead to a better understanding of the heat induced alterations in conformation and activity for this oligomeric protein.