A Ca2+-binding Chimera of Human Lysozyme and Bovine α-Lactalbumin That Can Form a Molten Globule

Els Pardon,Petra Haezebrouck,Annie De Baetselier,Shaun D Hooke,Katherine T Fancourt,Johan Desmet,Christopher M Dobson,Herman Van Dael,Marcel Joniau

doi:10.1074/jbc.270.18.10514

Abstract

In contrast to lysozymes, which undergo two-state thermal denaturation, the Ca(2+)-free form of the homologous alpha-lactalbumins forms an intermediate "molten globule" state. To understand this difference, we have produced a chimera of human lysozyme and bovine alpha-lactalbumin. In the synthetic gene of the former the sequence coding for amino acid residues 76-102 was replaced by that for bovine alpha-lactalbumin 72-97, which represents the Ca(2+)-binding loop and the central helix C. The chimeric protein, LYLA1, expressed in Saccharomyces cerevisiae was homogeneous on electrophoresis and mass spectrometry. Its Ca2+ binding constant was 2.50 (+/- 0.04) x 10(8) M-1, and its muramidase activity 10% of that of human lysozyme. One-dimensional NMR spectroscopy indicated the presence of a compact, well structured protein. From two-dimensional NMR spectra, main chain resonances for 118 of a total of 129 residues could be readily assigned. Nuclear Overhauser effect analysis and hydrogen-deuterium exchange measurements indicated the presence and persistence of all expected secondary structure elements. Thermal denaturation, measured by circular dichroism, showed a single transition temperature for the Ca2+ form at 90 degrees C, whereas unfolding of the apo form occurred at 73 degrees C in the near-UV and 81 degrees C in the far-UV range. These observations illustrate that by transplanting the central part of bovine alpha-lactalbumin, we have introduced into human lysozyme two important properties of alpha-lactalbumins, i.e. stabilization through Ca2+ binding and molten globule behavior.

Highlights

In contrast to lysozymes, which undergo two-state thermal denaturation, the Ca2 + -free form of the homologous a-Iactalbumins forms an intermediate "molten globule" state
In the present contribution we describe a chimera, LYLA1, obtained by transplanting the central segment of bovine a-lactalbumin, which comprises the Ca 2+ -binding loop and the main helix C, into the homologous position in human lysozyme
When the degree of structural resemblance between human lysozyme (HLY) and baboon a-lactalbumin is calculated using the spatial coordinates of the Brookhaven Protein Data Bank [35], a root mean square value of 1.167 A is obtained for the main chain atoms, indicating a very pronounced three-dimensional homology

Summary

Introduction

In contrast to lysozymes, which undergo two-state thermal denaturation, the Ca2 + -free form of the homologous a-Iactalbumins forms an intermediate "molten globule" state. To understand this difference, we have produced a chimera of human lysozyme and bovine a-lactalbumin. Thermal denaturation, measured by circular dichroism, showed a single transition temperature for the Ca2 + form at 90 °C, whereas unfolding of the apo form occurred at 73 °C in the near-UV and 81 °C in the far-UV range These observations illustrate that by transplanting the central part of bovine a-lactalbumin, we have introduced into human lysozyme two important properties of o-laetalbumins, i,e.

Objectives

Results

Conclusion