Hemagglutinin (HA) is a major surface membrane glycoprotein responsible for the binding of influenza virus to sialic acid-containing receptors in target cells. Fusogenic activity is triggered by a pH-dependent conformational change of HA in the acidic milieu of the endosomes. HA is a trimeric glycoprotein (ca. 220,000 MW) comprising an ectodomain of identical subunits, each of which contains two polypeptides (HA1 and HA2) linked by a disulfide bond. The conformational and thermal stability of HA purified from influenza strain X31 has been investigated by differential scanning calorimetry (DSC), circular dichroism (CD), fluorescence, ultracentrifugation, and electron microscopy. HA has been found to have a rosette structure with 5-6 trimers/rosette (31-35 S) at pH 7.4 to 5.4 in a mixed buffer containing 50 mM phosphate-50 mM acetate with 100 mM NaCl and 1 mM EDTA. Below pH 5.4, HA preparations are heterogeneous and unstable, and intact influenza virus has been found also to be rapidly inactivated at below pH 5.4 by Blumenthal et al. Analyses of DSC profiles of HA at pH 7.4 in the absence and presence of 1% octylglucoside show three domains with Tm = 66 and 65 C, respectively, and an overall unfolding enthalpy of 900 kcal/mol of trimer even though HA is dissociated to trimers (9.4 S) in the presence of the detergent. In both cases, the cooperative ratio CR (calorimetric to van't Hoff enthalpy) is 3.0, suggesting that HA subunits unfold independently in the trimer. Moreover, intermolecular interactions between trimers in the rosette structure appears to contribute little to the thermal unfolding parameters. As the pH is decreased from pH 7.4 to 5.4 (with a 30 min preincubation of samples at 37 C), both transition temperatures (Tm) and unfolding enthalpies decrease from 66 to 47 C and from 900 to 240 kcal/mol trimer, respectively, with corresponding decreases in CR from 3.0 to 1.2. The acid-induced destabilization corresponds to tertiary structure loss, as measured by near UV CD and intrinsic tryptophanyl residue fluorescence. Interestingly, temperature-dependent far UV CD measurements indicated that HA secondary structure is actually stabilized (from 66 to > 90 C) as the protein is acidified (pH 7 to 5). The proton-induced destabilization of tertiary structure and apparent stabilization of secondary structure are novel features of HA.Spectral and DSC experiments have been performed this year to redetermine some of the parameters for thermal unfolding HA with fresh preparations of HA. Also, the partial specific volume of HA at pH 7.4 has been determined to be 0.73 ml/g by simultaneous sedimentation equilibrium of HA rosettes in aqueous and deuterium oxide buffer.
| Remeta, David P; Krumbiegel, Mathias; Minetti, Conceicao A S A et al. (2002) Acid-induced changes in thermal stability and fusion activity of influenza hemagglutinin. Biochemistry 41:2044-54 |
