α-Hemoglobin Stabilizing Protein (AHSP), a Kinetic Scheme of the Action of a Human Mutant, AHSPV56G

Thomas Brillet,Véronique Baudin-Creuza,Corinne Vasseur,Elisa Domingues-Hamdi,Laurent Kiger,Henri Wajcman,Serge Pissard,Michael C Marden

doi:10.1074/jbc.m109.098491

Abstract

A kinetic analysis has been made of the interaction of alpha-Hb chains with a mutant alpha-hemoglobin stabilizing protein, AHSP(V56G), which is the first case of an AHSP mutation associated with clinical symptoms of mild thalassemia syndrome. The chaperone AHSP is thought to protect nascent alpha chains until final binding to the partner beta-Hb. Rather than protecting alpha chains, the mutant chaperone is partially unfolded but recovers its secondary structure via interaction with alpha-Hb. For both AHSP(WT) and AHSP(V56G), the binding to alpha-Hb is quite rapid relative to the alpha-beta reaction, as expected because the chaperone binding must be quite competitive to complete the alpha chain folding process before alpha-Hb binds irreversibly to beta-Hb. The main kinetic difference is a dissociation rate of AHSP(V56G).alpha-Hb some four times faster relative to AHSP.alpha-Hb. Considering a role of protein folding, the AHSP(V56G) apparently does not bind long enough (0.5 s versus 2 s for the WT) to complete the structural modifications. The overall replacement reaction (AHSP.alpha-Hb + beta-Hb --> AHSP + alphabeta) can be quite long, especially if there is an excess of AHSP relative to beta-Hb monomers.

Highlights

abnormality of the ␣-hemoglobin stabilizing protein (AHSP) is a small 102-residue protein expressed only in erythroid cells, which adopts a three helix bundle [3]
The temperature corresponding to 50% unfolded molecule is the melting temperature, Tm. Both AHSPV56G and AHSPWT were expressed in E. coli
We observed an identical expression of AHSPV56G and AHSPWT indicating a normal level of biosynthesis in bacteria

Summary

Introduction

AHSP is a small 102-residue protein expressed only in erythroid cells, which adopts a three helix bundle [3]. If the chaperone acts as a buffer for an excess of ␣ chains, an appropriate affinity is necessary, but there would be freedom in the protein-protein interaction rates This role would allow storage under a harmless form of the excess ␣-Hb, known to be naturally synthesized at an excess of 10% relative to ␤-Hb. an active role of AHSP for the correct folding of ␣ chains places strict conditions on the protein association and dissociation rates. Using the fluorescence energy transfer technique, we investigated the dynamic association and dissociation of AHSPWT and AHSPV56G with ␣-Hb. The protein secondary structure and thermal stability of the WT and mutant AHSP were studied by circular dichroism (CD) spectroscopy

Objectives

Results

Conclusion