Dissimilar flexibility of α and β subunits of human adult hemoglobin influences the protein dynamics and its alteration induced by allosteric effectors

Abstract

The general question by what mechanism an “effector” molecule and the hemes of hemoglobin interact over widely separated intramolecular distances to change the oxygen affinity has been extensively investigated, and still has remained of central interest. In the present work we were interested in clarifying the general role of the protein matrix and its dynamics in the regulation of human adult hemoglobin (HbA). We used a spectroscopy approach that yields the compressibility (κ) of the protein matrix around the hemes of the subunits in HbA and studied how the binding of heterotropic allosteric effectors modify this parameter. κ is directly related to the variance of volume fluctuation, therefore it characterizes the molecular dynamics of the protein structure. For the experiments the heme groups either in the α or in the β subunits of HbA were replaced by fluorescent Zn-protoporphyrinIX, and series of fluorescence line narrowed spectra were measured at varied pressures. The evaluation of the spectra yielded the compressibility that showed significant dynamic asymmetry between the subunits: κ of the α subunit was 0.17±0.05/GPa, while for the β subunit it was much higher, 0.36±0.07/GPa. The heterotropic effectors, chloride ions, inositol hexaphosphate and bezafibrate did not cause significant changes in κ of the α subunits, while in the β subunits the effectors lead to a significant reduction down to 0.15±0.04/GPa. We relate our results to structural data, to results of recent functional studies and to those of molecular dynamics simulations, and find good agreements. The observed asymmetry in the flexibility suggests a distinct role of the subunits in the regulation of Hb that results in the observed changes of the oxygen binding capability.

Interaction of human hemoglobin and semi-hemoglobins with the Staphylococcus aureus hemophore IsdB: a kinetic and mechanistic insight

“…To get insights about the complex intrinsic flexibility we analyzed all MD trajectories by means of Essential Dynamics (ED), able to identify the essential motions of the protein backbone. The well-known flexible C/E and C/D loops in Hb α chain and β chain 36 , respectively, were removed from the ED analyses to not bias the identification of other dynamic elements. In all complexes, the ED was carried out separately for each individual α and β globin chain, for the αβ dimer (Hb) and for IsdB.…”

“…We chose as reference MetHb, whose ED shows a quite stable structure, with the highest flexibility localized at the terminal region of helix F and at the E/F loop of chain β, being notably more flexible than α chain 36 (Fig. 7A).…”

Biomolecules under Pressure: Phase Diagrams, Volume Changes, and High Pressure Spectroscopic Techniques

“…The fluctuations and compressibility are connected by the fluctuation–dissipation theorem of statistical physics. The compressibility of proteins can be obtained from various methods: volumetric experiments, ultrasound velocity measurements, site selective hole burning, or fluorescence line narrowing spectroscopies [ 102 , 103 , 104 , 105 , 106 ]. There is a delicate balance in the proteins between the flexibility needed for the enzymatic activity and the rigidity needed for preserving the stable structure [ 20 ].…”

Slow dynamics measured by phosphorescence lifetime reveals global conformational changes in human adult hemoglobin induced by allosteric effectors

“…Several models have been developed to explain the functioning of Hb and the homotropic and heterotropic allosteric effectors. In our previous paper on HbA [ 1 ], we provided a concise overview of these models. Here is a reminder in the form of a short list of the most prominent ones:…”

“…The fitted parameters changed due to the presence of allosteric effectors in line with results of previous experimental studies. The correlation among the changes of oxygen affinity, the activation energy and entropy, and the subunit compressibility [ 1 ] suggests that allosteric effectors tightly couple the whole protein and point out the importance of global dynamics in the regulation of Hb function. This coupling to the global dynamics suggests that allostery generally per se is made possible by the existence of global structural motions (fluctuations), which encompass the whole (or a very large portion) of the protein molecule.…”