Quaternary structure of hemoglobin in solution

Abstract

Many important proteins perform their physiological functions under allosteric control, whereby the binding of a ligand at a specific site influences the binding affinity at a different site. Allosteric regulation usually involves a switch in protein conformation upon ligand binding. The energies of the corresponding structures are comparable, and, therefore, the possibility that a structure determined by x-ray diffraction in the crystalline state is influenced by its intermolecular contacts, and thus differs from the solution structure, cannot be excluded. Here, we demonstrate that the quaternary structure of tetrameric human normal adult carbonmonoxy-hemoglobin can readily be determined in solution at near-physiological conditions of pH, ionic strength, and temperature by NMR measurement of 15 N-1 H residual dipolar couplings in weakly oriented samples. The structure is found to be a dynamic intermediate between two previously solved crystal structures, known as the R and R2 states. Exchange broadening at the subunit interface points to a rapid equilibrium between different structures that presumably include the crystallographically observed states.A basic assumption in correlating protein structure and function is that the structure of a protein in the crystalline state is the same as that under physiological solution conditions. Although the weak intermolecular forces in a highly hydrated crystal are unlikely to shift ordered elements of a protein relative to one another, this does not necessarily hold true for flexible proteins that can switch between different conformations under allosteric control. For example, recent NMR data indicate that allosteric activation of the signaling protein NtrC simply involves the shift of a preexisting conformational equilibrium (1). The activated R-state conformation of the allosteric enzyme aspartate transcarbamoylase is another example where multiple conformations are sampled. The unliganded trimeric catalytic domain of this enzyme is fully active, but, except for increased intra-domain flexibility in the crystalline state, the trimer resembles the inactive T state of the holoenzyme (2). In addition, Alber, Schachman, and coworkers (3) found that upon ligation, this trimeric domain resembles the ligated holoenzyme, previously thought to represent the R state. This finding strongly suggests that the activated R state must be capable of traversing multiple conformations. The presence of such dynamic processes can easily be frozen out in the crystalline state.Human normal adult hemoglobin (Hb A) is the classic textbook example of a multimeric, allosteric protein and of the exquisite control a protein can exert over ligand binding. Hb A consists of four subunits: two ␣-chains of 141 amino acid residues each and two ␤-chains of 146 residues each. The oxygenation of Hb A in solution or inside red blood cells is cooperative; i.e., the binding of the first oxygen ligand to a Hb subunit enhances the binding of subsequent oxygen molecules to the remaining subunits. Oxygenation of ...

Unsuspected pathway of the allosteric transition in hemoglobin

“…Along the new pathway, it is still valid (9,14) to consider hemoglobin as a dimer of αβ-dimers in which the C2 symmetry between the two αβ-dimers is essentially maintained. This behavior is consistent with a recent NMR study showing that carbon monoxyhemoglobin, while undergoing its quaternary transition between the R and R2 states (R2 is a low-salt form), retains C 2 symmetry in solution (29). It also is consistent with the Monod-WymanChangeux model of cooperativity (12), in which the protein retains its symmetry at the quaternary level.…”

Insights into the Solution Structure of Human Deoxyhemoglobin in the Absence and Presence of an Allosteric Effector

“…We have already reported that the solution structure of HbCO A is a dynamic ensemble of the R and R2 crystal structures (23). While additional research is needed to gain a deeper insight, there is sufficient information to suggest that a dynamic ensemble may exist in the deoxy form as well.In conclusion, the plasticity and dynamic picture of hemoglobin is consistent with the emerging view of allostery as a change in population distribution of an ensemble of structures, rather than the equilibrium of only two discrete conformations, upon binding of ligand (6,8,23,(42)(43)(44)(45). Hence, in order to understand how hemoglobin functions as an efficient oxygen carrier in our physiological system, we need to know the structure, dynamics, and function of this protein in solution.…”

“…Fitting was done using singular value decomposition (SVD) (37) or non-linear least-squares methods. Analysis included a comparison of experimental RDCs with back-calculated RDC values as described previously (23). Both root-mean square deviation (RMSD) and chi square (χ 2 ) values have been used to characterize the quality of the fit between the experimental and back-calculated RDCs.…”

Estimating hydration changes upon biomolecular reactions from osmotic stress, high pressure, and preferential hydration experiments

“…The calculated number of adsorbed water molecules upon haemoglobin's oxygen uptake varies from Ϫ65 to ϩ20 (Table 2) smaller than ϩ65 Ϯ 4 (4) inferred from OSA. It is emphasised that the multiple results of ⌬NЈ 21 obtained from my theory is due to the uncertainty regarding the structures of deoxygenated and oxygenated states (37,38). The expulsion of camphor from cytochrome P450cam, according to Eq.…”

“…They are summarised in Table 1. For hemoglobin, the conformations for deoxygenated and oxygenated structures are still under debate (37,38). Therefore, I have followed LiCata and Allewell (17) and calculated ⌬V E s under all possible conformational changes between the deoxygenated and oxygenated structures determined thus far (see Table 1).…”