Probing the energetics of proteins through structural perturbation: sites of regulatory energy in human hemoglobin.

Abstract

The sites of energy transduction within the human hemoglobin molecule for the regulation of oxygen affinity have been determined by an extensive study of the molecule's energetic response to structural alteration at individual amino acid residues. For 22 mutant and chemically modified hemoglobins we have determined the total free energy used by the tetrameric molecule for alteration ofoxygen affinity at the four binding steps. The results imply that the regulation of oxygen binding affinity is due to energy changes which are mostly localized at the a'(32 interface. They also indicate a high degree of "internal cooperativity" within this contact region-i.e., the structural perturbations at individual residue sites are energetically coupled. Cooperativity in ligand binding is thus a reflection of cooperativity at a deeper level-that of the protein-protein interactions within the al(92-interfacial domain.A fundamental problem of protein structure and function is the issue ofhow "local" properties ofindividual amino acid residues are related to "system" properties which reflect behavior ofthe molecule as a whole. Well-known manifestations ofthis problem include (a) the acid-base titration behavior of proteins, (b) the cooperative folding of tertiary structures, (c) the nucleated polymerization of self-assembling aggregates, and (d) the cooperative binding ofligands in allosteric systems. A large number of studies, including both experimental and theoretical work, have provided insights into the nature of these problems (cf. refs. 1-15).One strategy for the exploration ofstructure-energy coupling and its role in biological function lies in perturbing a protein molecule through alteration of individual amino acid residues (i.e., deletion, substitution, or chemical modification) and determining the effects of these alterations upon appropriately selected system properties. By studying the effects on the system properties ofa series of such changes, distributed throughout the molecular structure, one can determine which regions of the molecule are especially sensitive to structural perturbation. The power of this approach will be maximized when the system properties chosen reflect the energy states of the molecule as a whole and are at the same time directly related to its biological functions.In this paper we present results of such a study with human hemoglobin. Using 23 different hemoglobins we have determined the effects of structural perturbation upon the energy invested by-the molecule in altering the affinity at successive oxygenation steps. The results provide a structural "map" of energetic sensitivity related to the regulation of function.The problem of structure-energy correlation in the human hemoglobin molecule is centered on the classic problem of resolving the cooperative mechanism of oxygen binding. A complete understanding of this problem must include (a) the structural and energetic changes at the heme site that accompany the binding of oxygen, (b) the changes of tertiary structure and energy ...

Implication of the α1β1 interface in the hemoglobin affinity changes

“…For both hemoglobins, the spectra of oxygen derivatives are invariant over the pH range in which the proteins are stable (pH 6 -9). Our spectral invariance confirms the lack of an enhanced dissociation into dimers in hemoglobin San Diego [16]. …”

Experimental resolution of cooperative free energies for the ten ligation states of human hemoglobin.

“…USA 82 (1985) V.Yj oxyhemoglobin (20), and human carboxyhemoglobin (21) are closely similar in regard to quaternary structure, although minor tertiary structure differences exist. (ii) The total cooperative free energy (over all four steps) differs by only a few tenths of a kcal/mol (out of -6) for these three ligands (9). (iii) The pattern of energetic effects we find for cyanomethemoglobin is supported by results of kinetic and spectral studies with the ligand NO (22,23) and by cryogenic isoelectric focusing studies with CO (24), all of which indicate species [21] and [22] to dominate the distribution of doubly ligated tetramers and to have different properties from the other members of this set.…”

“…In an extensive series of measurements of the rate constants for association of normal, mutant, and chemically modified hemoglobins over a wide range of pH, this value was found to be invariant (9,15). The value of kf used in this study was also found to be in good agreement with that calculated from the values of 4K21 and kr determined independently for cyanomet hemoglobin with all four subunits ligated.…”

A quantitative model for the cooperative mechanism of human hemoglobin.

“…refs. [6][7][8][9][10][11][12][13][14][15]. These studies, and work from other laboratories, have resulted in findings that impose stringent constraints on the nature of interactions responsible for cooperativity.…”

“…In the past, the structural analyses have provided an admirable wealth of detailed (atomic-level) information, whereas the corresponding development of energetic information has been severely lagging. Thus models based upon detailed structural assumptions-e.g., the Perutz mechanism (21, 22)-have only recently become amenable to critical tests (3)(4)(5)(12)(13)(14). An example is the detailed model of Szabo and Karplus (23), which we have now tested against the extensive sets of highly accurate pH-dependent oxygenation data recently obtained (15).…”