The rate of the H-D exchange of the peptide NH atoms of the different forms of human Hb was studied at the range of pH 5-10 and temperature 10-63 degrees C by the IR spectroscopy. The pH-dependence of the H-D exchange rate is accordance with the EX2 mechanism. Two pH-dependent conformers of ligand forms of Hb existes at 10-30 degrees C with lower probability of local fluctuations of the alkaline conformer. The difference between two conformers vanishes at 40 degrees C with the appearance of the third conformer with higher probability of local fluctuations. The deoxyHb at 20 degrees C and pH range 6-9 has no pH-dependent conformers and the probability of local fluctuations is considerably reduced in comparison to the acid conformer of ligand Hb. Upon the destabilization of the ligand Hb structure by the pH decreasing to 5.0 at 20 degrees C or the temperature increasing up to 50-60 degrees C at pH 7.1 the global fluctuations of the native structure are intensified providing the H-D exchange of the slowest exchanging NH atoms. The nature of the local and global fluctuations and possible similarity between the two pH-dependent conformers of ligand Hb and its functional R and R2 states revealed by the X-ray analysis and NMR spectroscopy were discussed.

Deuterium↗

PubMedDate not supplied

[The conformational dynamic of the tetramer hemoglobin molecule as revealed by hydrogen exchange. II. Influence of the intersubunit contact splitting].

The rate of the H-D exchange of the peptide NH atoms of the isolated alpha and beta subunits of human Hb were studied at the pH range 5.5-9.0 and 20 degrees C by the IR spectroscopy. The factor retardation of the exchange rate of subunits -P in the range -10(2)-10(7). In comparison with tetramer Hb the probability of local fluctuations (1/P) is increased to a slightly greater extent for the monomeric alpha subunits then for the tetramer beta subunits. Unlike Hb oxygenation of subunits does not influence on the probability of the local fluctuations and subunits have no the pH-dependent change of the value 1/P observable for the ligand Hb. The possible mechanisms of the overall intensification of the local fluctuations upon the splitting of the Hb tetrameric contacts between subunits are discussed with the inviting of the structural crystallographic data.

Animals↗

PubMedDate not supplied

[The conformational dynamics of the tetramer hemoglobin molecule as revealed by hydrogen exchange. III. Influence of the heme removal].

Two main types of conformational fluctuations--local and global are characteristic of the native protein structure and revealed by hydrogen exchange. The probability of those fluctuations changes to a different extent upon hemoglobin oxygenation, changing of pH, splitting of the intersubunit contacts. To compare with the influence of the heme removal the rate of the H-D exchange of the peptide NH atoms of the human apoHb was studied at the pH range 5.5-9.0 and temperature 10-38 degrees C by the IR spectroscopy. The removal of the heme increases the rate of the H-D exchange of the 80% peptide NH atoms with the factor retardation of the exchange rate (P) in the range approximately 10(2)-10(8). For the most of the peptide NH atoms the probability of the local fluctuations weakly depends on the temperature, the enthalpy changes upon all such local conformational transitions deltaH(op) degrees are 0-15 kcal/M. Characterized by the stronger temperature dependence the global fluctuations are not arised upon the temperature increases up to 38 degrees C at pH 7.0 inspite of in these conditions the slow denaturation and aggregation of apoHb begin to occur. Upon the destabilization of the apoHb structure by the simultaneous decreasing of pH to 5.5 and temperature to 10 degrees C the global fluctuations of the apoHb native structure described by deltaH(op)o < 0 begin to intensify. The mechanism of the overall intensification of the local fluctuations upon the heme removal, the peculiarity of the heat denaturation of apoHb in conditions, close to that existing upon the selfassembly of Hb in vivo, and analogy between low temperature global fluctuations and cold denaturation of globular proteins are discussed.

Apoproteins↗

PubMedDate not supplied

[Dynamic structure of globular proteins: conformational rigidity and fluctuational motility].

The data on medium-scale fluctuation of globular proteins revealed by hydrogen exchange, proteolytic degradation and 1H NMR are considered. It is shown that the main features of those conformational transitions including their cooperativity can be described within the model that is based on mobile equilibrium defects of a solid characterized by the integrity of properties of the polymeric monocrystal and the molecular organic crystal. The extent of the fluctuational motility of the protein structure is regulated by strain energy. The functional isomerization of the protein is realized as generalized collective fluctuation induced by destabilization of the structure. The hydration of the backbone may play an important role providing the conformational rigidity of the protein, the extent of hydration being varied upon temperature changes and in the presence of organic co-solvents. Unlike the solid, the liquid-like state of the protein globula is characterized by high fluctuational motility and very narrow distribution of probabilities of medium-scale fluctuations.

Kinetics↗