Ligand binding and proton exchange dynamics in site-specific mutants of human myoglobin

Thesis/Dissertation · Wed Jan 01 04:00:00 EST 1992

OSTI ID:7233884

Lambright, D G

Site specific mutagenesis was used to make substitutions of four residues in the distal heme pocket of human myoglobin: Val68, His64, Lys45, and Asp60. Strongly diffracting crystals of the conservative mutation K45R in the met aquo form were grown in the trigonal space group P3[sub 2]21 and the X-ray crystal structure determined at 1.6 [angstrom] resolution. The overall structure is similar to that of sperm whale met aquo myoglobin. Several of the mutant proteins were characterized by 2-D NMR spectroscopy. The NMR data suggest the structural changes are localized to the region of the mutation. The dynamics of ligand binding to myoglobin mutants were studied by transient absorption spectroscopy following photolysis of the CO complexes. Transient absorption kinetics and spectra on the ns to ms timescale were measured in aqueous solution from 280 K to 310 K and in 75% glycerol: water from 250 K to 310 K. Two significant basis spectra were obtained from singular value decomposition of the matrix of time dependent spectra. The information was used to obtain approximations for the extent of ligand rebinding and the kinetics of conformational relaxation. Except for K45R, substitutions at Lys45 or Asp60 produce changes in the kinetics for ligand rebinding. Replacement of Lys45 with Arg increases the rate of ligand rebinding from the protein matrix by a factor of 2, but does not alter the rates for ligand escape or entry into the protein or the dynamics of the conformational relaxation. Substitutions at His64 and Val68 influence the kinetics of ligand rebinding and the dynamics of conformational relaxation. The results do not support the hypothesis that ligand migration between the heme pocket and solvent is determined solely by fluctuations of Arg45 and His64 between open and closed conformations of the heme pocket but can be rationalized if ligand diffusion through the protein matrix involves multiple competing pathways.

• Research Organization: Stanford Univ., CA (United States)
• OSTI ID: 7233884
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
CHEMICAL BONDS
CONFORMATIONAL CHANGES
GLOBINS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HUMAN POPULATIONS
KINETICS
LIGANDS
MOLECULAR BIOLOGY
MOLECULAR STRUCTURE
MUTAGENESIS
MUTANTS
MYOGLOBIN
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PIGMENTS
POPULATIONS
PORPHYRINS
PROTEINS
REACTION KINETICS