Speed of intersubunit communication in proteins.
To determine the speed of communication between protein subunits, time-resolved absorption spectra were measured following partial photodissociation of the carbon monoxide complex of hemoglobin. The experiments were carried out using linearly polarized, 10-ns laser pulses, with the polarization of the excitation pulse both parallel and perpendicular to the polarization of the probe pulse. The substantial contribution to the observed spectra from photoselection effects was eliminated by isotropically averaging the polarized spectra, allowing a detailed comparison of the kinetics as a function of the degree of photolysis. These results show that prior to 1 microsecond both geminate ligand rebinding and conformational relaxation are independent of the number of ligands dissociated from the hemoglobin tetramer, as expected for a two-state allosteric model. After this time the kinetics depend on the ligation state of the tetramer. The conformational relaxation at 10 microseconds can be interpreted in terms of the two-state allosteric model as arising from the R to T quaternary conformational change of both unliganded and singly liganded molecules. These results suggest that communication between subunits requires about 1 microsecond and that the mechanism of the communication which occurs after this time is via the R to T conformational change. The optical anisotropy provides a novel means of accurately determining the extinction coefficients of the transient photoproduct. The decay in the optical anisotropy, moreover, provides an accurate determination of the rotational correlation time of 36 +/- 3 ns.
Jones
C M
CM
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Ansari
A
A
Henry
E R
ER
Christoph
G W
GW
Hofrichter
J
J
Eaton
W A
WA
D002263:Carboxyhemoglobin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006441:Hemoglobin A / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008962:Models, Theoretical; D010108:Oxyhemoglobins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010782:Photolysis; D011506:Proteins / Q000737:chemistry*; D013997:Time Factors
D046911:Macromolecular Substances; D010108:Oxyhemoglobins; D011506:Proteins; D006441:Hemoglobin A; D002263:Carboxyhemoglobin; C029774:oxyhemoglobin A