ABSTRACT
The aggregation state of the gonadotropin receptor has been examined by coupling fluorescence energy transfer donor and acceptor fluorophores to hCG and LH. Energy transfer is observed at low (4 degrees C) but not at high (37 degrees C) temperature. Energy transfer could also be detected with receptor solubilized in the presence of hormone at the lower temperature only. Solubilization of receptor in the absence of hormone and subsequent addition of hormone conjugates revealed no energy transfer. These results are consistent with stabilization of receptor complexes at low temperatures, but presumptive hormone induced receptor dissociation under physiological conditions.
Subject(s)
Receptors, Cell Surface/metabolism , Chemical Phenomena , Chemistry , Chorionic Gonadotropin/metabolism , Energy Transfer , Fluorescein-5-isothiocyanate , Fluoresceins , Humans , Luteinizing Hormone/metabolism , Receptors, LHRH , Rhodamines , Spectrometry, Fluorescence , Surface Properties , Temperature , ThiocyanatesABSTRACT
A major problem encountered with the use of electronic spectroscopy in the analysis of biological materials in the ultraviolet, visible, and infrared region involves the limited range of the physical state of samples that can be examined. In an attempt to expand this range, photoacoustic spectra of both solid- and solution-state proteins have been obtained in the near-infrared region. Solid proteins generate detailed spectra in the region 1.0-2.6 micron, resulting primarily from hydrogenic overtone and combinational modes. Harmonics and combinations of amide group frequencies which display significant spectral complexity are observed between 1.4 and 1.7 micron, although they appear to manifest only limited conformational sensitivity. Solution spectra in D2O are of much lower resolution. Assignments of peaks for both solution- and solid-state proteins are presented and the advantages and disadvantages of the use of near-infrared photoacoustic spectroscopy with proteins are discussed.
Subject(s)
Proteins/analysis , Spectrophotometry, Infrared/methods , Amino Acids/analysis , Peptides/analysis , Photochemistry , Protein ConformationABSTRACT
The compound N-cyclohexyl-2-pyrrolidone contains a substantial apolar region as well as a peptide bond-like moiety. This solvent, therefore, provides a useful model for protein interiors. Under certain conditions of temperature and salt concentration, cyclohexylpyrrolidone forms a two-phase system with water. This permits partition coefficients and subsequent free energies of transfer of amino acid side chains from cyclohexylpyrrolidone to water to be simply determined. Free energies of transfer measured in this manner for 21 amino acids are found to be substantially less than those obtained from the commonly used ethanol/water solubility model. This suggests less of a contribution of hydrophobic interactions to the stabilization of protein structure than is conventionally assumed.
Subject(s)
Proteins/metabolism , Pyrrolidinones , Amino Acids , Chemical Phenomena , Chemistry , Models, Biological , Protein Conformation , ThermodynamicsABSTRACT
An experimental approach to interpretation of the anomalous absorption and circular dichroism (CD) spectra of hemoglobin in situ in red blood cells is reported. Absorption flattening effects have been overcome by use of high cell concentratons in very short light path cuvettes. Differential scattering contributions to circular dichroism have been resolved using a CD instrument capable of variable detection geometry. Scattering effects have also been resolved using media of high refractive index to match that of the red blood cell. The results are in agreement with a parellel calculational analysis of red blood cell CD spectra, which predicted the relative magnitudes of the flattening and differential scattering CD contributions. An experimental absorption spectrum has been obtained for hemoglobin in the red blood cell with scattering and flattening eliminated. This quantitatively simulates the spectrum of a hemoglobin solution. The methods described should be widely applicable to conformational studies of macromolecules in their native environment.
