Thermodynamic measurements of the contributions of helix-connecting loops and of retinal to the stability of bacteriorhodopsin.

Thermodynamic studies of bacteriorhodopsin (BR) have been undertaken in order to investigate the factors that stabilize the structure of a membrane protein. The stability of the native, intact protein was compared to that of protein with retinal removed, and/or cleaved in one or two of the loops connecting the transmembrane helices. The stability was assessed using differential scanning calorimetry and thermal denaturation curves obtained from ultraviolet circular dichroism and absorption spectroscopy. Retinal binding and the loop connections were each found to make a small contribution to stability, and even a sample that was cleaved twice as well as bleached to remove retinal denatured well above room temperature. Removal of retinal destabilized the protein more than cleaving once, and about as much as cleaving twice. Retinal binding and the connections in the loops were found to stabilize BR in independent ways. Cleavage of the molecule into fragments did not reduce the intermolecular cooperativity of the denaturation. Dilution of the protein by addition of excess lipid in order to eliminate the purple membrane crystal lattice also did not alter the cooperativity. These results are used to compare the relative importance of various contributors to the stability of BR.

[1]  H. Khorana,et al.  Regeneration of the native bacteriorhodopsin structure from two chymotryptic fragments. , 1983, The Journal of biological chemistry.

[2]  U. Sonnewald,et al.  Reconstitution of an active lactose carrier in vivo by simultaneous synthesis of two complementary protein fragments , 1990, Journal of bacteriology.

[3]  T. Kurosaki,et al.  A subunit common to an IgG Fc receptor and the T-cell receptor mediates assembly through different interactions. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[4]  H. Khorana,et al.  Structure and thermal stability of monomeric bacteriorhodopsin in mixed pospholipid/detergent micelles , 1989, Proteins.

[5]  Y. Ovchinnikov,et al.  Recent findings in the structure—functional characteristics of bacteriorhodopsin , 1977, FEBS letters.

[6]  D. Engelman,et al.  Glycophorin A dimerization is driven by specific interactions between transmembrane alpha-helices. , 1992, The Journal of biological chemistry.

[7]  R. Callender,et al.  Effect of lipid-protein interaction on the color of bacteriorhodopsin. , 1989, Biochimica et biophysica acta.

[8]  J. L. López-Lacomba,et al.  Differential scanning calorimetry of the irreversible thermal denaturation of thermolysin. , 1988, Biochemistry.

[9]  D. Engelman,et al.  Bacteriorhodopsin can be refolded from two independently stable transmembrane helices and the complementary five-helix fragment. , 1992, Biochemistry.

[10]  C. Brouillette,et al.  pH dependence of bacteriorhodopsin thermal unfolding. , 1987, Biochemistry.

[11]  T O Yeates,et al.  Structure of the reaction center from Rhodobacter sphaeroides R-26: membrane-protein interactions. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[12]  G. Feher,et al.  The bacterial photosynthetic reaction center as a model for membrane proteins. , 1989, Annual review of biochemistry.

[13]  J. M. Sanchez-Ruiz,et al.  Theoretical analysis of Lumry-Eyring models in differential scanning calorimetry. , 1992, Biophysical journal.

[14]  H. Khorana,et al.  Structure-function studies of bacteriorhodopsin XV. Effects of deletions in loops B-C and E-F on bacteriorhodopsin chromophore and structure. , 1991, The Journal of biological chemistry.

[15]  H. Khorana,et al.  Refolding of an integral membrane protein. Denaturation, renaturation, and reconstitution of intact bacteriorhodopsin and two proteolytic fragments. , 1981, The Journal of biological chemistry.

[16]  H. Khorana,et al.  Regeneration of native bacteriorhodopsin structure from fragments. , 1984, The Journal of biological chemistry.

[17]  H. Khorana,et al.  Substitution of membrane-embedded aspartic acids in bacteriorhodopsin causes specific changes in different steps of the photochemical cycle. , 1989, Biochemistry.

[18]  David C. Lee,et al.  Structure of bacteriorhodopsin investigated using Fourier transform infrared spectroscopy and proteolytic digestion , 1987 .

[19]  T. Mitsui,et al.  Formation of the two-dimensional hexagonal lattice of bacteriorhodopsin in reconstituted brown membrane. , 1978, Biochimica et biophysica acta.

[20]  A. V. Kiselev,et al.  Products of limited proteolysis of bacteriorhodopsin generate a membrane potential , 1978, FEBS letters.

[21]  D. Engelman.  An Implication of the Structure of Bacteriorhodopsin: Globular Membrane Proteins are Stabilized by Polar Interactions. , 1982, Biophysical journal.

[22]  J. Sturtevant,et al.  Phase transitions of the purple membranes of Halobacterium halobium. , 1978, Biochemistry.

[23]  W. van Osdol,et al.  Calorimetrically determined dynamics of complex unfolding transitions in proteins. , 1990, Annual review of biophysics and biophysical chemistry.

[24]  B. Wallace,et al.  Differential absorption flattening optical effects are significant in the circular dichroism spectra of large membrane fragments. , 1987, Biochemistry.

[25]  D. Engelman,et al.  Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process. , 1987, Journal of molecular biology.

[26]  K. Matthews,et al.  Thermal denaturation of the core protein of lac repressor. , 1985, Biochemistry.

[27]  R. Henderson,et al.  Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. , 1990, Journal of molecular biology.

[28]  T. Mitsui,et al.  Phase transitions of the purple membrane and the brown holo-membrane X-ray diffraction, circular dichroism spectrum and absorption spectrum studies , 1981 .

[29]  D. Engelman,et al.  Membrane protein folding and oligomerization: the two-stage model. , 1990, Biochemistry.

[30]  B. Bormann,et al.  Synthetic peptides mimic the assembly of transmembrane glycoproteins. , 1989, The Journal of biological chemistry.

[31]  R. Wenger,et al.  Refolding of bacteriorhodopsin. Protease V8 fragmentation and chromophore reconstitution from proteolytic V8 fragments. , 1988, European journal of biochemistry.

[32]  H. Khorana,et al.  Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation. , 1991, The Journal of biological chemistry.

[33]  H. Kaback,et al.  In vivo expression of the lacY gene in two segments leads to functional lac permease. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[34]  R. Henderson,et al.  Temperature-dependent aggregation of bacteriorhodopsin in dipalmitoyl- and dimyristoylphosphatidylcholine vesicles. , 1978, Journal of molecular biology.

[35]  M. El-Sayed,et al.  Sensitivity of the retinal circular dichroism of bacteriorhodopsin to the mutagenetic single substitution of amino acids: tyrosine , 1990, FEBS letters.

[36]  T. Steitz,et al.  Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. , 1986, Annual review of biophysics and biophysical chemistry.

[37]  B. Litman.  RHODOPSIN: ITS MOLECULAR SUBSTRUCTURE AND PHOSPHOLIPID INTERACTIONS * , 1979, Photochemistry and photobiology.

[38]  B. Wallace,et al.  Differential light scattering and absorption flattening optical effects are minimal in the circular dichroism spectra of small unilamellar vesicles. , 1984, Biochemistry.

[39]  C. Pace,et al.  Thermodynamics of ribonuclease T1 denaturation. , 1992, Biochemistry.

[40]  J. Sturtevant.  Biochemical Applications of Differential Scanning Calorimetry , 1987 .

[41]  V. Marchesi,et al.  Subunit structure of human erythrocyte glycophorin A. , 1976, Biochemistry.

[42]  N. Allewell,et al.  High-resolution differential scanning calorimetric analysis of the subunits of Escherichia coli aspartate transcarbamoylase. , 1985, Biochemistry.