Role of the transmembrane domain in the stability of TrwB, an integral protein involved in bacterial conjugation.
TrwB is an integral membrane protein encoded by the conjugative plasmid R388. TrwB binds ATP and is essential for R388-directed bacterial conjugation. The protein consists of a cytosolic domain, which contains an ATP-binding site, and a transmembrane domain. The complete protein has been purified in the presence of detergents, and in addition, the cytosolic domain has also been isolated in the form of a soluble truncated protein, TrwBDeltaN70. The availability of intact and truncated forms of the protein provides a convenient system to study the role of the transmembrane domain in the stability of TrwB. Protein denaturation was achieved by heat, in the presence of guanidinium HCl, or under low salt conditions. In all three cases TrwB was significantly more stable than TrwBDeltaN70 with other conditions being the same. IR spectroscopy of the native and truncated forms revealed significant differences between them. In addition, it was found that TrwBDeltaN70 was stabilized in dispersions of non-ionic detergent, suggesting the presence of hydrophobic patches on the surface of the truncated protein. IR spectroscopy also confirmed the conformational stability provided by the detergent. These results suggest that in integral membrane proteins consisting of a transmembrane and a cytosolic domain, the transmembrane portion may have a role beyond the mere anchoring of the protein to the cell membrane. In addition, this study indicates that the truncated soluble parts of two-domain membrane proteins may not reflect the physiological conformation of their native counterparts.
Hormaeche
Itsaso
I
Unidad de Biofísica (Centro Mixto Consejo Superior de Investigaciones Científicas-Universidad del País Vasco), and Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain.
Iloro
Ibón
I
Arrondo
José L R
JL
Goñi
Félix M
FM
de la Cruz
Fernando
F
Alkorta
Itziar
I
D001483:Base Sequence; D003227:Conjugation, Genetic / Q000502:physiology*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D005960:Glucosides / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005960:Glucosides; C403125:trwB protein, E coli; C040358:dodecyl maltoside; D019791:Guanidine