Quantitative approaches to utilizing mutational analysis and disulfide crosslinking for modeling a transmembrane domain.
The transmembrane domain of chemoreceptor Trg from Escherichia coli contains four transmembrane segments in its native homodimer, two from each subunit. We had previously used mutational analysis and sulfhydryl cross-linking between introduced cysteines to obtain data relevant to the three-dimensional organization of this domain. In the current study we used Fourier analysis to assess these data quantitatively for periodicity along the sequences of the segments. The analyses provided a strong indication of alpha-helical periodicity in the first transmembrane segment and a substantial indication of that periodicity for the second segment. On this basis, we considered both segments as idealized alpha-helices and proceeded to model the transmembrane domain as a unit of four helices. For this modeling, we calculated helical crosslinking moments, parameters analogous to helical hydrophobic moments, as a quantitative way of condensing and utilizing a large body of crosslinking data. Crosslinking moments were used to define the relative separation and orientation of helical pairs, thus creating a quantitatively derived model for the transmembrane domain of Trg. Utilization of Fourier transforms to provide a quantitative indication of periodicity in data from analyses of transmembrane segments, in combination with helical crosslinking moments to position helical pairs should be useful in modeling other transmembrane domains.
Lee
G F
GF
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660, USA.
Hazelbauer
G L
GL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002633:Chemotaxis; D003432:Cross-Linking Reagents; D004252:DNA Mutational Analysis; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005583:Fourier Analysis; D008565:Membrane Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D010507:Periodicity; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000737:chemistry*
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; C041476:Trg protein, E coli