QUANTITATIVE APPROACHES TO UTILIZING MUTATIONAL ANALYSIS AND DISULFIDE CROSS-LINKING FOR MODELING A TRANSMEMBRANE DOMAIN

The transmembrane domain of chemoreceptor Trg from Escherichia coli co ntains four transmembrane segments in its native homodimer, two from e ach subunit. We had previously used mutational analysis and sulfhydryl crosslinking between introduced cysteines to obtain data relevant to the three-dimensional organization of this domain. In the current stud y we used Fourier analysis to assess these data quantitatively for per iodicity along the sequences of the segments. The analyses provided a strong indication of alpha-helical periodicity in the first transmembr ane segment and a substantial indication of that periodicity for the s econd segment. On this basis, we considered both segments as idealized alpha-helices and proceeded to model the transmembrane domain as a un it of four helices. For this modeling, we calculated helical crosslink ing moments, parameters analogous to helical hydrophobic moments, as a quantitative way of condensing and utilizing a large body of crosslin king data. Crosslinking moments were used to define the relative separ ation and orientation of helical pairs, thus creating a quantitatively derived model for the transmembrane domain of Trg. Utilization of Fou rier transforms to provide a quantitative indication of periodicity in data from analyses of transmembrane segments, in combination with hel ical crosslinking moments to position helical pairs should be useful i n modeling other transmembrane domains.