We present a new method for determining the orientation of alpha-helical sections of proteins or peptides in membrane. To apply this method, membranes containing proteins must be prepared in a multilayer array. Circular dichroism (CD) spectra of the multilayer sample are then measured at the normal as well as oblique incident angles with respect to the bilayer planes; we call such spectra oriented circular dichroism (OCD). The procedure of OCD measurement, particularly the ways to avoid the spectral artifacts due to the effects of dielectric interfaces, linear dichroism and birefringence, and the method of data analysis are described in detail. To illustrate the method, we analyze the OCD of alamethicin in diphytanoylphosphatidylcholine multilayers. We conclude unambiguously that the helical section of alamethicin is parallel to the membrane normal when the sample is in the full-hydration state, but the helical section rotates to the plane of membrane when the sample is in a low-hydration state. We also obtained the parallel and perpendicular CD spectra of alpha-helix, and found them to be in agreement with previous theoretical calculations based on the exciton theory. These spectra are useful for analyzing protein orientations in future experiments.

We have studied the solution structure of skeletal muscle troponin C complexed with troponin I in the presence of calcium using small-angle X-ray and neutron scattering. 4Ca2+.troponin C in the complex has an extended dumbbell shape with a radius of gyration of 23.9 +/- 0.5 A and a maximum linear dimension of approximately 72 A, similar to the values obtained from the crystal structure coordinates of troponin C (Herzberg & James, 1985). Troponin I is even more extended than troponin C with a radius of gyration of 41 +/- 2 A and a maximum linear dimension of approximately 118 A. The centers-of-mass for each component of the complex are approximately coincident (< 10-A separation) as are their long axes, and the troponin I component encompasses the 4Ca2+.troponin C. These data provide new insights into the nature of the conformational arrangement of this important Ca(2+)-sensitive molecular switch.

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We report here a model structure for 4Ca2+.troponin C.troponin I derived from small-angle X-ray and neutron scattering data using a Monte Carlo modeling method. In this model, troponin I appears as a spiral structure that wraps around 4Ca2+.troponin C which adopts an extended dumbbell conformation similar to that observed in the crystal structures of troponin C. The troponin I spiral has the approximate dimensions of an alpha-helix and winds through the hydrophobic "cups" in each globular domain of troponin C. The model is consistent with a body of previously published biochemical data on the interactions between troponin C and troponin I, and suggests the molecular mechanism for the Ca(2+)-sensitive switch that regulates the muscle contraction/relaxation cycle involves a signal transmitted via the central spiral region of troponin I.

Moffitt’s exciton theory for α helices, an important cornerstone of the circular dichroism (CD) theory for biopolymers, was recently cast in doubt by a linear dichroism measurement of electric field oriented polypeptides [Yamaoka et al., J. Am Chem. Soc. 108, 4619 (1986)]. In particular the prediction that the polarization of an exciton split component at 208 nm should be parallel to the α-helical axis was not borne out. This revealed the inadequacy of previous experiments which used long polypeptides to prove the theory. We performed two experiments to measure the effect of orientation of α helices on CD, one with a short membrane peptide, alamethicin, oriented in defect-free multibilayers and another with long polypeptides oriented with electric field. We found the result of the experiment with alamethicin to be consistent with the exciton theory. An elaborate procedure was established to measure the CD of protein molecules embedded in lipid multibilayers with light incident on bilayers at various angles (the helical sections of alamethicin are perpendicularly embedded in bilayers). Thus we are able to measure the polarization of the 208 nm band and prove that it is indeed parallel to the α-helical axis. The problem of electric field oriented polypeptides is discussed in paper II.