Nuclear Magnetic Studies of Alamethicin

Alamethicin is a polypeptide with many interesting biological properties, in particular it shows voltage dependent conductance in lipid bilayers and also under the correct conditions can mimic nerve action potentials. It was chosen for study because of these properties and also because it is of suitable size (MW 2,000), it had apparently been fully characterised and sequenced and it binds metal ions and hence its conformation could be studied using the lanthanide ions.
It was shown by workers at Porton Microbiological Research Establishment that 'alamethicin' is a mixture of several similar chromato graphic ally separable components the major ones being 'F30' ca 85% and 'F50' 12%. All previous literature work has been done on the unseparated mixture of these various components. Most of our work was performed on F30 which has an ionisable carboxyl group (GLN 18). Porton supplied the alamethicin F30 and F50 for our studies. A very pure sample of F30 was prepared in Oxford by Dr. J. D. Priddle.
The original aims of the project had to be substantially modified when it was found that the well-established and widely-quoted sequence of alamethicin proposed by J. W. Payne et al, and confirmed by A. A. Kiryushkin et al, was in fact incorrect. This discovery was made from the preliminary Nmr spectra of F30 (and F50). These spectra showed two unexpected sets of peaks which could not come from any groups in the published sequence, Nmr spectra of a sample of the 'Upjohn' alamethicin used in almost all previous published studies of alamethicin showed these same extra peaks were present and hence the problem was not unique to our alamethicin samples.
The nature of both of these extra moieties was determined by detailed Nmr studies of their properties in the intact F30, in partial hydrolysates and in complete hydrolysates of the F30. Both of the extra moieties were successfully isolated after they had been hydrolysed off the alamethicin, and hence their identity could be determined beyond all doubt. It was also shown that these extra moieties were covalently bound into the alamethicin and the nature of the linkages involved was established as a result of the experiments above.
The only weakness in the published sequence is the supposed GLU 17 γ carboxyl to PRO 1 peptide linkage which cyclised the alamethicin 'ring'. The presence of this link was never directly demonstrated. It was shown from our studies that one of the extra moieties, phenylalaninol was attached to the GLU 17 γ carboxyl group by an amide linkage, and the hydroxyl group of the phenlalaninol was free and hence the 'ring' was not closed and so alamethicin is linear. The other extra moiety, was known from the lanthanide studies to be a very long way from the phenylalaninol and this other moiety was shown to be attached to the PRO 1 hence blocking the N-terminus.
This finding that alamethicin F30 is not cyclic but is a linear peptide with the C- and N- termini blocked by groups which are undetected by conventional procedures is not in fact in disagreement with any of the previous sequence/composition studies of alamethicin. This finding is obviously of importance in trying to understand the interesting conduction properties shown by alamethicin.
A similar set of experiments on F50 showed that it too is linear and has the C- and N- termini blocked in an identical fashion to F30. The rest of the sequence of F50 is virtually identical to that of F30.
The conformational studies of alamethicin were performed in deuterated methanol as here, in contrast to aqueous solvents, alamethicin is monomeric and does not aggregate. The pH in methanol was measured using a directly calibrated indicator system. Control of pH is important as the lanthanide ions bind strongly to both ionised and unionised F30.
The method of determining the conformation involves binding a lanthanide ion to a single site in the molecule and then observing the two principal effects which the paramagnetic ions may produce on the Nmr spectrum of the molecule, namely changes in the resonance energy of the nuclei (i. e. shifts of the lines) and relaxation of the nuclei (broadening of the lines). It was shown that the required conditions of fast exchange, 1:1 complex and no contact effects were present and hence the magnitude of these effects depends on various geometric factors relating the bound metal ion to the observed nucleus.
The magnitude of the shift effect gives distance and angular information, however the simplifying case of axial symmetry which has been found in all previous studies by our group using lanthanide ions was not present in this case. It was shown that the case of non-axial symmetry is fundamentally insoluble and so it was only possible to obtain qualitative information about the conformation from these studies.
The magnitude of the relaxation effect depends only on distance. Analysis of the relaxation data enabled more detailed conformational information to be obtained, although for various reasons no attempt was made to obtain absolute distances. The relaxation results agreed with the shift results and this is an important check on the correctness of the conformation as the geometric dependence of the shift and relaxation effects is very different.
These studies showed that the conformation was pH dependent, i.e. depended on whether the GLN 18 carboxyl group was ionised or not, and that the binding was strong to both ionised and unionised forms.
Studies on the minor component F50 were very much less detailed but showed that the conformation of the F50-lanthanide complex was very similar to that of the unionised F30-lanthanide complex as might be expected.