Signal sequences conformational studies

In the 13C-NMR spectra, PEG gives a singlet signal at 8 71.5 ppm due to the inner chain carbons 228). In the conformational studies of PEG-bound sequences of ala-methiein, this signal has been eliminated from the spectrum by making use of a double resonance experiment by selective saturation of the methylene carbons 209). Distinct differences between random-coil and a-helical conformations were observed from these high-resolution 13C-NMR techniques. 

The experiments described in Sections VI,A,B show that two physical properties of the synthetic LamB signal peptides correlate with their in vivo export function tendency to adopt an a-helical conformation in hydrophobic environments, and tendency to insert into lipid mono-layers. These properties may be involved in the same step in the secretion process, or in different steps. An a-helical conformation may be required to generate a structure sufficiently hydrophobic to allow mono-layer insertion. Alternatively, these properties may reflect separate roles of the signal sequence in protein secretion. For instance, an a-helical conformation may be necessary for binding to a proteinaceous site, while the ability to interact with lipids may be important for another step in the secretion process. We have studied the conformations of the synthetic LamB signal peptides in phospholipid vesicles and monolayers by CD and IR spectroscopy. 

The removal of the signal sequence which directs the transport of secreted proteins is a critical step in protein export. Extensive studies by Randall, Hardy and coworkers have demonstrated that the leader sequence prevents the proper folding of the mature polypeptide within the cytoplasm (55,54). For the E.coli 6-lactamase, the presence of the leader sequence decelerates the folding kinetics but does not prevent the formation of the enzymatically active conformation (55). Bowden and Georgiou (56) showed that the mode of translocation of 6-lactamase across the cytoplasmic membrane of Escherichia coli exerts a profound effect on the folding of the mature protein following secretion, presumably by affecting the unfolded state in the periplasmic space. 

Synthetic polypeptides consist of repeating sequences of certain amino acids and their structures are not as complicated as those of proteins. Eor this reason, synthetic polypeptides are sometimes used as model compounds for proteins. Their preferred conformations are classified as a helix, P sheet, 0) helix and so on. High-resolution solid-state C NMR spectroscopy has proved to be a very powerful tool for determining the structure of polypeptides in the crystalline state. Eor example, C CP MAS NMR spectra of solid poly(L-alanine) ([Ala] J show the Ca, Cp and C=0 carbon signals to be well resolved between the a helix and P sheet forms. The chemical shifts of the Ca and C=0 carbons of the a helix are displaced sig nilicantly to high frequency by 4.2 and 4.6 ppm, re spectively, relative to those of the P sheet form, while the shift of the Cp carbon of the a heUx is displaced to low frequency by about 5 ppm with respect to that of the P sheet. Eor this reason, the value of the C shift can be used to describe the local conformation. In addition, the C shifts of randomly coiled [Ala] in trifluoroacetic acid solution have values between those of the a helix and P sheet forms. The absolute C shifts of the Ca and Cp carbons are affected by the chemical structure of the individual amino acid residues and can be used effectively for conformational studies of particular amino acid residues in polypeptides and proteins. On the other hand, the C=0 shifts do not seem to be affected by residue structure and can be used for diagnosing the main chain conformation. 

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