Conformation signal sequences

Figure 7 Maturation of insulin. Insulin is synthesized as preproinsulin that contains an N-terminal signal sequence. After translocating into the ER, the signal sequence is cleaved off by the signal peptidase and the resulting proinsulin folds into a stable conformation. Three disulfide bonds are formed between cysteine side chains. The connecting sequence (Chain C) is cleaved off in the Golgi by proprotein convertases to form the mature and active insulin molecule, which is then secreted.

Localization signals of Golgi GlycTs are more complex, and seem conformation dependent. Sequences in the cytoplasmic tails, transmembrane regions and... 

The importance of signal-sequence conformation for proper function has been tested by determining the effect on activity of sequence changes that are predicted to change conformational tendencies these are discussed below. Only in the case of the LamB mutants (see below and Section III,F) have correlations been made between the actual and predicted conformational preferences of the altered and native sequences. 

Brown et al. (1984) introduced insertions of three or four amino acids into the yeast invertase signal sequence near its amino terminus. The insertions were predicted to stabilize an a helix, favor a )3 turn, or to destabilize both a-helix and 3-sheet formation. None of these alterations prevented proper secretion of the protein. Thus, the amino-terminal domain of the signal sequence is relatively unconstrained as to conformation. 

The c region is more sensitive to conformational alterations. The . coli wild-type lipoprotein signal sequence is predicted to form a )3 turn at positions —7 to -4. Alanine was substituted for serine (position —6), which occurs frequently in ji turns, or threonine (position -5), which is also found in turns, but less often than serine, or both (Vlasuk et al.,... 

Substitution of Ala for Thr -5 alone did not cause a predicted loss of -turn conformation, while replacement of Ser —6 or both Ser —6 and Thr —5 yielded a structure predicted to lack a )3 turn. The phenotypes of the mutants correlate with the predicted presence or absence of the turn. Those predicted to lack a turn accumulate membrane-bound precursor lipoprotein, which is slowly processed to mature lipoprotein. Thus, the absence of a region favoring /3 turn in the c domain can inhibit removal of the signal sequence. 

Wickner (1980) proposed an alternative mechanism of protein secretion, called the membrane trigger hypothesis. This model proposes that the signal sequence influences the precursor protein or a domain of the precursor to fold into a conformation that can spontaneously partition into the hydrophobic part of the bilayer. In prokaryotes, the membrane potential causes the protein to traverse the bilayer. The protein then regains a water-soluble conformation, and is expelled into the medium. Signal peptidase removes the signal sequence during or after this process. Thus, secretory proteins or domains are transported across the membrane posttranslationally without the aid of a proteinaceous secretory apparatus. An energy source, such as the membrane potential, is required for secretion. 

These experiments have not demonstrated that the conformational preferences of signal sequences are important to their ability to export proteins. To address this problem, we synthesized the family of E. coli K-receptor protein wild-type and mutant signal sequences (shown in Fig. 5 and described in Section III,H) and determined their conformations in various polar and apolar environments by CD (Briggs and Gierasch, 1984 Briggs, 1986). The solvents for these experiments included aqueous buffer and TFE, as described above. In addition, sodium dodecyl sulfate (SDS) micelles and phospholipid vesicles were used as membrane model systems. 

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. 

An even more striking comparison can be made between the wild-type signal peptide s conformation when adsorbed to the monolayer and its conformation in aqueous solution. In both of these environments, the peptide should be solvated by water, but its conformations are very different. The peptide is 100% )3 structure when adsorbed to the mono-layer, while it is 80% random in aqueous buffer. Thus, it appears that contact with the lipid surface induces substantial amounts of secondary structure in a molecule that takes on little structure in an aqueous environment. This finding implies that the initial binding of a signal sequence to a membrane may induce a particular structure, which may be important to the mechanism of signal-sequence function. 

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