Transit peptide cleavage site

A fragment of the lettuce VDE cDNA was amplified from lettuce cDNA by PCR and used to isolate a near full-length cDNA for lettuce VDE (Bugos and Yamamoto, 1996). The cDNA encoded a polypeptide of 473 amino acids with a calculated molecular mass of 54.4 kDa. This was confirmed from in vitro transcription/translation of the VDE cDNA which produced a 55 kDa polypeptide when analyzed by SDS-PAGE. The deduced preprotein contained a 125 amino acid bipartite transit peptide for transport into the chloroplast and thylakoid lumen. Based on the N-terminal sequence of the purified VDE from lettuce, which indicates the transit peptide cleavage site, the mature VDE protein is encoded by 348 amino acids with a calculated molecular mass of... 

Sequencing of the labelled mature soy a-CT and BCCP, isolated from pea stroma after import of corresponding precursors, allowed us to determine the position of the transit peptide cleavage site, which is for a-CT2... 

Emanuelsson, O., Nielsen, H., and von Heijne, G. (1999). ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sd. 8, 978—984. 

Y Gavel, GA von Hejne. A conserved cleavage-site motif in chloroplast transit peptides. FEBS Lett 261 455-458, 1990. 

This moiety may be viewed as a carbon analogue of the transition state in peptide cleavage. The fragment is apparently close enough in structure to such an intermediate as to fit the cleavage site in peptidase enzymes. Once bound, this inactivates the enzyme as it lacks the scissile carbon-nitrogen bond. All five newer HIV protease inhibitors incorporate this structural unit. 

Table 1. Amino acid sequence of the 20 kDa apo-protein of CP24 deduced from isolated cDNA. Arrow indicates cleavage site for transit peptide. Boxed areas indicate areas of homology with LHCII and LHCI. Underlined stretches indicate putative membrane spanning regions.

The distribution of tm for 6 = 170 (P) and 8 = 100 (ot) is shown in Fig. 1 for selected members of the families of chloroplastic precursors. The amino acid sequences of the transit peptides are shown in the bottom panel. The plots are aligned according to the cleavage site at the end of the transit peptide. Proteins destined for the thyla-koid lumen (i.e., plastocyanin and the polypeptides of the oxygen evolving complex) must traverse the thylakoid membrane in addition to the envelope membranes. For these precursors, the first 40-60 amino acids comprise the stromal targeting domain, whereas the last 20-25 residues form the thylakoid transport domain (9). Thus, it is the first domain that is relevant for comparison with the other transit peptides. 

FIGURE 1. Hydrophobic moment of transit peptides for periodicity, S = 170 (left) and 100 (right), as a function of residue position from the cleavage site, stromal (A-E) and thylakoid (G-J) as described in text. Sequences are shown, along with possible intermediate stromal cleavage site in Silene PC (i). 

Consistent with the plastidial location of monoterpene biosynthesis [8], immunocy to chemical studies with limonene synthase confirmed the localization of this enzyme to the leucoplasts of peppermint oil glands [87]. Plastid targeting requires the translation of a preprotein bearing an N-terminal transit peptide that directs the newly synthesized enzyme to the plastid for proteolytic processing to the mature form [88]. The limonene synthase cDNA encodes such a preprotein, with an N-terminal sequence that exhibits the typical properties of a transit peptide (rich in serine and threonine (25-30%), rich in small hydrophobic amino acids with few acidic residues, and a propensity to form amphiphilic helices [89,90]). A precise cleavage site between the transit peptide and mature protein is not obvious in the limonene synthase preprotein, and mass spectral... 

Researchers have tested whether the membrane lipid environment affects the dynamics and cleavability of a model peptide corresponding to the amino acid sequence 684-726 of the amyloid precursor protein APP reconstituted in liposomes. SS NMR with H-Ala713, which is located within the putative transmembrane domain, suggested that the peptide observes less rotational motion in egg phosphatidylcholine (egg PC) membranes than in DMPC bilayers above the main phase transition temperature. They conclude stating that the dynamics of APP(684-726) on the transmembrane level as well as the motion of the ot-cleavage site and its hydrolysis by a model enzyme are dependent on the bilayer characteristics. ... 

The structural analysis of the trypsin inhibitor from bovine pancreas (BPTI) in complex with trypsin shows that the inhibitor occupies and blocks the substrate binding pocket in a highly complementary maimer (fig. 2.9). In the trypsin-BPTI complex, the catalytically essential Ser-OH of trypsin contacts a CO group of the inhibitor in a manner very similar to the tetrahedral transition state of amide or ester bond hydrolysis (see fig. 2.9b). The inhibitor can be likened to a pseudo-substrate and, as such, is bound with high affinity. The cleavage of the peptide bond is, however, not possible due to other circumstances, such as the fact that water is prevented from reaching the active site with the inhibitor boimd. 

Bovine pancreatic chymotrypsin (Mr 25,191) is a protease, an enzyme that catalyzes the hydrolytic cleavage of peptide bonds. This protease is specific for peptide bonds adjacent to aromatic amino acid residues (Trp, Phe, Tyr). The three-dimensional structure of chymotrypsin is shown in Figure 6-18, with functional groups in the active site emphasized. The reaction catalyzed by this enzyme illustrates the principle of transition-state stabilization and also provides a classic example of general acid-base catalysis and covalent catalysis. 

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