A prototype computer system forde novoprotein design (original) (raw)
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The Journal of Cell Biology, 1991
Using a photocross-linking approach we have investigated the cytosolic and membrane components involved in the targeting and insertion of signalanchor proteins into the membrane of the ER. The nascent chains of both type I and type II signal-anchor proteins can be cross-linked to the 54-kD subunit of the signal recognition particle. Upon addition of rough microsomes the type I and type II signal-anchor proteins interact with a number of components. Both types of protein interact with an integral membrane protein, the signal sequence receptor, previously identified by its proximity to preprolactin during its translocation (Wiedmann, M., T. V. Kurzchalia, E.
The Biochemical journal, 1987
An azidophenacyl derivative of a chemically synthesized consensus signal peptide has been prepared. The peptide, when photoactivated in the presence of rough or high-salt-stripped microsomes from pancreas, leads to inhibition of their activity in cotranslational processing of secretory pre-proteins translated from their mRNA in vitro. The peptide binds specifically with high affinity to components in the microsomal membranes from pancreas and liver, and photoreaction of a radioactive form of the azidophenacyl derivative leads to covalent linkage to yield two closely related radiolabelled proteins of Mr about 45,000. These proteins are integrated into the membrane, with large 30,000-Mr domains embedded into the phospholipid bilayer to which the signal peptide binds. A smaller, endopeptidase-sensitive, domain is exposed on the cytoplasmic surface of the microsomal vesicles. The specificity and selectivity of the binding of azidophenacyl-derivatized consensus signal peptide was demonst...
Towards a Determination of the SecA Signal Peptide Binding Site
SecA is an essential motor protein found in bacteria that uses ATP hydrolysis to transport preproteins across or into the cytoplasmic membrane. Before this translocation occurs, SecA must recognize and bind an N-terminal signal sequence of the newly synthesized preprotein. The location of this binding site on SecA and the orientation of the bound signal peptide is currently a matter of debate. Attempts at obtaining a high resolution X-ray crystal structure of signal peptide bound to SecA in order to elucidate the binding site have been inhibited by signal sequence hydrophobicity and aggregation. In this study, we have created a mutant E.coli SecA protein with the signal sequence KRR-LamB attached to the C-terminus using DNA recombination technology with the aim to obtain an X-ray crystal structure of SecA self-binding the attached signal peptide. Fluorescence anisotropy assays with a dyelabeled signal peptide and SecA or SecA-KRR-LamB proteins were used to analyze the extent of self-binding in two SecA-KRR-LamB constructs. Both the full length SecA-KRR-LamB and the truncated version lacking the C-terminus exhibit significant self-binding of the attached signal peptide. The functionality of the SecAattached KRR-LamB signal peptide was tested by engineering a SecA-KRR-LamB-PhoA chimera in which the alkaline phosphatase gene lacking its endogenous signal sequence was fused downstream of the SecA-attached KRR-LamB signal sequence, and the ability of the latter signal peptide to transport alkaline phosphatase across the membrane where it is enzymatically active in vivo was assayed. Both the full-length and truncated construct show self-binding and are able to facilitate translocation of the fused preprotein substrate to different extents. First and foremost, I would like to thank my advisor, Professor Donald Oliver, for giving me the opportunity to work in his lab and produce the work that turned into this thesis. I greatly appreciate his academic insight, life advice, and overall support. A special thank you goes to the readers of my thesis-Professors Manju Hingorani and Ishita Mukerjifor taking the time to look over my work and provide feedback. I highly value their input. Thank you to the MB&B department as a whole-faculty, staff, students-it has been an enriching few years. To the members of the Oliver Lab, past and present-thanks for everything. My lab experience would not have been nearly as positive without you in it. Qi, it has been an exceptional pleasure collaborating with you on this project, I hope you didn't mind my constant questions too much. Zeliang, Christine, Tithi, Sudipta, Jenn, Lorry and Stephanie, thank you. This process would not have been tolerable without the Cave-Zack, Ryu, Jaewon, and Corey-and of course all those who associate with the Cave. It has been a blast living with you guys. Thanks to my family for backing me in whatever I do, especially my Aunt Ellen and Uncle Lou. I appreciate beyond words your constant support, enthusiasm, and belief. Finally, to Kat, the person who has kept me reasonably sane throughout my two years of often frustrating lab work and my semester of writing. Thank you. I can't imagine these past years without you.
Requirements for the membrane insertion of signal-anchor type proteins
The Journal of Cell Biology, 1991
Proteins which are inserted and anchored in the membrane of the ER by an uncleaved signalanchor sequence can assume two final orientations. Type I signal-anchor proteins translocate the NH2 terminus across the membrane while type 1I signalanchor proteins translocate the COOH terminus. We investigated the requirements for cytosolic protein components and nucleotides for the membrane targeting and insertion of single-spanning type I signalanchor proteins. Besides the ribosome, signal recognition particle (SRP), GTE and rough microsomes (RMs) no other components were found to be re-quired. The GTP analogue GMPPNP could substitute for GTP in supporting the membrane insertion of IMC-CAT. By using a photocrosslinking assay we show that for secreted, type I and type II signalanchor proteins the presence of both GTP and RMs is required for the release of the nascent chain from the 54-kD subunit of SRP. For two of the proteins studied the release of the nascent chain from SRP54 was accompanied by a new interaction with components of the ER. We conclude that the GTP-dependent release of the nascent chain from SRP54 occurs in an identical manner for each of the proteins studied.
The Journal of Cell Biology, 1989
Salt-extracted microsomal membranes (K-RM) contain an activity that is capable of releasing the signal recognition particle (SRP)-mediated elongation arrest of the synthesis of secretory polypeptides (Walter, P., and G. Blobel, 1981, J. Cell Biol., 91:557-561). This arrestreleasing activity was shown to be a function of an integral microsomal membrane protein, termed the SRP receptor (Gilmore, R., P. Walter, and G. Blobel, 1982, J. Cell BioL, 95:470-477). We attempted to solubilize the arrest-releasing activity of the SRP receptor by mild protease digestion of K-RM using either trypsin or elastase. We found, however, that neither a trypsin, nor an elastase "solubilized" supernatant fraction exhibited the arrest-releasing activity. Only when either the trypsin-or elastase-derived supernatant fraction was combined with the trypsinized membrane fraction, which by itself was also inactive, was the arrest-releasing activity restored. Release of the elongation arrest was followed by the translocation of the secretory protein across the microsomal membrane and the removal of the signal peptide. Thus, although we have been unable to proteolytically sever the arrest-releasing activity from K-RM and thereby to uncouple the release of the elongation arrest from the process of chain translocation, we have been able to proteolytically dissect and reconstitute the arrest-releasing activity. Furthermore, we found that the arrest-releasing activity of the SRP receptor can be inactivated by alkylation of K-RM with N-ethylmaleimide.
Proteins: Structure, Function, and Bioinformatics, 2012
A protein destined for export from the cell cytoplasm is synthesized as a preprotein with an aminoterminal signal peptide. In Escherichia coli, signal peptides that guide preproteins into the SecYEG protein conduction channel are typically subsequently removed by signal peptidase I. To understand the mechanism of this critical step, we have assessed the conformation of the signal peptide when bound to signal peptidase by solution NMR. We employed a soluble form of signal peptidase without its two transmembrane domains (SPase I Δ2-75) and the E. coli alkaline phosphatase signal peptide. Using a transferred NOE approach, we found clear evidence of weak peptide-enzyme complex formation. The peptide adopts a "U-turn" shape originating from the proline residues within the primary sequence that is stabilized by its interaction with the peptidase and leaves key residues of the cleavage region exposed for proteolysis. In dodecylphosphocholine (DPC) micelles the signal peptide also adopts a U-turn shape comparable to that observed in association with the enzyme. In both environments this conformation is stabilized by the signal peptide phenylalanine side chain-interaction with enzyme or lipid mimetic. Moreover, in the presence of DPC, the N-terminal core region residues of the peptide adopt a helical motif and, based on PRE (paramagnetic relaxation enhancement) experiments, are shown to be buried within the membrane. Taken together, this is consistent with proteolysis of the preprotein occurring while the signal peptide remains in the bilayer and the enzyme active site functioning at the membrane surface.
The Sec61p Complex Is a Dynamic Precursor Activated Channel
Molecular Cell, 2003
transit through the ER membrane first suggested a predominantly hydrophilic microenvironment for the 1 Biophysik, Universitä t Osnabrü ck FB Biologie/Chemie transport substrates (Gilmore and Blobel, 1985). Subsequently, the mammalian ER was shown by electrophysi-D-49034 Osnabrü ck 2 Medizinische Biochemie und Molekularbiologie ological techniques to contain ion channels with a conductance of 220pS that can be activated by the release Universitä t des Saarlandes D-66421 Homburg of nascent polypeptides in transit (Simon et al., 1989; Simon and Blobel, 1991). Therefore, these ion channels Germany were proposed to be involved in protein translocation, i.e., termed protein-conducting channels. Subsequently, the protein-conducting channels or pores were shown Summary by fluorescence quenching experiments to have a diameter of 4 to 6 nm in the active or open state and to Previous studies have shown that the rough endoplasbe sealed by both the translating ribosomes and the mic reticulum (ER) contains nascent precursor polylumenal chaperone BiP (Hamman et al., 1997 and 1998; peptide gated channels. Circumstantial evidence sug-Liao et al. , 1997). However, in all these studies the nature gests that these channels are formed by the Sec61p of the protein-conducting channel remained elusive. On complex. We reconstituted the purified Sec61p comthe other hand, reconstitution into proteoliposomes plex in a lipid bilayer and characterized its dynamics identified the Sec61p complex as the central component and regulation. The Sec61p complex is sufficient to of the protein translocase (Gö rlich and Rapoport, 1993). form the precursor polypeptide activated channel un-Furthermore, cryo-and freeze fracture-electron microder co-and posttranslational transport conditions. Acscopic analysis of the Sec61p complexes as present in tivity of the Sec61p channel in both transport modes intact membranes, or in purified form in artificial memis induced by direct interaction with precursor protein.
Signal Sequences for Early Events in Protein Secretion and Membrane Assembly
Annals of the New York Academy of Sciences, 1980
The earliest step in the secretory pathway involves synthesis of. secretory proteins on membrane-bound ribosomes with vectorial translocation of the nascent growing chains across the membrane of the endoplasmic reticulum.' In the signal hypothesis' it was proposed that the initial event in translocation was interaction of a discrete portion of the nascent chain, termed the signal sequence, with receptors in the membrane of the endoplasmic reticulum (ER). As a result of this interaction, a functional ribosome-membrane junction was formed in continuity with a temporary proteinaceous pore in the membrane through which the growing chain passed. Upon completion of synthesis, the ribosomal subunits detach to enter the free cytoplasmic pool and the pore in the membrane disaggregates with the newly synthesized protein localized exclusively within the cisternal space of the ER. Similarly, it has been proposed' that transmembrane proteins, which span the bilayer asymmetrically, are inserted into the membrane according to a modified version of the signal hypothesis (FIGURE 1B). This model differs from that of secretory proteins in that translocation does not proceed to completion but rather, is arrested at a specific point by another sequence-termed a stop-transfer sequence. As a result, all copies of a given protein species are deposited in an identical fashion in the 356 0077-8923/80/0343-0356 SO1.75/0 0 1980, NYAS
Structure and biosynthesis of the signal-sequence receptor
European Journal of Biochemistry, 1990
The signal-sequence receptor (SSR) has previously been shown to be a component of the environment which nascent polypeptides meet on passage through the endoplasmic reticulum (ER) membrane. We report here on the primary structure of the SSR as deduced from cDNA clones and from direct protein sequencing. The glycoprotein is synthesized with a cleavable amino-terminal signal sequence and contains only one classical membrane-spanning segment. Its insertion into the ER membrane during biosynthesis depends on the function of the signal-recognition particle. SSR shows a remarkable charge distribution with the amino terminus being highly negatively charged, and the cytoplasmic carboxyl terminus positively charged. The SSR can be phosphorylated in its cytoplasmic tail both in intact cells and in a cell-free system, suggesting a regulation of its function. The localization of the protein in the ER membrane was confirmed by immunofluorescence microscopy.