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Journal articles on the topic 'Protein folding; Proline isomerisation'

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Published: 4 June 2021
Last updated: 12 February 2022

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1

Wedemeyer, William J., Ervin Welker, and Harold A. Scheraga. "Proline Cis−Trans Isomerization and Protein Folding†." Biochemistry 41, no. 50 (December 2002): 14637–44. http://dx.doi.org/10.1021/bi020574b.

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2

Lin, Lung-Nan, Hideyo Hasumi, and John F. Brandts. "Catalysis of proline isomerization during protein-folding reactions." Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 956, no. 3 (October 1988): 256–66. http://dx.doi.org/10.1016/0167-4838(88)90142-2.

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3

Troilo, Francesca, Francesca Malagrinò, Lorenzo Visconti, Angelo Toto, and Stefano Gianni. "The Effect of Proline cis-trans Isomerization on the Folding of the C-Terminal SH2 Domain from p85." International Journal of Molecular Sciences 21, no. 1 (December 23, 2019): 125. http://dx.doi.org/10.3390/ijms21010125.

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SH2 domains are protein domains that modulate protein–protein interactions through a specific interaction with sequences containing phosphorylated tyrosines. In this work, we analyze the folding pathway of the C-terminal SH2 domain of the p85 regulatory subunit of the protein PI3K, which presents a proline residue in a cis configuration in the loop between the βE and βF strands. By employing single and double jump folding and unfolding experiments, we demonstrate the presence of an on-pathway intermediate that transiently accumulates during (un)folding. By comparing the kinetics of folding of the wild-type protein to that of a site-directed variant of C-SH2 in which the proline was replaced with an alanine, we demonstrate that this intermediate is dictated by the peptidyl prolyl cis-trans isomerization. The results are discussed in the light of previous work on the effect of peptidyl prolyl cis-trans isomerization on folding events.
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4

Lee, Schuyler, Chao Wang, Haolin Liu, Jian Xiong, Renee Jiji, Xia Hong, Xiaoxue Yan, et al. "Hydrogen bonds are a primary driving force forde novoprotein folding." Acta Crystallographica Section D Structural Biology 73, no. 12 (November 10, 2017): 955–69. http://dx.doi.org/10.1107/s2059798317015303.

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The protein-folding mechanism remains a major puzzle in life science. Purified soluble activation-induced cytidine deaminase (AID) is one of the most difficult proteins to obtain. Starting from inclusion bodies containing a C-terminally truncated version of AID (residues 1–153; AID153), an optimizedin vitrofolding procedure was derived to obtain large amounts of AID153, which led to crystals with good quality and to final structural determination. Interestingly, it was found that the final refolding yield of the protein is proline residue-dependent. The difference in the distribution ofcisandtransconfigurations of proline residues in the protein after complete denaturation is a major determining factor of the final yield. A point mutation of one of four proline residues to an asparagine led to a near-doubling of the yield of refolded protein after complete denaturation. It was concluded that the driving force behind protein folding could not overcome thecis-to-transproline isomerization, orvice versa, during the protein-folding process. Furthermore, it was found that successful refolding of proteins optimally occurs at high pH values, which may mimic protein foldingin vivo. It was found that high pH values could induce the polarization of peptide bonds, which may trigger the formation of protein secondary structures through hydrogen bonds. It is proposed that a hydrophobic environment coupled with negative charges is essential for protein folding. Combined with our earlier discoveries on protein-unfolding mechanisms, it is proposed that hydrogen bonds are a primary driving force forde novoprotein folding.
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5

Arnold, Ulrich, and Ronald T. Raines. "Replacing a single atom accelerates the folding of a protein and increases its thermostability." Organic & Biomolecular Chemistry 14, no. 28 (2016): 6780–85. http://dx.doi.org/10.1039/c6ob00980h.

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6

Kubyshkin, Vladimir, Rebecca Davis, and Nediljko Budisa. "Biochemistry of fluoroprolines: the prospect of making fluorine a bioelement." Beilstein Journal of Organic Chemistry 17 (February 15, 2021): 439–60. http://dx.doi.org/10.3762/bjoc.17.40.

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Due to the heterocyclic structure and distinct conformational profile, proline is unique in the repertoire of the 20 amino acids coded into proteins. Here, we summarize the biochemical work on the replacement of proline with (4R)- and (4S)-fluoroproline as well as 4,4-difluoroproline in proteins done mainly in the last two decades. We first recapitulate the complex position and biochemical fate of proline in the biochemistry of a cell, discuss the physicochemical properties of fluoroprolines, and overview the attempts to use these amino acids as proline replacements in studies of protein production and folding. Fluorinated proline replacements are able to elevate the protein expression speed and yields and improve the thermodynamic and kinetic folding profiles of individual proteins. In this context, fluoroprolines can be viewed as useful tools in the biotechnological toolbox. As a prospect, we envision that proteome-wide proline-to-fluoroproline substitutions could be possible. We suggest a hypothetical scenario for the use of laboratory evolutionary methods with fluoroprolines as a suitable vehicle to introduce fluorine into living cells. This approach may enable creation of synthetic cells endowed with artificial biodiversity, containing fluorine as a bioelement.
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7

Osváth, Szabolcs, and Martin Gruebele. "Proline Can Have Opposite Effects on Fast and Slow Protein Folding Phases." Biophysical Journal 85, no. 2 (August 2003): 1215–22. http://dx.doi.org/10.1016/s0006-3495(03)74557-3.

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8

Radzicka, Anna, Scott A. Acheson, and Richard Wolfenden. "Cis/trans isomerization at proline: Desolvation and its consequences for protein folding." Bioorganic Chemistry 20, no. 4 (December 1992): 382–86. http://dx.doi.org/10.1016/0045-2068(92)90048-8.

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9

Chattopadhyay, Madhab K., Renée Kern, Michel-Yves Mistou, Abhaya M. Dandekar, Sandra L. Uratsu, and Gilbert Richarme. "The Chemical Chaperone Proline Relieves the Thermosensitivity of a dnaK Deletion Mutant at 42°C." Journal of Bacteriology 186, no. 23 (December 1, 2004): 8149–52. http://dx.doi.org/10.1128/jb.186.23.8149-8152.2004.

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ABSTRACT Since, like other osmolytes, proline can act as a protein stabilizer, we investigated the thermoprotectant properties of proline in vitro and in vivo. In vivo, elevated proline pools in Escherichia coli (obtained by altering the feedback inhibition by proline of γ-glutamylkinase, the first enzyme of the proline biosynthesis pathway) restore the viability of a dnaK-deficient mutant at 42°C, suggesting that proline can act as a thermoprotectant for E. coli cells. Furthermore, analysis of aggregated proteins in the dnaK-deficient strain at 42°C by two-dimensional gel electrophoresis shows that high proline pools reduce the protein aggregation defect of the dnaK-deficient strain. In vitro, like other “chemical chaperones,” and like the DnaK chaperone, proline protects citrate synthase against thermodenaturation and stimulates citrate synthase renaturation after urea denaturation. These results show that a protein aggregation defect can be compensated for by a single mutation in an amino acid biosynthetic pathway and that an ubiquitously producible chemical chaperone can compensate for a defect in one of the major chaperones involved in protein folding and aggregation.
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10

Wruck, Florian, Alexandros Katranidis, Knud H. Nierhaus, Georg Büldt, and Martin Hegner. "Translation and folding of single proteins in real time." Proceedings of the National Academy of Sciences 114, no. 22 (May 15, 2017): E4399—E4407. http://dx.doi.org/10.1073/pnas.1617873114.

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Protein biosynthesis is inherently coupled to cotranslational protein folding. Folding of the nascent chain already occurs during synthesis and is mediated by spatial constraints imposed by the ribosomal exit tunnel as well as self-interactions. The polypeptide’s vectorial emergence from the ribosomal tunnel establishes the possible folding pathways leading to its native tertiary structure. How cotranslational protein folding and the rate of synthesis are linked to a protein’s amino acid sequence is still not well defined. Here, we follow synthesis by individual ribosomes using dual-trap optical tweezers and observe simultaneous folding of the nascent polypeptide chain in real time. We show that observed stalling during translation correlates with slowed peptide bond formation at successive proline sequence positions and electrostatic interactions between positively charged amino acids and the ribosomal tunnel. We also determine possible cotranslational folding sites initiated by hydrophobic collapse for an unstructured and two globular proteins while directly measuring initial cotranslational folding forces. Our study elucidates the intricate relationship among a protein’s amino acid sequence, its cotranslational nascent-chain elongation rate, and folding.
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11

Kumat, T. K. S., D. Samuel, G. Jayaraman, T. Srimathi, and C. Yu. "The role of proline in the prevention of aggregation during protein folding in vitro." IUBMB Life 46, no. 3 (October 1998): 509–17. http://dx.doi.org/10.1080/15216549800204032.

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12

Thomas, Colin A., Erach R. Talaty, and James G. Bann. "3S-Fluoroproline as a probe to monitor proline isomerization during protein folding by 19F-NMR." Chemical Communications, no. 23 (2009): 3366. http://dx.doi.org/10.1039/b821952d.

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13

Hurle, Mark R., Stephen Anderson, and Irwin D. Kuntz. "Confirmation of the predicted source of a slow folding reaction: proline 8 of bovine pancreatic trypsin inhibitor." "Protein Engineering, Design and Selection" 4, no. 4 (1991): 451–55. http://dx.doi.org/10.1093/protein/4.4.451.

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14

Kamen, Douglas E., and Robert W. Woody. "Folding Kinetics of the Protein Pectate Lyase C Reveal Fast-Forming Intermediates and Slow Proline Isomerization†,‡." Biochemistry 41, no. 14 (April 2002): 4713–23. http://dx.doi.org/10.1021/bi0115129.

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15

KEMPER, B. "Structural basis for the role in protein folding of conserved proline-rich regions in cytochromes P450." Toxicology and Applied Pharmacology 199, no. 3 (September 2004): 305–15. http://dx.doi.org/10.1016/j.taap.2003.11.030.

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16

Moparthi, Satish Babu, Per Hammarström, and Uno Carlsson. "A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding." Protein Science 18, no. 2 (February 2009): 475–79. http://dx.doi.org/10.1002/pro.28.

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17

Moparthi, Satish Babu, Per Hammarström, and Uno Carlsson. "A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding." Protein Science 18, no. 6 (June 2009): 1332. http://dx.doi.org/10.1002/pro.129.

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18

Waudby, Christopher A., Tomasz Wlodarski, Maria-Evangelia Karyadi, Anaïs M. E. Cassaignau, Sammy H. S. Chan, Anne S. Wentink, Julian M. Schmidt-Engler, et al. "Systematic mapping of free energy landscapes of a growing filamin domain during biosynthesis." Proceedings of the National Academy of Sciences 115, no. 39 (September 10, 2018): 9744–49. http://dx.doi.org/10.1073/pnas.1716252115.

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Cotranslational folding (CTF) is a fundamental molecular process that ensures efficient protein biosynthesis and minimizes the formation of misfolded states. However, the complexity of this process makes it extremely challenging to obtain structural characterizations of CTF pathways. Here, we correlate observations of translationally arrested nascent chains with those of a systematic C-terminal truncation strategy. We create a detailed description of chain length-dependent free energy landscapes associated with folding of the FLN5 filamin domain, in isolation and on the ribosome, and thus, quantify a substantial destabilization of the native structure on the ribosome. We identify and characterize two folding intermediates formed in isolation, including a partially folded intermediate associated with the isomerization of a conserved cis proline residue. The slow folding associated with this process raises the prospect that neighboring unfolded domains might accumulate and misfold during biosynthesis. We develop a simple model to quantify the risk of misfolding in this situation and show that catalysis of folding by peptidyl-prolyl isomerases is sufficient to eliminate this hazard.
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19

Meng, Fan-Guo, Yong-Doo Park, and Hai-Meng Zhou. "Role of proline, glycerol, and heparin as protein folding aids during refolding of rabbit muscle creatine kinase." International Journal of Biochemistry & Cell Biology 33, no. 7 (July 2001): 701–9. http://dx.doi.org/10.1016/s1357-2725(01)00048-6.

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20

Jakob, Roman P., Bettina K. Zierer, Ulrich Weininger, Stefanie D. Hofmann, Stefan H. Lorenz, Jochen Balbach, Holger Dobbek, and Franz X. Schmid. "Elimination of a cis-Proline-Containing Loop and Turn Optimization Stabilizes a Protein and Accelerates Its Folding." Journal of Molecular Biology 399, no. 2 (June 2010): 331–46. http://dx.doi.org/10.1016/j.jmb.2010.04.007.

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21

Ruiz, Carlos A., Susana G. Rossi, and Richard L. Rotundo. "Rescue and Stabilization of Acetylcholinesterase in Skeletal Muscle by N-terminal Peptides Derived from the Noncatalytic Subunits." Journal of Biological Chemistry 290, no. 34 (July 2, 2015): 20774–81. http://dx.doi.org/10.1074/jbc.m115.653741.

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The vast majority of newly synthesized acetylcholinesterase (AChE) molecules do not assemble into catalytically active oligomeric forms and are rapidly degraded intracellularly by the endoplasmic reticulum-associated protein degradation pathway. We have previously shown that AChE in skeletal muscle is regulated in part post-translationally by the availability of the noncatalytic subunit collagen Q, and others have shown that expression of a 17-amino acid N-terminal proline-rich attachment domain of collagen Q is sufficient to promote AChE tetramerization in cells producing AChE. In this study we show that muscle cells, or cell lines expressing AChE catalytic subunits, incubated with synthetic proline-rich attachment domain peptides containing the endoplasmic reticulum retrieval sequence KDEL take up and retrogradely transport them to the endoplasmic reticulum network where they induce assembly of AChE tetramers. The peptides act to enhance AChE folding thereby rescuing them from reticulum degradation. This enhanced folding efficiency occurs in the presence of inhibitors of protein synthesis and in turn increases total cell-associated AChE activity and active tetramer secretion. Pulse-chase studies of isotopically labeled AChE molecules show that the enzyme is rescued from intracellular degradation. These studies provide a mechanistic explanation for the large scale intracellular degradation of AChE previously observed and indicate that simple peptides alone can increase the production and secretion of this critical synaptic enzyme in muscle tissue.
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22

Stefan, Christopher J., Mark C. Overton, and Kendall J. Blumer. "Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors." Molecular Biology of the Cell 9, no. 4 (April 1998): 885–99. http://dx.doi.org/10.1091/mbc.9.4.885.

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We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate agonist-induced structural changes leading to G protein activation. Constitutive signaling by mutant receptors is suppressed upon coexpression with wild-type, but not G protein coupling-defective, receptors. Wild-type receptors may therefore sequester a limiting pool of G proteins; this apparent “precoupling” of receptors and G proteins could facilitate signal production at sites where cell surface projections form during mating partner discrimination. Finally, rather than being expressed mainly at the cell surface, constitutively active pheromone receptors accumulate in post-endoplasmic reticulum compartments. This is in contrast to other defective membrane proteins, which apparently are targeted by default to the vacuole. We suggest that the quality-control mechanism that retains receptors in post-endoplasmic reticulum compartments may normally allow wild-type receptors to fold into their native, fully inactive conformations before reaching the cell surface. This may ensure that receptors do not trigger a response in the absence of agonist.
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23

Kumawat, Manoj, Irungbam Karuna, Neeraj Ahlawat, and Sushma Ahlawat. "Identification of Salmonella Typhimurium Peptidyl-prolyl cis-trans Isomerase B (PPIase B) and Assessment of their Role in the Protein Folding." Protein & Peptide Letters 27, no. 8 (September 24, 2020): 744–50. http://dx.doi.org/10.2174/0929866527666200225124104.

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Background: Peptidyl-prolyl cis-trans isomerase (PPIases) enzyme plays a vital role in protein folding. It catalyses the cis-trans isomerisation of peptide bonds, an essential step for newly synthesized protein to acquire its correct functional conformation in both prokaryotes and eukaryotes. Objective: The present study showed the biochemical and molecular characterisation of cyclophilins (PpiB), a type of peptidyl-prolyl isomerases proteins from the pathogenic bacteria Salmonella Typhimurium. Methods: Salmonella Typhimurium is one of the leading serovars responsible for human and animal salmonellosis globally, with the majority of human cases originating through the food chain. Here successful expression and purification of PpiB protein have been demonstrated and LC-MS based analyses showed high protein score and similarity with other PPi protein. Further the enzymatic activity of the purified recombinant PpiB was determined using Succinyl-Ala-Phe-Pro- Phe-p nitroanilide as substrate and enzyme-catalysed reaction. Result: Km and Vmax were calculated and found to be Vm = 1.023 ± .06400 min/μg, Km = 0.6219 ± 0.1701 μM, respectively. We have reported for the first time the presence of Salmonella PPIase-B (PpiB) protein isoforms in salmonella genome having PPi activity. Conclusion: Taken together, our data clearly showed that Salmonella Cyclophilin B (PpiB) protein is active and involved in diverse biological processes and highly similar to the different domain of Cyclophilin proteins.
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24

Vicente, R. L., S. Marín, J. R. Valverde, C. Palomino, R. P. Mellado, and S. Gullón. "Functional identification of a Streptomyces lividans FKBP-like protein involved in the folding of overproduced secreted proteins." Open Biology 9, no. 10 (October 2019): 190201. http://dx.doi.org/10.1098/rsob.190201.

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Some bacterial peptidyl-prolyl cis/trans isomerases (PPIases) are involved in secretory protein folding after the translocation step. Streptomyces lividans has been used as a host for engineering extracellular overproduction of homologous and heterologous proteins in industrial applications. Although the mechanisms governing the major secretory pathway (Sec route) and the minor secretory pathway (Tat route) are reasonably well described, the function of proteins responsible for the extracellular secretory protein folding is not characterized as yet. We have characterized a Tat-dependent S . lividans FK506-binding protein-like lipoprotein (FKBP) that has PPIase activity. A mutant in the sli-fkbp gene induces a secretion stress response and affects secretion and activity of the Sec-dependent protein α-amylase. Additionally, propagation in high copy number of the sli-fkbp gene has a positive effect on the activity of both the overproduced α-amylase and the overproduced Tat-dependent agarase, both containing proline cis isomers. Targeted proteomic analyses showed that a relevant group of secreted proteins in S. lividans TK21 are affected by Sli-FKBP, revealing a wide substrate range. The results obtained indicate that, regardless of the secretory route used by proteins in S. lividans , adjusting the expression of sli-fkbp may facilitate folding of dependent proteins when engineering Streptomyces strains for the overproduction of homologous or heterologous secretory proteins.
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25

Vivoli-Vega, Mirella, Prandvera Guri, Fabrizio Chiti, and Francesco Bemporad. "Insight into the Folding and Dimerization Mechanisms of the N-Terminal Domain from Human TDP-43." International Journal of Molecular Sciences 21, no. 17 (August 29, 2020): 6259. http://dx.doi.org/10.3390/ijms21176259.

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TAR DNA-binding protein 43 (TDP-43) is a 414-residue long nuclear protein whose deposition into intraneuronal insoluble inclusions has been associated with the onset of amyotrophic lateral sclerosis (ALS) and other diseases. This protein is physiologically a homodimer, and dimerization occurs through the N-terminal domain (NTD), with a mechanism on which a full consensus has not yet been reached. Furthermore, it has been proposed that this domain is able to affect the formation of higher molecular weight assemblies. Here, we purified this domain and carried out an unprecedented characterization of its folding/dimerization processes in solution. Exploiting a battery of biophysical approaches, ranging from FRET to folding kinetics, we identified a head-to-tail arrangement of the monomers within the dimer. We found that folding of NTD proceeds through the formation of a number of conformational states and two parallel pathways, while a subset of molecules refold slower, due to proline isomerism. The folded state appears to be inherently prone to form high molecular weight assemblies. Taken together, our results indicate that NTD is inherently plastic and prone to populate different conformations and dimeric/multimeric states, a structural feature that may enable this domain to control the assembly state of TDP-43.
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26

Martin, Andreas, and Franz X. Schmid. "A Proline Switch Controls Folding and Domain Interactions in the Gene-3-protein of the Filamentous Phage fd." Journal of Molecular Biology 331, no. 5 (August 2003): 1131–40. http://dx.doi.org/10.1016/s0022-2836(03)00864-7.

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27

Reader, John S., Nico A. J. Van Nuland, Gary S. Thompson, Stuart J. Ferguson, Christopher M. Dobson, and Sheena E. Radford. "A partially folded intermediate species of the β-sheet protein apo-pseudoazurin is trapped during proline-limited folding." Protein Science 10, no. 6 (June 2001): 1216–24. http://dx.doi.org/10.1110/ps.52801.

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28

Kizhatil, Krishnakumar, Adam Gromley, and Lorraine M. Albritton. "Two Point Mutations Produce Infectious Retrovirus Bearing a Green Fluorescent Protein-SU Fusion Protein." Journal of Virology 75, no. 23 (December 1, 2001): 11881–85. http://dx.doi.org/10.1128/jvi.75.23.11881-11885.2001.

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ABSTRACT Two second-site mutations in Moloney murine leukemia virus envelope surface protein (SU) were previously shown to rescue infection of two different SU mutants, a fusion-defective point mutant and a fusion-defective modified SU that exhibits weak subunit association. We report here that they also rescue infection of a third defective SU, one modified by insertion of the green fluorescent protein (GFP) between serine 6 and proline 7. GFP-SU assembled into virions and showed a strong association with the transmembrane protein (TM). However, these virions were noninfectious. GFP-SU expression was not maintained within cells, suggesting that the protein was toxic. Addition of the second-site mutations rendered the GFP-SU virus infectious and resulted in prolonged expression of the modified envelope protein. This virus showed a slight reduction in receptor binding but not in envelope protein processing, suggesting that addition of the GFP sequences results in subtle structural changes. Extrapolating these data, we see that the fundamental problem with the GFP-SU envelope protein appears to be a folding problem, suggesting that the second-site mutations rescue GFP-SU primarily by a mechanism that involves stabilizing the envelope protein structure.
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29

Bubeck, P., S. Pistor, J. Wehland, and B. M. Jockusch. "Ligand recruitment by vinculin domains in transfected cells." Journal of Cell Science 110, no. 12 (June 15, 1997): 1361–71. http://dx.doi.org/10.1242/jcs.110.12.1361.

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Vinculin, a prominent protein component of microfilament-membrane attachment sites, consists of three major domains: an N-terminal, compact head and a C-terminal rod-like tail that are connected by a flexible, proline-rich hinge. In vitro, the protein has been shown to interact with numerous ligands, including other components of the microfilament system. To characterize the ligand recruitment ability of the different vinculin domains in a cellular environment, we used a novel approach of comprising chimeric proteins of either the vinculin head, hinge or tail regions, fused to the membrane anchor sequence of ActA, a surface protein of the intracellular bacterial pathogen Listeria monocytogenes. When PtK2 cells were transfected with the corresponding constructs, the ActA membrane anchor directed the chimeric polypeptides to mitochondrial membranes. In this position, they accumulated microfilament proteins, as seen by immunofluorescence analysis. A chimera comprising the full length vinculin clone recruited a substantial amount of the cellular F-actin, the vasodilator stimulated phosphoprotein (VASP) and paxillin, but little alpha-actinin and talin. The presence of only the vinculin head directed some of the fusion protein to focal contacts, and alpha-actinin recruitment was still ineffective. Prominent recruitment of F-actin and of VASP required the presence of the tail and proline-rich hinge, respectively. Reducing the vinculin tail to short pieces harboring only one of the two F-actin binding sequences, which were defined by in vitro experiments, resulted in loss of activity, possibly by incorrect polypeptide folding. The proline-rich hinge domain could be exchanged for the analogous region of the ActA protein, and the number of such proline-clusters, containing an FPPPP motif, correlated with the extent of VASP recruitment. The results show that this system can be used to analyze in vivo the activity of vinculin domains responsible for the assembly of various cytoskeletal ligands.
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30

Ghosh, Shereen Georges, Lu Wang, Martin W. Breuss, Joshua D. Green, Valentina Stanley, Xiaoxu Yang, Danica Ross, et al. "Recurrent homozygous damaging mutation in TMX2, encoding a protein disulfide isomerase, in four families with microlissencephaly." Journal of Medical Genetics 57, no. 4 (October 5, 2019): 274–82. http://dx.doi.org/10.1136/jmedgenet-2019-106409.

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BackgroundProtein disulfide isomerase (PDI) proteins are part of the thioredoxin protein superfamily. PDIs are involved in the formation and rearrangement of disulfide bonds between cysteine residues during protein folding in the endoplasmic reticulum and are implicated in stress response pathways.MethodsEight children from four consanguineous families residing in distinct geographies within the Middle East and Central Asia were recruited for study. All probands showed structurally similar microcephaly with lissencephaly (microlissencephaly) brain malformations. DNA samples from each family underwent whole exome sequencing, assessment for repeat expansions and confirmatory segregation analysis.ResultsAn identical homozygous variant in TMX2 (c.500G>A), encoding thioredoxin-related transmembrane protein 2, segregated with disease in all four families. This variant changed the last coding base of exon 6, and impacted mRNA stability. All patients presented with microlissencephaly, global developmental delay, intellectual disability and epilepsy. While TMX2 is an activator of cellular C9ORF72 repeat expansion toxicity, patients showed no evidence of C9ORF72 repeat expansions.ConclusionThe TMX2 c.500G>A allele associates with recessive microlissencephaly, and patients show no evidence of C9ORF72 expansions. TMX2 is the first PDI implicated in a recessive disease, suggesting a protein isomerisation defect in microlissencephaly.
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31

Hill, Melissa K., Miranda Shehu-Xhilaga, Suzanne M. Crowe, and Johnson Mak. "Proline Residues within Spacer Peptide p1 Are Important for Human Immunodeficiency Virus Type 1 Infectivity, Protein Processing, and Genomic RNA Dimer Stability." Journal of Virology 76, no. 22 (November 15, 2002): 11245–53. http://dx.doi.org/10.1128/jvi.76.22.11245-11253.2002.

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ABSTRACT The full-length human immunodeficiency virus type 1 (HIV-1) mRNA encodes two precursor polyproteins, Gag and GagProPol. An infrequent ribosomal frameshifting event allows these proteins to be synthesized from the same mRNA in a predetermined ratio of 20 Gag proteins for each GagProPol. The RNA frameshift signal consists of a slippery sequence and a hairpin stem-loop whose thermodynamic stability has been shown in in vitro translation systems to be critical to frameshifting efficiency. In this study we examined the frameshift region of HIV-1, investigating the effects of altering stem-loop stability in the context of the complete viral genome and assessing the role of the Gag spacer peptide p1 and the GagProPol transframe (TF) protein that are encoded in this region. By creating a series of frameshift region mutants that systematically altered the stability of the frameshift stem-loop and the protein sequences of the p1 spacer peptide and TF protein, we have demonstrated the importance of stem-loop thermodynamic stability in frameshifting efficiency and viral infectivity. Multiple changes to the amino acid sequence of p1 resulted in altered protein processing, reduced genomic RNA dimer stability, and abolished viral infectivity. The role of the two highly conserved proline residues in p1 (position 7 and 13) was also investigated. Replacement of the two proline residues by leucines resulted in mutants with altered protein processing and reduced genomic RNA dimer stability that were also noninfectious. The unique ability of proline to confer conformational constraints on a peptide suggests that the correct folding of p1 may be important for viral function.
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Hong, Suntaek, Gyu Choi, Sunyoung Park, An-Sik Chung, Eric Hunter, and Sung S. Rhee. "Type D Retrovirus Gag Polyprotein Interacts with the Cytosolic Chaperonin TRiC." Journal of Virology 75, no. 6 (March 15, 2001): 2526–34. http://dx.doi.org/10.1128/jvi.75.6.2526-2534.2001.

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ABSTRACT The carboxy terminus-encoding portion of the gag gene of Mason-Pfizer monkey virus (M-PMV), the prototype immunosuppressive primate type D retrovirus, encodes a 36-amino-acid, proline-rich protein domain that, in the mature virion, becomes the p4 capsid protein. The p4 domain has no known role in M-PMV replication. We found that two mutants with premature termination codons that remove half or all of the p4 domain produced lower levels of stable Gag protein and of self-assembled capsids. Interestingly, yeast two-hybrid screening revealed that p4 specifically interacted with TCP-1γ, a subunit of the chaperonin TRiC (TCP-1 ring complex). TRiC is a cytosolic chaperonin that is known to be involved in both folding and subunit assembly of a variety of cellular proteins. TCP-1γ also associated with high specificity with the M-PMV pp24/16-p12 domain and human immunodeficiency virus p6. Moreover, in cells, Gag polyprotein associated with the TRiC chaperonin complex and this association depended on ATP hydrolysis. In the p4 truncation mutants, the Gag-TRiC association was significantly reduced. These results strongly suggest that cytosolic chaperonin TRiC is involved in Gag folding and/or capsid assembly. We propose that TRiC associates transiently with nascent M-PMV Gag molecules to assist in their folding. Consequently, properly folded Gag molecules carry out the intermolecular interactions involved in self-assembly of the immature capsid.
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33

Johnson, Colin P., Massimiliano Gaetani, Vanessa Ortiz, Nishant Bhasin, Sandy Harper, Patrick G. Gallagher, David W. Speicher, and Dennis E. Discher. "Pathogenic proline mutation in the linker between spectrin repeats: disease caused by spectrin unfolding." Blood 109, no. 8 (December 27, 2006): 3538–43. http://dx.doi.org/10.1182/blood-2006-07-038588.

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Abstract Pathogenic mutations in α and β spectrin result in a variety of syndromes, including hereditary elliptocytosis (HE), hereditary pyropoikilocytosis (HPP), and hereditary spherocytosis (HS). Although some mutations clearly lie at sites of interaction, such as the sites of spectrin α-βtetramer formation, a surprising number of HE-causing mutations have been identified within linker regions between distal spectrin repeats. Here we apply solution structural and single molecule methods to the folding and stability of recombinant proteins consisting of the first 5 spectrin repeats of α-spectrin, comparing normal spectrin with a pathogenic linker mutation, Q471P, between repeats R4 and R5. Results show that the linker mutation destabilizes a significant fraction of the 5-repeat construct at 37°C, whereas the WT remains fully folded well above body temperature. In WT protein, helical linkers propagate stability from one repeat to the next, but the mutation disrupts the stabilizing influence of adjacent repeats. The results suggest a molecular mechanism for the high frequency of disease caused by proline mutations in spectrin linkers.
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34

Davis, Elaine C., Thomas J. Broekelmann, Yuji Ozawa, and Robert P. Mecham. "Identification of Tropoelastin as a Ligand for the 65-kD FK506-binding Protein, FKBP65, in the Secretory Pathway." Journal of Cell Biology 140, no. 2 (January 26, 1998): 295–303. http://dx.doi.org/10.1083/jcb.140.2.295.

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The folding and trafficking of tropoelastin is thought to be mediated by intracellular chaperones, although the identity and role of any tropoelastin chaperone remain to be determined. To identify proteins that are associated with tropoelastin intracellularly, bifunctional chemical cross-linkers were used to covalently stabilize interactions between tropoelastin and associated proteins in the secretory pathway in intact fetal bovine auricular chondrocytes. Immunoprecipitation of tropoelastin from cell lysates after cross-linking and analysis by SDS-PAGE showed the presence of two proteins of ∼74 kD (p74) and 78 kD (p78) that coimmunoprecipitated with tropoelastin. Microsequencing of peptide fragments from a cyanogen bromide digest of p78 identified this protein as BiP and sequence analysis identified p74 as the peptidyl-prolyl cis–trans isomerase, FKPB65. The appearance of BiP and FKBP65 in the immunoprecipitations could be enhanced by the addition of brefeldin A (BFA) and N-acetyl-leu-leu-norleucinal (ALLN) to the culture medium for the final 4 h of labeling. Tropoelastin accumulates in the fused ER/Golgi compartment in the presence of BFA if its degradation is inhibited by ALLN (Davis, E.C., and R.P. Mecham. 1996. J. Biol. Chem. 271:3787–3794). The use of BFA and other secretion-disrupting agents suggests that the association of tropoelastin with FKBP65 occurs in the ER. Results from this study provide the first identification of a ligand for an FKBP in the secretory pathway and suggest that the prolyl cis–trans isomerase activity of FKBP65 may be important for the proper folding of the proline-rich tropoelastin molecule before secretion.
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35

Georgescu, Roxana E., Jian-Hua Li, Michel E. Goldberg, Maria Luisa Tasayco, and Alain F. Chaffotte. "Proline Isomerization-Independent Accumulation of an Early Intermediate and Heterogeneity of the Folding Pathways of a Mixed α/β Protein,Escherichia coliThioredoxin†." Biochemistry 37, no. 28 (July 1998): 10286–97. http://dx.doi.org/10.1021/bi9805083.

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36

Takahashi, Ryuji, Ryuhei Yoshida, Kosuke Maki, and Kunihiro Kuwajima. "1P114 Folding mechanisms of a proline-free variant of staphylococcal nuclease studied by mutagenesis approach(3. Protein folding and misfolding (1),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)." Seibutsu Butsuri 46, supplement2 (2006): S175. http://dx.doi.org/10.2142/biophys.46.s175_2.

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37

Kim, Sung-Hye, Yong-Bin Yan, and Hai-Meng Zhou. "Role of osmolytes as chemical chaperones during the refolding of aminoacylase." Biochemistry and Cell Biology 84, no. 1 (February 1, 2006): 30–38. http://dx.doi.org/10.1139/o05-148.

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The refolding and reactivation of aminoacylase is particularly difficult because of serious off-pathway aggregation. The effects of 4 osmolytes — dimethylsulphoxide, glycerol, proline, and sucrose — on the refolding and reactivation of guanidine-denaturated aminoacylase were studied by measuring aggregation, enzyme activity, intrinsic fluorescence spectra, 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra, and circular dishroism (CD) spectra. The results show that all the osmolytes not only inhibit aggregation but also recover the activity of aminoacylase during refolding in a concentration-dependent manner. In particularly, a 40% glycerol concentration and a 1.5 mol/L sucrose concentration almost completely suppressed the aminoacylase aggregation. The enzyme activity measurements revealed that the influence of glycerol is more significant than that of any other osmolyte. The intrinsic fluorescence results showed that glycerol, proline, and sucrose stabilized the aminoacylase conformation effectively, with glycerol being the most effective. All 4 kinds of osmolytes reduced the exposure of the hydrophobic surface, indicating that osmolytes facilitate the formation of protein hydrophobic collapse. The CD results indicate that glycerol and sucrose facilitate the return of aminoacylase to its native secondary structure. The results of this study suggest that the ability of the various osmolytes to facilitate the refolding and renaturation of aminoacylase is not the same. A survey of the results in the literature, as well as those presented here, suggests that although the protective effect of osmolytes on protein activity and structure is equal for different osmolytes, the ability of osmolytes to facilitate the refolding of various proteins differs from case to case. In all cases, glycerol was found to be the best stabilizer and a folding aid.Key words: protein aggregation, aminoacylase, chaperone, osmolytes, protein refolding.
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38

Wu, Ying, and C. Robert Matthews. "Proline Replacements and the Simplification of the Complex, Parallel Channel Folding Mechanism for the Alpha Subunit of Trp Synthase, a TIM Barrel Protein." Journal of Molecular Biology 330, no. 5 (July 2003): 1131–44. http://dx.doi.org/10.1016/s0022-2836(03)00723-x.

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39

Syx, Delfien, Yoshihiro Ishikawa, Jan Gebauer, Sergei P. Boudko, Brecht Guillemyn, Tim Van Damme, Sanne D’hondt, et al. "Aberrant binding of mutant HSP47 affects posttranslational modification of type I collagen and leads to osteogenesis imperfecta." PLOS Genetics 17, no. 2 (February 1, 2021): e1009339. http://dx.doi.org/10.1371/journal.pgen.1009339.

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Heat shock protein 47 (HSP47), encoded by the SERPINH1 gene, is a molecular chaperone essential for correct folding of collagens. We report a homozygous p.(R222S) substitution in HSP47 in a child with severe osteogenesis imperfecta leading to early demise. p.R222 is a highly conserved residue located within the collagen interacting surface of HSP47. Binding assays show a significantly reduced affinity of HSP47-R222S for type I collagen. This altered interaction leads to posttranslational overmodification of type I procollagen produced by dermal fibroblasts, with increased glycosylation and/or hydroxylation of lysine and proline residues as shown by mass spectrometry. Since we also observed a normal intracellular folding and secretion rate of type I procollagen, this overmodification cannot be explained by prolonged exposure of the procollagen molecules to the modifying hydroxyl- and glycosyltransferases, as is commonly observed in other types of OI. We found significant upregulation of several molecular chaperones and enzymes involved in procollagen modification and folding on Western blot and RT-qPCR. In addition, we showed that an imbalance in binding of HSP47-R222S to unfolded type I collagen chains in a gelatin sepharose pulldown assay results in increased binding of other chaperones and modifying enzymes. The elevated expression and binding of this molecular ensemble to type I procollagen suggests a compensatory mechanism for the aberrant binding of HSP47-R222S, eventually leading to overmodification of type I procollagen chains. Together, these results illustrate the importance of HSP47 for proper posttranslational modification and provide insights into the molecular pathomechanisms of the p.(R222S) alteration in HSP47, which leads to a severe OI phenotype.
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40

Matsushima, Norio, Shintaro Takatsuka, Hiroki Miyashita, and Robert H. Kretsinger. "Leucine Rich Repeat Proteins: Sequences, Mutations, Structures and Diseases." Protein & Peptide Letters 26, no. 2 (February 20, 2019): 108–31. http://dx.doi.org/10.2174/0929866526666181208170027.

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Mutations in the genes encoding Leucine Rich Repeat (LRR) containing proteins are associated with over sixty human diseases; these include high myopia, mitochondrial encephalomyopathy, and Crohn’s disease. These mutations occur frequently within the LRR domains and within the regions that shield the hydrophobic core of the LRR domain. The amino acid sequences of fifty-five LRR proteins have been published. They include Nod-Like Receptors (NLRs) such as NLRP1, NLRP3, NLRP14, and Nod-2, Small Leucine Rich Repeat Proteoglycans (SLRPs) such as keratocan, lumican, fibromodulin, PRELP, biglycan, and nyctalopin, and F-box/LRR-repeat proteins such as FBXL2, FBXL4, and FBXL12. For example, 363 missense mutations have been identified. Replacement of arginine, proline, or cysteine by another amino acid, or the reverse, is frequently observed. The diverse effects of the mutations are discussed based on the known structures of LRR proteins. These mutations influence protein folding, aggregation, oligomerization, stability, protein-ligand interactions, disulfide bond formation, and glycosylation. Most of the mutations cause loss of function and a few, gain of function.
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41

Chan, W., E. Kordeli, and V. Bennett. "440-kD ankyrinB: structure of the major developmentally regulated domain and selective localization in unmyelinated axons." Journal of Cell Biology 123, no. 6 (December 15, 1993): 1463–73. http://dx.doi.org/10.1083/jcb.123.6.1463.

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440-kD ankyrinB is an alternatively spliced variant of 220-kD ankyrinB, with a predicted 220-kD sequence inserted between the membrane/spectrin binding domains and COOH-terminal domain (Kunimoto, M., E. Otto, and V. Bennett. 1991. J. Cell Biol. 236:1372-1379). This paper presents the sequence of 2085 amino acids comprising the alternatively spliced portion of 440-kD ankyrinB, and provides evidence that much of the inserted sequence has the configuration of an extended random coil. Notable features of the inserted sequence include a hydrophilicity profile that contains few hydrophobic regions, and 220 predicted sites for phosphorylation by protein kinases (casein kinase 2, protein kinase C, and proline-directed protein kinase). Secondary structure and folding of the inserted amino acid residues were deduced from properties of recombinant polypeptides. Frictional ratios of 1.9-2.4 were calculated from Stokes radii and sedimentation coefficients, for polypeptides comprising 70% of the inserted sequence, indicating a highly asymmetric shape. Circular dichroism spectra of these polypeptides indicate a nonglobular structure with negligible alpha-helix or beta sheet folding. These results suggest a ball-and-chain model for 440-kD ankyrinB with a membrane-associated globular head domain and an extended filamentous tail domain encoded by the inserted sequence. Immunofluorescence and immunoblot studies of developing neonatal rat optic nerve indicate that 440-kD ankyrinB is selectively targeted to premyelinated axons, and that 440-kD ankyrinB disappears from these axons coincident with myelination. Hypomyelinated nerve tracts of the myelin-deficient Shiverer mice exhibit elevated levels of 440-kD ankyrinB. 440-kD ankyrinB thus is a specific component of unmyelinated axons and expression of 440-kD ankyrinB may be downregulated as a consequence of myelination.
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42

PAGE, Antony P., Kenneth MacNIVEN, and Michael O. HENGARTNER. "Cloning and biochemical characterization of the cyclophilin homologues from the free-living nematode Caenorhabditis elegans." Biochemical Journal 317, no. 1 (July 1, 1996): 179–85. http://dx.doi.org/10.1042/bj3170179.

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Cyclosporin A (CsA) is the most widely used immunosuppressive agent, whose properties are exerted via an interaction with cyclophilin, resulting in down-regulation of signal-transduction events in the T-cell. Cyclophilin is identical with peptidylprolyl cis–trans isomerase (PPI; EC 5.2.1.8), an enzyme which catalyses the isomerization between the two proline conformations in proteins, thereby acting as a catalyst in protein-folding events. Several reports indicate that CsA has potent anti-parasitic activity, effective against both protozoan and helminth species. In order to understand the various biological roles that cyclophilins play we have initiated a study of these proteins in the genetically tractable nematode Caenorhabditis elegans. Here we describe the cloning and characterization of 11 cyclophilin genes (cyp-1 to -11) derived from this nematode; this is currently the greatest number of isoforms described in a single species. Southern blotting and physical mapping indicated that these genes are dispersed throughout the nematode genome. A high degree of conservation exists between several isoforms, which also share characteristics with the ubiquitous isoforms previously described. The remaining isoforms are divergent, having altered CsA-binding domains and additional non-cyclophilin domains, which may impart compartmental specificity. Ten of these isoforms have been expressed in Escherichia coli, and the resultant fusion proteins have been examined biochemically for PPI activity, which they all possess. Isomerase activity is highest in the conserved and lowest in divergent isoforms, perhaps indicating a more specific substrate for the latter. Analysis of the C. elegans cyp genes will provide answers as to the roles played by cyclophilins in protein folding and signal transduction.
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43

Roderick, H. Llewelyn, James D. Lechleiter, and Patricia Camacho. "Cytosolic Phosphorylation of Calnexin Controls Intracellular Ca2+ Oscillations via an Interaction with Serca2b." Journal of Cell Biology 149, no. 6 (June 12, 2000): 1235–48. http://dx.doi.org/10.1083/jcb.149.6.1235.

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Calreticulin (CRT) and calnexin (CLNX) are lectin chaperones that participate in protein folding in the endoplasmic reticulum (ER). CRT is a soluble ER lumenal protein, whereas CLNX is a transmembrane protein with a cytosolic domain that contains two consensus motifs for protein kinase (PK) C/proline- directed kinase (PDK) phosphorylation. Using confocal Ca2+ imaging in Xenopus oocytes, we report here that coexpression of CLNX with sarco endoplasmic reticulum calcium ATPase (SERCA) 2b results in inhibition of intracellular Ca2+ oscillations, suggesting a functional inhibition of the pump. By site-directed mutagenesis, we demonstrate that this interaction is regulated by a COOH-terminal serine residue (S562) in CLNX. Furthermore, inositol 1,4,5-trisphosphate– mediated Ca2+ release results in a dephosphorylation of this residue. We also demonstrate by coimmunoprecipitation that CLNX physically interacts with the COOH terminus of SERCA2b and that after dephosphorylation treatment, this interaction is significantly reduced. Together, our results suggest that CRT is uniquely regulated by ER lumenal conditions, whereas CLNX is, in addition, regulated by the phosphorylation status of its cytosolic domain. The S562 residue in CLNX acts as a molecular switch that regulates the interaction of the chaperone with SERCA2b, thereby affecting Ca2+ signaling and controlling Ca2+-sensitive chaperone functions in the ER.
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44

VANHOVE, Marc, Gilliane GUILLAUME, Philippe LEDENT, John H. RICHARDS, Roger H. PAIN, and Jean-Marie FRÈRE. "Kinetic and thermodynamic consequences of the removal of theCys-77–Cys-123 disulphide bond for the folding of TEM-1 β-lactamase." Biochemical Journal 321, no. 2 (January 15, 1997): 413–17. http://dx.doi.org/10.1042/bj3210413.

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Class A α-lactamases of the TEM family contain a single disulphide bond which connects cysteine residues 77 and 123. To clarify the possible role of the disulphide bond in the stability and folding kinetics of the TEM-1 α-lactamase, this bond was removed by introducing a Cys-77 → Ser mutation, and the enzymically active mutant protein was studied by reversible guanidine hydrochloride-induced denaturation. The unfolding and refolding rates were monitored using tryptophan fluorescence. At low guanidine hydrochloride concentrations, the refolding of the wild-type and mutant enzymes followed biphasic time courses. The characteristics of the two phases were not significantly affected by the mutation. Double-jump experiments, in which the protein was unfolded in a high concentration of guanidine hydrochloride for a short time period and then refolded by diluting out the denaturant, indicated that, for both the wild-type and mutant enzymes, the two refolding phases could be ascribed to proline isomerization reactions. Equilibrium unfolding experiments monitored by fluorescence spectroscopy and far-UV CD indicated a three-state mechanism (N ↔ H ↔ U). Both the folded mutant protein (N) and, to a lesser extent, the thermodynamically stable intermediate, H, were destabilized relative to the fully unfolded state, U. Removal of the disulphide bond resulted in a decrease of 14.2 kJ/mol (3.4 kcal/mol) in the global free energy of stabilization. Similarly, the mutation also induced a drastic increase in the rate of thermal inactivation.
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45

Lang, Ping, Can-kui Zhang, Fenny Dane, Shasha Meng, Robert Ebel, and Narendra Singh. "(87) Molecular Characterization of Cold Acclimation in Poncirus and Citrus." HortScience 41, no. 4 (July 2006): 1021E—1022. http://dx.doi.org/10.21273/hortsci.41.4.1021e.

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Commercial citrus species, some of the most important fruit crops worldwide, are sensitive to sub-freezing temperatures. Poncirus trifoliata, a species closely related to commercial citrus and tolerant to –30 °C, has been used in breeding programs or as a rootstock to impart greater freeze tolerance. Gene expression of P. trifoliata and C. unshiu (Satsuma mandarin) were investigated and compared under slow and fast cold-acclimation regimes. The mRNA differential display-polymerase chain reaction (DDRT-PCR) and cDNA-AFLP, coupled with quantitative relative RT-PCR or real-time PCR were used. Many unique gene fragments were isolated and found to be up- or down–regulated as a result of exposure to low temperature. The up-regulated fragments in Poncirus show high similarities to genes involved in osmotic regulation (betaine/proline transporter, water channel protein, and nitrate transporter), oxidative stress (aldoketo reductase, early light induced protein), and protein interaction (tetratricopeptide-repeat protein, F-box protein, and ribosomal protein L15). In C. unshiu the up-regulated genes show high similarities to genes involved in transcription (zinc finger and GTP-binding protein-related), signal transduction (14–3–3 protein and extension-like protein), protein synthesis and amino acid translocation (permease and ribosomal proteins), chromosome folding (chromosome condensation, structural maintenance of chromosomes-like protein), and carbohydrate metabolism (glycosyl transferase). Several genes involved in photosynthesis, defense and cell wall metabolism were down regulated. Characterization of cold responsive genes will be discussed.
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46

Lee, D. H., M. Y. Sherman, and A. L. Goldberg. "Involvement of the molecular chaperone Ydj1 in the ubiquitin-dependent degradation of short-lived and abnormal proteins in Saccharomyces cerevisiae." Molecular and Cellular Biology 16, no. 9 (September 1996): 4773–81. http://dx.doi.org/10.1128/mcb.16.9.4773.

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In Escherichia coli and mitochondria, the molecular chaperone DnaJ is required not only for protein folding but also for selective degradation of certain abnormal polypeptides. Here we demonstrate that in the yeast cytosol, the homologous chaperone Ydj1 is also required for ubiquitin-dependent degradation of certain abnormal proteins. The temperature-sensitive ydj1-151 mutant showed a large defect in the overall breakdown of short-lived cell proteins and abnormal polypeptides containing amino acid analogs, especially at 38 degrees C. By contrast, the degradation of long-lived cell proteins, which is independent of ubiquitin, was not altered nor was cell growth affected. The inactivation of Ydj1 markedly reduced the rapid, ubiquitin-dependent breakdown of certain beta-galactosidase (beta-gal) fusion polypeptides. Although degradation of N-end rule substrates (arginine-beta-gal and leucine-beta-gal) and the B-type cyclin Clb5-beta-gal occurred normally, degradation of the abnormal polypeptide ubiquitin-proline-beta-gal (Ub-P-beta-gal) and that of the short-lived normal protein Gcn4 were inhibited. As a consequence of reduced degradation of Ub-P-beta-gal, the beta-gal activity was four to five times higher in temperature-sensitive ydj1-151 mutant cells than in wild-type cells; thus, the folding and assembly of this enzyme do not require Ydj1 function. In wild-type cells, but not in ydj1-151 mutant cells, this chaperone is associated with the short-lived substrate Ub-P-beta-gal and not with stable beta-gal constructs. Furthermore, in the ydj1-151 mutant, the ubiquitination of Ub-P-beta-gal was blocked and the total level of ubiquitinated protein in the cell was reduced. Thus, Ydj1 is essential for the ubiquitin-dependent degradation of certain proteins. This chaperone may facilitate the recognition of unfolded proteins or serve as a cofactor for certain ubiquitin-ligating enzymes.
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47

Zeng, Jingjue, Xudong Zhu, Muhammad S. Haider, Xicheng Wang, Cheng Zhang, and Chen Wang. "Genome-Wide Identification and Analysis of the Type-B Authentic Response Regulator Gene Family in Peach (Prunus persica)." Cytogenetic and Genome Research 151, no. 1 (2017): 41–49. http://dx.doi.org/10.1159/000458170.

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The type-B authentic response regulator (ARR-B) family members serve as DNA-binding transcriptional regulators, whose activities are probably regulated by phosphorylation/dephosphorylation, resulting in the rapid induction of type-A ARR genes. Type-B ARRs are believed to be involved in many biological processes, including cytokinin signaling, plant growth, and stress responses through a chaperone or by isomerization of proline residues during protein folding. The public availability of complete peach genome sequences allows the identification of 23 ARR-B genes by HMMER and blast analysis. Scaffold locations of these genes in the peach genome were determined, and the protein domain and motif organization of peach type-B ARRs were analyzed. The phylogenetic relationships between peach type-B ARRs were also assessed. The expression profiles of peach ARR-B genes revealed that most of the type-B ARRs showed high expression levels in tissues undergoing rapid cell division and may engage more cytokinins, like half-opened flowers, fruits at expansion stages, and young leaves. These findings not only contribute to a better understanding of the complex regulation of the peach ARR-B gene family, but also provide valuable information for future research in peach functional genomics.
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48

Jaouen, Thomas, Emmanuelle D�, Sylvie Chevalier, and Nicole Orange. "Pore Size Dependence on Growth Temperature Is a Common Characteristic of the Major Outer Membrane Protein OprF in Psychrotrophic and Mesophilic Pseudomonas Species." Applied and Environmental Microbiology 70, no. 11 (November 2004): 6665–69. http://dx.doi.org/10.1128/aem.70.11.6665-6669.2004.

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ABSTRACT Pseudomonas species adapt well to hostile environments, which are often subjected to rapid variations. In these bacteria, the outer membrane plays an important role in the sensing of environmental conditions such as temperature. In previous studies, it has been shown that in the psychrotrophic strain P. fluorescens MF0, the major porin OprF changes its channel size according to the growth conditions and could affect outer membrane permeability. Studies of the channel-forming properties of OprFs from P. putida 01G3 and P. aeruginosa PAO1 in planar lipid bilayers generated similar results. The presence of a cysteine- or proline-rich cluster in the central linker region is not essential for channel size modulations. These findings suggest that OprF could adopt two alternative conformations in the outer membrane and that folding is thermoregulated. In contrast, no difference according to growth temperature was observed for structurally different outer membrane proteins, such as OprE3 from the Pseudomonas OprD family of specific porins. Our results are consistent with the fact that the decrease in channel size observed at low growth temperature is a particular feature of the OprF porin in various psychrotrophic and mesophilic Pseudomonas species isolated from diverse ecological niches. The ability to reduce outer membrane permeability at low growth temperature could provide these bacteria with adaptive advantages.
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49

Kubyshkin, Vladimir, and Nediljko Budisa. "The Alanine World Model for the Development of the Amino Acid Repertoire in Protein Biosynthesis." International Journal of Molecular Sciences 20, no. 21 (November 5, 2019): 5507. http://dx.doi.org/10.3390/ijms20215507.

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A central question in the evolution of the modern translation machinery is the origin and chemical ethology of the amino acids prescribed by the genetic code. The RNA World hypothesis postulates that templated protein synthesis has emerged in the transition from RNA to the Protein World. The sequence of these events and principles behind the acquisition of amino acids to this process remain elusive. Here we describe a model for this process by following the scheme previously proposed by Hartman and Smith, which suggests gradual expansion of the coding space as GC–GCA–GCAU genetic code. We point out a correlation of this scheme with the hierarchy of the protein folding. The model follows the sequence of steps in the process of the amino acid recruitment and fits well with the co-evolution and coenzyme handle theories. While the starting set (GC-phase) was responsible for the nucleotide biosynthesis processes, in the second phase alanine-based amino acids (GCA-phase) were recruited from the core metabolism, thereby providing a standard secondary structure, the α-helix. In the final phase (GCAU-phase), the amino acids were appended to the already existing architecture, enabling tertiary fold and membrane interactions. The whole scheme indicates strongly that the choice for the alanine core was done at the GCA-phase, while glycine and proline remained rudiments from the GC-phase. We suggest that the Protein World should rather be considered the Alanine World, as it predominantly relies on the alanine as the core chemical scaffold.
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50

Vijaya, S., N. Elango, F. Zavala, and B. Moss. "Transport to the cell surface of a peptide sequence attached to the truncated C terminus of an N-terminally anchored integral membrane protein." Molecular and Cellular Biology 8, no. 4 (April 1988): 1709–14. http://dx.doi.org/10.1128/mcb.8.4.1709.

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Attempts to construct hybrid proteins that are transported to the plasma membrane are frequently unsuccessful because of perturbations in polypeptide folding. In seeking to minimize this problem, we have used the less common type of integral membrane protein, which has an uncleaved signal-anchor domain and an extracellular carboxyl portion, to transport a peptide sequence of interest to the cell surface. A set of plasmids was constructed that contained the gene encoding respiratory syncytial virus glycoprotein G (RSVG) interrupted immediately after one of several proline codons by a synthetic sequence containing unique restriction endonuclease sites and a stop codon. The shortened RSVG gene was flanked by vaccinia virus DNA to permit cloning and expression in a vaccinia virus vector. An open reading frame encoding four copies of the immunodominant repeating epitope of the circumsporozoite protein of Plasmodium falciparum was inserted into the tails of the truncated RSVG genes. Recombinant vaccinia viruses were isolated and shown to express hybrid proteins that reacted with a monoclonal antibody directed to the repeating circumsporozoite epitope. Moreover, immunofluorescence studies indicated that the peptide was on the external cell surface and available to react with antibodies. Expression of the hybrid protein also occurred in rabbits inoculated with the live recombinant vaccinia virus, as demonstrated by the generation of antibodies that bound to P. falciparum sporozoites in vitro.
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