Protein biosynthesis

Summary

Protein biosynthesis (or protein synthesis) is a core biological process, occurring inside cells, balancing the loss of cellular proteins (via degradation or export) through the production of new proteins. Proteins perform a number of critical functions as enzymes, structural proteins or hormones. Protein synthesis is a very similar process for both prokaryotes and eukaryotes but there are some distinct differences. Protein synthesis can be divided broadly into two phases—transcription and translation. During transcription, a section of DNA encoding a protein, known as a gene, is converted into a template molecule called messenger RNA (mRNA). This conversion is carried out by enzymes, known as RNA polymerases, in the nucleus of the cell. In eukaryotes, this mRNA is initially produced in a premature form (pre-mRNA) which undergoes post-transcriptional modifications to produce mature mRNA. The mature mRNA is exported from the cell nucleus via nuclear pores to the cytoplasm of the cell

Official source

https://en.wikipedia.org/wiki/Protein_biosynthesis

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Nature-inspired Circular-economy Recycling (NaCRe) for Proteins

Simone Giaveri

Since a few decades our planet has been loaded with billion tons of synthetic polymer-based materials, commonly named plastics. The large scale of plastic production, associated with its limited recyclability, are the driving force for the accumulation of such materials into the environment. Garbage patches, i.e. islands of plastics in the ocean, are striking evidences of this issue. A sustainable handling, that is production and disposal, of synthetic polymer-based materials is one of the greatest challenges that humanity has to face.Proteins and nucleic acids are natural polymers. Arguably, Nature produces an amount of such polymers that is higher with respect to man-made polymers. However, these materials do not accumulate into the environment, that is the approach used by Nature to handle these polymers is sustainable. The secret for its sustainaibility lies on the circularity in the materialsâ use. Indeed, proteins and nucleic acids are sequence-defined polymers that undergo depolymerization to monomers, and recycling into new materials by reassembling the so obtained monomers into arbitrarily different sequences. Organisms digest proteins into amino acids. These monomers are in turn polymerized to produce the protein of need, at the time of the protein synthesis.In this thesis we show that this process is achievable outside living organisms. Indeed, we depolymerized structurally different short peptides, and peptides mixtures into their constitutive amino acids, and recycled such monomers into biotechnologically relevant proteins (green, and red fluorescent proteins), by using an amino acid-free cell-free transcription-translation system. We further applied our methodology to recycle proteins with high relevance in materials engineering such as Î²-lactoglobulin films, used for water filtration, or silk fibroin solutions into green fluorescent protein. We were also successful in recycling mixtures composed of short peptides, and technologically relevant proteins into fluorescent proteins, as well as into bioactive enzymes (catechol 2,3-dioxygenase). Finally, we achieved multiple cycles of recycling (two cycles), and we demonstrated that the strategy can be expanded beyond the set of the twenty proteinogenic amino acids.Presented herein is a nature-inspired approach to recycling of soft materials, where unknown mixtures of polymers are recycled into the polymer of need, by the local community, at the time of recycling. The materials are costantly transformed into different ones, without any external feed, and there is no way to distinguish a new polymer from a recycled one. The strength of this method lies in its compatibility with the principles of circular economy.

EPFL2021

Structural characterisation and membrane insertion of M13 procoat, M13 coat and Pf3 coat proteins

Membrane proteins fulfill many central functions in the biological membrane. The insertion process of these proteins and their structure, which are intimately linked to their function, are not yet well understood. As a model we studied three proteins reconstituted into lipid bilayers (which serve as a model for the biological membrane): the major coat proteins of phages Pf3 and M13 and the precursor of the latter, M13 procoat protein, which spans the inner membrane two times and serves as a model for a multi-spanning membrane protein. Whereas Pf3 coat protein can insert into the membrane in its mature form, M13 coat protein needs the signal sequence present in the precursor to insert. A new, simple and efficient purification method has been developed for M13 procoat, M13 and Pf3 coat proteins. Homogenous preparations were obtained in isopropanol/0.l% TFA, where M13 coat protein is found to be dissolved in a monomeric form, and the two other proteins as dimers. The conformations of these particular proteins in different environments have been determined by circular dichroism and infrared spectroscopy. In organic solvents, the proteins adopt a conformation with an average helix content of 90%. In lipid bilayers composed of phospatidylcholine and phosphatidylglycerol lipids, the average helix content is 50% for M13 procoat protein, 60% for M13 coat protein and 75% for Pf3 coat protein. In order to characterize the orientational distribution the orientational order parameter Sα of the protein helices in planar lipid bilayers have been determined by polarized infrared measurements in the amide I spectral range. The helices of the three proteins are oriented preferentially parallel to the membrane normal, with Sα = 0.63 for M13 procoat protein, Sα = 0.58 for Pf3 coat protein and a distinctly higher value of Sα = 0.81 for M13 coat protein. The topology of M13 and Pf3 coat proteins was investigated using limited proteolysis in lipid vesicles with proteinase K combined with mass spectroscopy to analyze the lytic fragments. The C-terminal part of both proteins was found to be inaccessible to proteinase K, only 14 (Pf3) or 15 (M13) residues on the N-terminal were protease accessible from the outside of the vesicles. Furthermore, the standard free energy change, ΔGo, of a membrane-inserting protein with a leader sequence has been determined experimentally for the first time, using M13 procoat protein as an example. The partition coefficient for the distribution of this protein between the aqueous phase and the membrane phase of preformed lipid vesicles yielded a value of Γ = 6.5×105 M-1, corresponding to a ΔGo of -10.4 kcal/mol, based on measurements of the fluorescence energy transfer between the intrinsic tryptophan of the protein and a suitably labeled lipid membrane of POPC. For comparison, the partition coefficient of the M13 coat protein between the aqueous and the POPC lipid bilayer phase was determined to be distinctly lower: Γ = 1×105 M-1 (ΔGo = -9.3 kcal/mol). Proteinase K digestion experiments have been performed, showing that 20% of the procoat protein bound to lipid vesicles spontaneously integrate in a transbilayer form, whereas 80% remain inserted in the interfacial membrane region. By taking together these results, an upper limit for the free energy change of the transmembrane insertion of procoat protein was estimated to be -14.8 kcal/mol. In order to distinguish further the contribution arising from insertion of the procoat protein into the membrane interfacial region from that due to transmembrane insertion, the partition coefficient of a mutant procoat protein OM30R (which contains a positively charged amino acid in its mature hydrophobic segment (exchange of a Val to an Arg residue at position 30)) was determined, yielding Γ = 0.3×105 M-1 (ΔGo = -8.6 kcal/mol). Previously reported in vivo experiments have shown that the OM30R mutant protein is not translocated across Escherichia coli inner membrane, but only binds to the inner surface. The results presented here indicate that although the insertion of the procoat protein into the interfacial region of the lipid bilayer contributes the major part to ΔGo, it is the final energy gain of the interaction of the hydrophobic portions of the folded pre-protein with the lipid chains which drives the transmembrane insertion of the M13 procoat protein. Neither the leader sequence nor the mature coat protein alone yields this free energy gain. For the different proteins investigated here, spontaneous membrane insertion occurs only for fluid lipid bilayers, but not for membranes in the crystalline lipid phase. Furthermore, by using lipid bilayers with negative membrane surface charges, it was shown that both procoat and coat proteins are electrostatically attracted to the surface of the lipid membrane, though only to a small extent, with apparent partition coefficients of the same order of magnitude as for the phosphatidylcholine lipid membrane. This work has yielded new information about the structure and the insertion of the phage coat proteins. The new methods developed in this work might be of general applicability to study the structural dynamics of membrane protein assembly.

EPFL1996

Cells need to be able to precisely control their proteome composition in order to maintain homeostasis. This is of particular relevance in fast dividing cells, such as embryonic stem cells, since their global protein levels need to be doubled at each division to ensure reliable growth and pluripotency maintenance. However, how protein synthesis and degradation interplay to regulate global protein expression levels throughout the cell cycle remains unclear. In addition, while cell-to-cell variability at the level of transcription and protein expression is relatively well studied, it remains unknown to what extent protein degradation rates vary between single cells. Previous studies focusing on protein turnover have mainly relied on bulk biochemical approaches, thus only providing population averages and a low temporal resolution. In addition, many approaches to study the changes in protein turnover throughout the cell cycle have been dependent on cell cycle synchonizing drugs, which might interfere with protein synthesis and also reduce cell viability. Therefore, we further developed and applied two fluorescence time-lapse imaging strategies to study the cell cycle dependence and cell-to-cell variability of protein synthesis and degradation in unperturbed dividing mouse embryonic stem (ES) cells: 1) a Mammalian Cell-optimized Tandem Fluorescent Timer (MCFT), consisting of the fusion of the fast maturing green fluorescent protein superfolder GFP (sfGFP) and the slower maturing red fluorescent protein mOrange2, which in combination with computational modeling allows for disentangling changes in protein synthesis from changes in protein degradation in single living cells, and 2) fluorescent pulse labeling of protein tags (SNAP/Halo-tags) to monitor protein half-lives, as well as transient changes in protein degradation rates in single cells without the use of protein synthesis inhibitors. We discovered a substantial cell cycle-dependence in both protein synthesis and degradation. In particular, a large fraction (40%) of proteins was stabilized around cytokinesis, an effect that has not been described previously. Interestingly, protein degradation rates were highly variable between single cells and were positively correlated with synthesis rates in individual cells to buffer protein expression level variability. Furthermore, degradation rates of different proteins co-varied between single cells, both in ES cells and fibroblasts, suggesting variability in proteasome activity as a source of global extrinsic noise in gene expression. We identified the proteasome component ADRM1 as a potential limiting factor that contributes to generating such noise, as its expression levels were negatively correlated with protein half-lives in single cells. Our work demonstrates the importance of single cell live imaging approaches to study dynamic processes and cell-to-cell variability and sheds more light on the complex interplay of protein synthesis and degradation in determining protein expression levels in single mammalian cells.

EPFL2018