The translocon is a complex of proteins associated with the translocation of polypeptides across membranes. In eukaryotes the term translocon most refers to the complex that transports nascent polypeptides with a targeting signal sequence into the interior space of the endoplasmic reticulum from the cytosol; this translocation process requires the protein to cross a hydrophobic lipid bilayer. The same complex is used to integrate nascent proteins into the membrane itself. In prokaryotes, a similar protein complex transports polypeptides across the plasma membrane or integrates membrane proteins. Bacterial pathogens can assemble other translocons in their host membranes, allowing them to export virulence factors into their target cells. In either case, the protein complex are formed from Sec proteins, with the hetrotrimeric Sec61 being the channel. In prokaryotes, the homologous channel complex is known as SecYEG; the translocation channel is a hetero-trimeric protein complex called SecYEG in prokaryotes and Sec61 in eukaryotes.
It consists of the subunits SecY, SecE, SecG. The structure of this channel, in its idle state, has been solved by X-ray crystallography in archaea. SecY is the large pore subunit. In a side view, the channel has an hourglass shape, with a funnel on each side; the extracellular funnel has a little "plug" formed out of an alpha-helix. In the middle of the membrane is a construction, formed from a pore ring of six hydrophobic amino acids that project their side chains inwards. During protein translocation, the plug is moved out of the way, a polypeptide chain is moved from the cytoplasmic funnel, through the pore ring, the extracellular funnel, into the extracellular space. Hydrophobic segments of membrane proteins exit sideways through the lateral gate into the lipid phase and become membrane-spanning segments. In E. coli, SecYEG complexes dimerize on the membrane. In eukaryotes, multiple copies of Sec61 come together and form a larger complex along with further components such as the oligosaccharyl transferase complex, the TRAP complex, the membrane protein TRAM.
For further components, such as signal peptidase complex and the SRP receptor it is not clear to what extent they only associate transiently to the translocon complex. The channel allows peptides to move in either direction, so additional systems in the translocon are required to move the peptide in a specific direction. There are three types of translocation: cotranslational translocation that happens as translation happens, two types of post-translational translocation that happens after translation, each seen in eukaryotes and bacteria. While eukaryotes unfold the protein with BiP and use other complexes to transport the peptide, bacteria use the SecA ATPase. In co-translational translocation, the translocon associates with the ribosome so that a growing nascent polypeptide chain is moved from the ribosome tunnel into the SecY channel; the translocon acts as a channel through the hydrophobic membrane of the endoplasmic reticulum. The emerging polypeptide is threaded through the channel as an unfolded string of amino acids driven by a Brownian Ratchet.
Once translation is finished, a signal peptidase cleaves off the short signal peptide from the nascent protein, leaving the polypeptide free in the interior of the endoplasmic reticulum. In eukaryotes, proteins due to be translocated to the endoplasmic reticulum are recognized by the signal-recognition particle, which halts translation of the polypeptide by the ribosome while it attaches the ribosome to the SRP receptor on the endoplasmic reticulum; this recognition event is based upon a specific N-terminal signal sequence, in the first few codons of the polypeptide to be synthesised. Bacteria use an SRP, together with a chaperone YidC, similar to the eukaryote TRAM; the translocon can translocate and integrate membrane proteins in the correct orientation into the membrane of the endoplasmic reticulum. The mechanism of this process is not understood, but involves the recognition and processing by the translocon of hydrophobic stretches in the amino acid sequence which are destined to become transmembrane helices.
The plug alters between its closed states to place helices in different orientations. In eukaryotes, post-translational translocation depends on BiP and other complexes, including the SEC62/SEC63 integral membrane protein complex. In this mode of translocation, Sec63 helps BiP hydrolyze ATP, which binds to the peptide and "pulls" it out; this process is repeated for other BiP molecules. In bacteria, the same process is done by a "pushing" ATPase known as SecA, sometimes assisted by the SecDF complex on the other side responsible for pulling; the SecA ATPase uses a "push-and-slide" mechanism to move a polypeptide through the channel. In the ATP-bound state, SecA interacts through a two-helix finger with a subset of amino acids in a substrate, pushing them into the channel; the interaction is weakened as SecA enters the ADP-bound state, allowing the polypeptide chain to slide passively in either direction. SecA grabs a further section of the peptide to repeat the process. Translocators can move polypeptides from the cisternal space of the endoplasmic reticulum to the cytosol.
ER-proteins are degraded in the cytosol by the 26S proteasome, a process known as Endoplasmic-reticulum-associated protein degradation, therefore have to be transported by an appropriate channel. This retrotranslocon is
The endoplasmic reticulum is a type of organelle found in eukaryotic cells that forms an interconnected network of flattened, membrane-enclosed sacs or tube-like structures known as cisternae. The membranes of the ER are continuous with the outer nuclear membrane; the endoplasmic reticulum occurs in most types of eukaryotic cells, but is absent from red blood cells and spermatozoa. There are two types of ER: smooth endoplasmic reticulum; the outer face of the rough endoplasmic reticulum is studded with ribosomes that are the sites of protein synthesis. The rough endoplasmic reticulum is prominent in cells such as hepatocytes; the smooth endoplasmic reticulum lacks ribosomes and functions in lipid synthesis but not metabolism, the production of steroid hormones, detoxification. The smooth ER is abundant in mammalian liver and gonad cells; the ER was observed with light microscope by Garnier in 1897, who coined the term "ergastoplasm". With electron microscopy, the lacy membranes of the endoplasmic reticulum were first seen in 1945 by Keith R. Porter, Albert Claude, Ernest F. Fullam.
The word "reticulum", which means "network", was applied by Porter in 1953 to describe this fabric of membranes. The general structure of the endoplasmic reticulum is a network of membranes called cisternae; these sac-like structures are held together by the cytoskeleton. The phospholipid membrane encloses the cisternal space, continuous with the perinuclear space but separate from the cytosol; the functions of the endoplasmic reticulum can be summarized as the synthesis and export of proteins and membrane lipids, but varies between ER and cell type and cell function. The quantity of both rough and smooth endoplasmic reticulum in a cell can interchange from one type to the other, depending on the changing metabolic activities of the cell. Transformation can include embedding of new proteins in membrane as well as structural changes. Changes in protein content may occur without noticeable structural changes; the surface of the rough endoplasmic reticulum is studded with protein-manufacturing ribosomes giving it a "rough" appearance.
The binding site of the ribosome on the rough endoplasmic reticulum is the translocon. However, the ribosomes are not a stable part of this organelle's structure as they are being bound and released from the membrane. A ribosome only binds to the RER; this special complex forms when a free ribosome begins translating the mRNA of a protein destined for the secretory pathway. The first 5–30 amino acids polymerized encode a signal peptide, a molecular message, recognized and bound by a signal recognition particle. Translation pauses and the ribosome complex binds to the RER translocon where translation continues with the nascent protein forming into the RER lumen and/or membrane; the protein is processed in the ER lumen by an enzyme. Ribosomes at this point may be released back into the cytosol; the membrane of the rough endoplasmic reticulum forms large double membrane sheets that are located near, continuous with, the outer layer of the nuclear envelope. The double membrane sheets are stacked and connected through several right or left-handed helical ramps, the so-called Terasaki ramps, giving rise to a structure resembling a multi-storey car park.
Although there is no continuous membrane between the endoplasmic reticulum and the Golgi apparatus, membrane-bound transport vesicles shuttle proteins between these two compartments. Vesicles are surrounded by coating proteins called COPI and COPII. COPII targets vesicles to the Golgi apparatus and COPI marks them to be brought back to the rough endoplasmic reticulum; the rough endoplasmic reticulum works in concert with the Golgi complex to target new proteins to their proper destinations. A second method of transport out of the endoplasmic reticulum involves areas called membrane contact sites, where the membranes of the endoplasmic reticulum and other organelles are held together, allowing the transfer of lipids and other small molecules; the rough endoplasmic reticulum is key in multiple functions: Manufacture of lysosomal enzymes with a mannose-6-phosphate marker added in the cis-Golgi network. Manufacture of secreted proteins, either secreted constitutively with no tag or secreted in a regulatory manner involving clathrin and paired basic amino acids in the signal peptide.
Integral membrane proteins that stay embedded in the membrane as vesicles exit and bind to new membranes. Rab proteins are key in targeting the membrane. Initial glycosylation as assembly continues; this is N-linked. N-linked glycosylation: If the protein is properly folded, Oligosaccharyltransferase recognizes the AA sequence NXS or NXT and adds a 14-sugar backbone to the side-chain nitrogen of Asn. In most cells the smooth endoplasmic reticulum is scarce. Instead there are areas where the ER is smooth and rough, this area is called the transitional ER; the transitional ER gets its name. These are areas where the transport vesicles that contain lipids and proteins made in the ER, detach from the ER and start moving to the Golgi apparatus. Specialized cells can have a lot of smooth endoplasmic reticulum and in these cells the smooth ER has many functions
Archaea constitute a domain of single-celled microorganisms. These microbes are prokaryotes. Archaea were classified as bacteria, receiving the name archaebacteria, but this classification is outdated. Archaeal cells have unique properties separating them from the other two domains of life and Eukarya. Archaea are further divided into multiple recognized phyla. Classification is difficult because most have not been isolated in the laboratory and were only detected by analysis of their nucleic acids in samples from their environment. Archaea and bacteria are similar in size and shape, although a few archaea have shapes quite unlike that of bacteria, such as the flat and square-shaped cells of Haloquadratum walsbyi. Despite this morphological similarity to bacteria, archaea possess genes and several metabolic pathways that are more related to those of eukaryotes, notably for the enzymes involved in transcription and translation. Other aspects of archaeal biochemistry are unique, such as their reliance on ether lipids in their cell membranes, including archaeols.
Archaea use more energy sources than eukaryotes: these range from organic compounds, such as sugars, to ammonia, metal ions or hydrogen gas. Salt-tolerant archaea use sunlight as an energy source, other species of archaea fix carbon, but unlike plants and cyanobacteria, no known species of archaea does both. Archaea reproduce asexually by budding; the first observed archaea were extremophiles, living in harsh environments, such as hot springs and salt lakes with no other organisms, but improved detection tools led to the discovery of archaea in every habitat, including soil and marshlands. They are part of the microbiota of all organisms, in the human microbiota they are important in the gut, on the skin. Archaea are numerous in the oceans, the archaea in plankton may be one of the most abundant groups of organisms on the planet. Archaea are a major part of Earth's life, may play roles in the carbon cycle and the nitrogen cycle. No clear examples of archaeal pathogens or parasites are known. Instead they are mutualists or commensals, such as the methanogens that inhabit the gastrointestinal tract in humans and ruminants, where their vast numbers aid digestion.
Methanogens are used in biogas production and sewage treatment, biotechnology exploits enzymes from extremophile archaea that can endure high temperatures and organic solvents. For much of the 20th century, prokaryotes were regarded as a single group of organisms and classified based on their biochemistry and metabolism. For example, microbiologists tried to classify microorganisms based on the structures of their cell walls, their shapes, the substances they consume. In 1965, Emile Zuckerkandl and Linus Pauling proposed instead using the sequences of the genes in different prokaryotes to work out how they are related to each other; this phylogenetic approach is the main method used today. Archaea – at that time only the methanogens were known – were first classified separately from bacteria in 1977 by Carl Woese and George E. Fox based on their ribosomal RNA genes, they called these groups the Urkingdoms of Archaebacteria and Eubacteria, though other researchers treated them as kingdoms or subkingdoms.
Woese and Fox gave the first evidence for Archaebacteria as a separate "line of descent": 1. Lack of peptidoglycan in their cell walls, 2. Two unusual coenzymes, 3. Results of 16S ribosomal RNA gene sequencing. To emphasize this difference, Otto Kandler and Mark Wheelis proposed reclassifying organisms into three natural domains known as the three-domain system: the Eukarya, the Bacteria and the Archaea, in what is now known as "The Woesian Revolution"; the word archaea comes from the Ancient Greek ἀρχαῖα, meaning "ancient things", as the first representatives of the domain Archaea were methanogens and it was assumed that their metabolism reflected Earth's primitive atmosphere and the organisms' antiquity, but as new habitats were studied, more organisms were discovered. Extreme halophilic and hyperthermophilic microbes were included in Archaea. For a long time, archaea were seen as extremophiles that only exist in extreme habitats such as hot springs and salt lakes, but by the end of the 20th century, archaea had been identified in non-extreme environments as well.
Today, they are known to be a large and diverse group of organisms abundantly distributed throughout nature. This new appreciation of the importance and ubiquity of archaea came from using polymerase chain reaction to detect prokaryotes from environmental samples by multiplying their ribosomal genes; this allows the detection and identification of organisms that have not been cultured in the laboratory. The classification of archaea, of prokaryotes in general, is a moving and contentious field. Current classification systems aim to organize archaea into groups of organisms that share structural features and common ancestors; these classifications rely on the use of the sequence of ribosomal RNA genes to reveal relationships between organisms. Most of the culturable and well-investigated species of archaea are members of two main phyla, the Euryarchaeota and Crenarchaeota. Other groups have been tentatively created. For example, the peculiar species Nanoarchaeum equitans, discovered in 2003, has been given its own phylum, the Nanoarchaeota.
A new phylum Korarchaeota has been proposed. It contains a sm
The SecY protein is the main transmembrane subunit of the bacterial Sec or Type II secretory pathway and a protein-secreting ATPase complex known as a SecYEG translocon. Homologs of the SecYEG complex are found in eukaryotes, where the subunit is known as Sec61α, in archaea. Secretion of some proteins carrying a signal-peptide across the inner membrane in Gram-negative bacteria occurs via the preprotein translocase pathway. Proteins are produced in the cytoplasm as precursors, require a chaperone subunit to direct them to the translocase component within the membrane. From there, the mature proteins are either targeted to the outer membrane, or remain as periplasmic proteins; the translocase protein subunits are encoded on the bacterial chromosome. The translocase pathway comprises 7 proteins, including a chaperone protein, an ATPase, an integral membrane complex, two additional membrane proteins that promote the release of the mature peptide into the periplasm; the chaperone protein SecB is a acidic homotetrameric protein that exists as a "dimer of dimers" in the bacterial cytoplasm.
SecB maintains preproteins in an unfolded state after translation, targets these to the peripheral membrane protein ATPase SecA for secretion. The structure of the Escherichia coli SecYEG assembly revealed a sandwich of two membranes interacting through the extensive cytoplasmic domains; each membrane is composed of dimers of SecYEG. The monomeric complex contains 15 transmembrane helices; the eubacterial SecY protein interacts with the signal sequences of secretory proteins as well as with two other components of the protein translocation system: SecA and SecE. SecY is an integral plasma membrane protein of 419 to 492 amino acid residues that contains 10 transmembrane, 6 cytoplasmic and 5 periplasmic regions. Cytoplasmic regions 2 and 3, TM domains 1, 2, 4, 5, 7 and 10 are well conserved: the conserved cytoplasmic regions are believed to interact with cytoplasmic secretion factors, while the TM domains may participate in protein export. SecY is encoded in the chloroplast genome of some algae where it could be involved in a prokaryotic-like protein export system across the two membranes of the chloroplast endoplasmic reticulum, present in chromophyte and cryptophyte algae.
SecY-related translocase InterPro: IPR014269 SEC61A1.
Protein Data Bank
The Protein Data Bank is a database for the three-dimensional structural data of large biological molecules, such as proteins and nucleic acids. The data obtained by X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy, submitted by biologists and biochemists from around the world, are accessible on the Internet via the websites of its member organisations; the PDB is overseen by an organization called the Worldwide Protein Data Bank, wwPDB. The PDB is a key in areas such as structural genomics. Most major scientific journals, some funding agencies, now require scientists to submit their structure data to the PDB. Many other databases use protein structures deposited in the PDB. For example, SCOP and CATH classify protein structures, while PDBsum provides a graphic overview of PDB entries using information from other sources, such as Gene ontology. Two forces converged to initiate the PDB: 1) a small but growing collection of sets of protein structure data determined by X-ray diffraction.
In 1969, with the sponsorship of Walter Hamilton at the Brookhaven National Laboratory, Edgar Meyer began to write software to store atomic coordinate files in a common format to make them available for geometric and graphical evaluation. By 1971, one of Meyer's programs, SEARCH, enabled researchers to remotely access information from the database to study protein structures offline. SEARCH was instrumental in enabling networking, thus marking the functional beginning of the PDB; the Protein Data Bank was announced in October 1971 in Nature New Biology as a joint venture between Cambridge Crystallographic Data Centre, UK and Brookhaven National Laboratory, USA. Upon Hamilton's death in 1973, Tom Koeztle took over direction of the PDB for the subsequent 20 years. In January 1994, Joel Sussman of Israel's Weizmann Institute of Science was appointed head of the PDB. In October 1998, the PDB was transferred to the Research Collaboratory for Structural Bioinformatics; the new director was Helen M. Berman of Rutgers University.
In 2003, with the formation of the wwPDB, the PDB became an international organization. The founding members are PDBe, RCSB, PDBj; the BMRB joined in 2006. Each of the four members of wwPDB can act as deposition, data processing and distribution centers for PDB data; the data processing refers to the fact that annotate each submitted entry. The data are automatically checked for plausibility; the PDB database is updated weekly. The PDB holdings list is updated weekly; as of 17 October 2018, the breakdown of current holdings is as follows: 120,052 structures in the PDB have a structure factor file. 9,734 structures have an NMR restraint file. 3,486 structures in the PDB have a chemical shifts file. 2,531 structures in the PDB have a 3DEM map file deposited in EM Data BankThese data show that most structures are determined by X-ray diffraction, but about 10% of structures are now determined by protein NMR. When using X-ray diffraction, approximations of the coordinates of the atoms of the protein are obtained, whereas estimations of the distances between pairs of atoms of the protein are found through NMR experiments.
Therefore, the final conformation of the protein is obtained, in the latter case, by solving a distance geometry problem. A few proteins are determined by cryo-electron microscopy; the significance of the structure factor files, mentioned above, is that, for PDB structures determined by X-ray diffraction that have a structure file, the electron density map may be viewed. The data of such structures is stored on the "electron density server". In the past, the number of structures in the PDB has grown at an exponential rate, passing the 100 registered structures milestone in 1982, the 1,000 in 1993, the 10,000 in 1999, the 100,000 in 2014. However, since 2007, the rate of accumulation of new protein structures appears to have plateaued; the file format used by the PDB was called the PDB file format. This original format was restricted by the width of computer punch cards to 80 characters per line. Around 1996, the "macromolecular Crystallographic Information file" format, mmCIF, an extension of the CIF format started to be phased in.
MmCIF is now the master format for the PDB archive. An XML version of this format, called PDBML, was described in 2005; the structure files can be downloaded in any of these three formats. In fact, individual files are downloaded into graphics packages using web addresses: For PDB format files, use, e.g. http://www.pdb.org/pdb/files/4hhb.pdb.gz or http://pdbe.org/download/4hhb For PDBML files, use, e.g. http://www.pdb.org/pdb/files/4hhb.xml.gz or http://pdbe.org/pdbml/4hhbThe "4hhb" is the PDB identifier. Each structure published in PDB receives a four-character alphanumeric identifier, its PDB ID; the structure files may be viewed using one of several free and open source computer programs, including Jmol, Pymol, VMD, Rasmol. Other non-free, shareware programs
Biochemistry, sometimes called biological chemistry, is the study of chemical processes within and relating to living organisms. Biochemical processes give rise to the complexity of life. A sub-discipline of both biology and chemistry, biochemistry can be divided in three fields. Over the last decades of the 20th century, biochemistry has through these three disciplines become successful at explaining living processes. All areas of the life sciences are being uncovered and developed by biochemical methodology and research. Biochemistry focuses on understanding how biological molecules give rise to the processes that occur within living cells and between cells, which in turn relates to the study and understanding of tissues and organism structure and function. Biochemistry is related to molecular biology, the study of the molecular mechanisms by which genetic information encoded in DNA is able to result in the processes of life. Much of biochemistry deals with the structures and interactions of biological macromolecules, such as proteins, nucleic acids and lipids, which provide the structure of cells and perform many of the functions associated with life.
The chemistry of the cell depends on the reactions of smaller molecules and ions. These can be inorganic, for example water and metal ions, or organic, for example the amino acids, which are used to synthesize proteins; the mechanisms by which cells harness energy from their environment via chemical reactions are known as metabolism. The findings of biochemistry are applied in medicine and agriculture. In medicine, biochemists investigate the cures of diseases. In nutrition, they study how to maintain health wellness and study the effects of nutritional deficiencies. In agriculture, biochemists investigate soil and fertilizers, try to discover ways to improve crop cultivation, crop storage and pest control. At its broadest definition, biochemistry can be seen as a study of the components and composition of living things and how they come together to become life, in this sense the history of biochemistry may therefore go back as far as the ancient Greeks. However, biochemistry as a specific scientific discipline has its beginning sometime in the 19th century, or a little earlier, depending on which aspect of biochemistry is being focused on.
Some argued that the beginning of biochemistry may have been the discovery of the first enzyme, diastase, in 1833 by Anselme Payen, while others considered Eduard Buchner's first demonstration of a complex biochemical process alcoholic fermentation in cell-free extracts in 1897 to be the birth of biochemistry. Some might point as its beginning to the influential 1842 work by Justus von Liebig, Animal chemistry, or, Organic chemistry in its applications to physiology and pathology, which presented a chemical theory of metabolism, or earlier to the 18th century studies on fermentation and respiration by Antoine Lavoisier. Many other pioneers in the field who helped to uncover the layers of complexity of biochemistry have been proclaimed founders of modern biochemistry, for example Emil Fischer for his work on the chemistry of proteins, F. Gowland Hopkins on enzymes and the dynamic nature of biochemistry; the term "biochemistry" itself is derived from a combination of chemistry. In 1877, Felix Hoppe-Seyler used the term as a synonym for physiological chemistry in the foreword to the first issue of Zeitschrift für Physiologische Chemie where he argued for the setting up of institutes dedicated to this field of study.
The German chemist Carl Neuberg however is cited to have coined the word in 1903, while some credited it to Franz Hofmeister. It was once believed that life and its materials had some essential property or substance distinct from any found in non-living matter, it was thought that only living beings could produce the molecules of life. In 1828, Friedrich Wöhler published a paper on the synthesis of urea, proving that organic compounds can be created artificially. Since biochemistry has advanced since the mid-20th century, with the development of new techniques such as chromatography, X-ray diffraction, dual polarisation interferometry, NMR spectroscopy, radioisotopic labeling, electron microscopy, molecular dynamics simulations; these techniques allowed for the discovery and detailed analysis of many molecules and metabolic pathways of the cell, such as glycolysis and the Krebs cycle, led to an understanding of biochemistry on a molecular level. Philip Randle is well known for his discovery in diabetes research is the glucose-fatty acid cycle in 1963.
He confirmed. High fat oxidation was responsible for the insulin resistance. Another significant historic event in biochemistry is the discovery of the gene, its role in the transfer of information in the cell; this part of biochemistry is called molecular biology. In the 1950s, James D. Watson, Francis Crick, Rosalind Franklin, Maurice Wilkins were instrumental in solving DNA structure and suggesting its relationship with genetic transfer of information. In 1958, George Beadle and Edward Tatum received the Nobel Prize for work in fungi showing that one gene produces one enzyme. In 1988, Colin Pitchfork was the first person convicted of murder with DNA evidence, which led to the growth of forensic science. More Andrew Z. Fire and Craig C. Mello received the 2006 Nobel Prize for discovering the role of RNA interference, in the silencing of gene expression. Around two dozen of the 92