Protein G B1 domain

The solution structure of the isolated fragments 1-20 (beta-hairpin), 21-40 (alpha-helix) and 41-56 (beta-hairpin), corresponding to all the secondary structure elements of the protein G B1 domain, have been studied by circular dichroism and nuclear magnetic resonance techniques. In the protein G B1-(1-20) fragment turn-like folded structures were detected in water though low populated. In the presence of 30% aqueous trifluoroethanol there is a complex conformational behaviour in which a helical structure at the N-terminal half is formed in equilibrium with random and native-like beta-hairpin structures. The peptide corresponding to the alpha-helix is predominantly unstructured in water, while in 30% trifluoroethanol it highly populates a native alpha-helical conformation, including a (i,i + 5) interaction between hydrophobic residues at its C-terminus. The third peptide was previously reported to form a monomeric native beta-hairpin structure in water . We show in this work that the beta-hairpin structure is further stabilized in 30% trifluoroethanol and destabilised in the presence of 6 M urea, though some folded structure persists even in these highly denaturing conditions. The conformational properties of these peptides suggests that the second beta-hairpin could be an important folding initiation site on which the rest of the chain folds. Reconstitution experiments failed to show evidence of interaction between the peptides. Algorithms designed to predict the helical and extended conformations of peptides in aqueous solution successfully describe the complicated behaviour of these peptides. Comparison of the predicted and the experimental results with those for a structurally related protein, ubiquitin, shows very strong similarities, the main difference being the switch of the most stable beta-hairpin from the N-terminus in ubiquitin to the C-terminus in protein G.

G protein receptor

G protein-coupled receptors (GPCRs), also known as seven transmembrane domain receptors, 7TM receptors, heptahelical receptors, and G protein-linked receptors (GPLR), comprise a large protein family of transmembrane receptors that sense molecules outside the cell and activate inside signal transduction pathways and, ultimately, cellular responses. G protein-coupled receptors are found only in eukaryotes, including yeast, plants, choanoflagellates, and animals. The ligands that bind and activate these receptors include light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters, and vary in size from small molecules to peptides to large proteins. G protein-coupled receptors are involved in many diseases, but are also the target of around half of all modern medicinal drugs

  Subscribe in a reader

These proteins receive chemical signals from outside the cell and pass the signal into the cell, so that the cell can respond to the signal. The structures of the endogenous ligands for GPCRs are exceptionally diverse. They include biogenic amines such as norephnephrine and serotonine, peptides, glycoproteins, lipids, nucleotides, ions, and proteases. The sensation of exogenous stimuli, such as light, odors, and taste, is also mediated by this class of receptors. Activation of the receptor causes an effector inside the cell to produce a second signal chemical, which eventually triggers the cell to react to the original external chemical signal.

A ligand, in this case Norepinepherine (NE), binds to the receptor and induces a conformational change. This conformational change activates the a/b complex. The complex is bound to GDP while it is inactive. GTP replaces GDP, thus activating the Alpha subunit. The activated Alpha subunit undergoes a conformational change and activates Adenylate Cyclase. Once the Adenylate Cyclase is activated, it is then able to convert ATP. The products of ATP conversion are c-AMP and two phosphate molecules. c-AMP is a second messenger used in many processes required for cell survival and growth.

What is Endocytosis

Endocytosis is a process whereby cells absorb material (molecules such as proteins) from the outside by engulfing it with their cell membrane. It is used by all cells of the body because most substances important to them are large polar molecules, and thus cannot pass through the hydrophobic plasma membrane. The function of endocytosis is the opposite of exocytosis.

Types

The absorption of material from the outside environment of the cell is commonly divided into two processes: phagocytosis and pinocytosis.

Phagocytosis (literally, cell-eating) is the process by which cells ingest large objects, such as cells which have undergone apoptosis, bacteria, or viruses. The membrane folds around the object, and the object is sealed off into a large vacuole known as a phagosome.

Pinocytosis (literally, cell-drinking) is a synonym for endocytosis. This process is concerned with the uptake of solutes and single molecules such as proteins.

Receptor-mediated endocytosis is a more specific active event where the cytoplasm membrane folds inward to form coated pits. These inward budding vesicles bud to form cytoplasmic vesicles.

Endocytosis pathways

There are three types of endocytosis: namely, macropinocytosis, clathrin-mediated endocytosis, and caveolar endocytosis.

Macropinocytosis is the invagination of the cell membrane to form a pocket which then pinches off into the cell to form a vesicle filled with extracellular fluid (and molecules within it). The filling of the pocket occurs in a non-specific manner. The vesicle then travels into the cytosol and fuses with other vesicles such as endosomes and lysosomes.

Clathrin-mediated endocytosis is the specific uptake of large extracellular molecules such as proteins, membrane localized receptors and ion-channels. These receptors are associated with the cytosolic protein clathrin which initiates the formation of a vesicle by forming a crystalline coat on the inner surface of the cell's membrane.

Caveolae consist of the protein caveolin-1 with a bilayer enriched in cholesterol and glycolipids. Caveolae are flask shaped pits in the membrane that resemble the shape of a cave (hence the name caveolae). Uptake of extracellular molecules are also believed to be specifically mediated via receptors in caveolae.

Clathrin-mediated endocytosis

The major route for endocytosis in most cells, and the best understood, is that mediated by the molecule clathrin. This large protein assists in the formation of a coated pit on the inner surface of the plasma membrane of the cell. This pit then buds into the cell to form a coated vesicle in the cytoplasm of the cell. In so doing, it brings into the cell not only a small area of the surface of the cell but also a small volume of fluid from outside the cell.

Vesicles selectively concentrate and exclude certain proteins during formation and are not representative of the membrane as a whole. AP2 adaptors are multisubunit complexes that performs this function at the plasma membrane. The best understood receptors which are found concentrated in coated vesicles of mammalian cells are the LDL receptor (which removes LDL from the blood circulation), the transferrin receptor (which brings ferric ions bound by transferrin into the cell) and certain hormone receptors (such as that for EGF).

At any one moment, about 2% of the plasma membrane of a fibroblast is made up of coated pits. As a coated pit has a life of about a minute before it buds into the cell, a fibroblast takes up its surface by this route about once every 50 minutes. Coated vesicles formed from the plasma membrane have a diameter of about 100nm and a life time measured in a few seconds. Once the coat has been shed, the remaining vesicle fuses with endosomes and proceeds down the endocytic pathway. The actual budding-in process, whereby a pit is converted to a vesicle, is carried out by clathrin assisted by a set of cytoplasmic proteins which includes dynamin and adaptors such as adaptin.

Coated pits and vesicles were first seen in thin sections of tissue in the electron microscope by Thomas Roth and Keith Porter in 1964. The importance of them for the clearance of LDL from blood was discovered by R. G Anderson, Michael S. Brown and Joseph L. Goldstein in 1976. Coated vesicles were first purified by Barbara Pearse, who discovered the clathrin coat molecule, also in 1976.

Biochemistry Lecture (Protein Purification)

Cell Nuclei Protein Extraction Video

Protein Synthesis Animation

Protein biosynthesis (Synthesis) is the process in which cells build proteins. The term is sometimes used to refer only to protein translation but more often it refers to a multi-step process, beginning with amino acid synthesis and transcription which are then used for translation. Protein biosynthesis, although very similar, differs between prokaryotes and eukaryotes.

Amino acid synthesis


Amino acids are the monomers which are polymerized to produce proteins. Amino acid synthesis is the set of biochemical processes (metabolic pathways) which build the amino acids from carbon sources like glucose. Not all amino acids may be synthesised by every organism, for example adult humans have to obtain 8 of the 20 amino acids from their diet.
The amino acids are then loaded onto tRNA molecules for use in the process of translation.


Transcription is the process by which an mRNA template, carrying the sequence of the protein, is produced for the translation step from the genome. Transcription makes the template from one strand of the DNA double helix, called the template strand. Transcription takes place in 3 stages.
Transcription starts with the process of initiation. RNA polymerase, the enzyme which produces RNA from a DNA template, binds to a specific region on DNA that designates the starting point of transcription. This binding region is called the promoter. As the RNA polymerase binds on to the promoter, the DNA strands are beginning to unwind.
The second process is elongation. RNA polymerase travels along the template (noncoding) strand, synthesizing a ribonucleotide polymer. RNA polymerase does not use the coding strand as a template because a copy of any strand produces a base sequence that is complementary to the strand which is being copied. Therefore DNA from the noncoding strand is used as a template to copy the coding strand.
The third stage is termination. As the polymerase reaches the termination stage, modifications are required for the newly transcribed mRNA to be able to travel to the other parts of the cell, including cytoplasm and endoplasmic reticulum, for translation. A 5' cap is added to the mRNA to protect it from degradation. In eukaryotes, poly-A-polymerase adds a poly-A tail onto the 3’ end for stabilization, protection from cytoplasmic hydrolytic enzymes, and as a template for further processes. Also in eukaryotes (higher organisms) the vital process of splicing occurs at this stage by the spliceosome enzyme. It removes the introns (non-coding bits of genetic material) and glues together the exons (the segments that code for a specific protein).
The mRNA now exits the nuclear pore to be translated.


Translation
Protein translation involves the transfer of information from the mRNA into a peptide, composed of amino acids. This process is mediated by the ribosome, with the adaptation of the RNA sequence into amino acids mediated by transfer RNA. Numerous initation and elongation factors also play a role.

Translation requires a lot of energy, with the hydrolysis of approximately 4 NTP --> NDP per amino acid added. (This includes the aminoacylation of the tRNA. Thus, gene expression is highly regulated to ensure that only proteins that are required are translated.

Translation involves 3 processes: initiation, elongation, and termination.

Initiation in Prokaryotes

The initiation of protein translation involves the assembly of the ribosome and addition of the first amino acid, methionine.
The 30S ribosomal subunit attaches to the mRNA, mediated by IF-1 and IF-3 (initiation factors). The 30S ribosome brings with it the P and A site, but the A site is blocked by IF-1 to prevent binding of tRNA. It aligns to the Shine-Dalgarno sequence, which positions the first codon (AUG) in the P site.
Next, the specific aminoacyl-tRNA for N-formylmethionine (F-Met) is brought into the P site by IF-2. The anticodon of this tRNA will bind to the AUG codon on the mRNA. Note: this is the only tRNA brought into the P site; all successive aminoacyl-tRNAs will be brought to the A site for peptide elongation.
The 50S ribosomal subunit is then brought in to complete the ribosome, and with it, IF-1, IF-2, and IF-3 come off the complex. The A and P site are completed, and the 50S subunit also brings the E (exit) site.

Initiation in Eukaryotes

The initiation of protein translation in eukaryotes is similar to that of prokaryotes with some modifications.
A complex of proteins will connect the 5'cap and 3'PolyA tail, and this complex will recruit the ribosome subunits.
There is no Shine-Dalgarno sequence in eukaryotes. Instead, the ribosome scans along the mRNA for the first methionine codon. Similarly, there is no N-formylmethionine in eukaryotic cells.

Elongation

Elongation of protein biosynthesis is fairly similar between prokaryotes and eukaryotes. The following is a description of elongation in prokaryotes.
Elongation proceeds after initiation with the binding of an aminoacyl-tRNA to the A site, which is the next codon in the mRNA. The aminoacyl-tRNA is brought to the ribosome through a series of interactions with EF-Tu (an elongation factor). This step involves the hydrolysis of GTP: EF-Tu-GTP --> EF-Tu-GDP (The hydrolyzed GDP is switched for GTP through another series of reactions with EF-Ts.)
The next aminoacyl-tRNA binds to the codon, and the C-terminus of the F-Met undergoes nucleophilic attack by the N-terminus of the second amino acid. The F-Met is now connected to the second amino acid through a peptide bond.
The first tRNA (for F-Met) is now uncharged. The entire ribosome complex moves along the mRNA through the action of another elongation factor (EF-G) and the hydrolysis of GTP --> GDP.
The first tRNA is now in the E site and comes off from the ribosome, while the second tRNA, with the nascent peptide chain, is in the P site. Step 1-4 will repeat as successive amino acids are added.


Termination

Termination of protein biosynthesis occurs when the ribosome comes across a stop codon, for which there is no tRNA. At this point, protein biosynthesis halts and one of three release factors will bind to the stop codon. (Note: In eukaryotes, there is only one release factor that will bind to all three stop codons.) This induces a nucleophilic attack of the C-terminus of the nascent peptide by water - this hydrolysis releases the peptide from the ribosome. The ribosome, release factor, and uncharged tRNA then dissociates and translation is complete.


Events following Protein Translation
The events following biosynthesis include post-translational modification and protein folding. During and after synthesis, polypeptide chains often fold to assume, so called, native secondary and tertiary structures. This is known as protein folding.

Many proteins undergo post-translational modification. This may include the formation of disulfide bridges or attachment of any of a number of biochemical functional groups, such as acetate, phosphate, various lipids and carbohydrates. Enzymes may also remove one or more amino acids from the leading (amino) end of the polypeptide chain, leaving a protein consisting of two polypeptide chains connected by disulfide bonds.