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Lecture 15

CHEM564 Lecture 15: 85 - 118 from amino acide to protein

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Christopher Cairo

85 Biomolecular chemistry 4. From amino acids to proteins Primary Source Material • Chapters 4 and 12 of Introduction to Genetic Analysis Anthony: J.F. Griffiths, Jeffrey H. Miller, David T. Suzuki, Richard C. Lewontin, William M. Gelbart (courtesy of the NCBI bookshelf). • Chapters 4, 4 and 6 of Biochemistry: Berg, Jeremy M.; Tymoczko, John L.; and Stryer, Lubert (courtesy of the NCBI bookshelf). • Chapters 3 and 7 of Molecular Cell Biology: Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. (courtesy of the NCBI bookshelf). • ExPASy: online course on Principles of Protein Structure • Many figures and the descriptions for the figures are from the educational resources provided at the Protein Data Bank ( • Most of these figures and accompanying legends have been written by David S. Goodsell of the Scripps Research Institute and are being used with permission. I highly recommend browsing the Molecule of the Month series at the PDB ( • Suggested reading: Sections 14.5 to 14.8 and Sections 2.1 to 2.4 of Mikkelsen and Cortón, Bioanalytical Chemistry 86 Where are we and how did we get here? We are here! • We are done with the Central Dogma and now we move into the realms of protein structure and function. The Central Dogma only relates to the flow of genetic information, not to the function of biological macromolecules. 87 Proteins come in all shapes and sizes • Proteins are diverse and versatile ‘nano’ structures and machines • Large number of potential combinations • There is a relatively large number number of amino acids (a.a.) which you can use to construct a protein. • Includes 20 common a.a.’s plus numerous post-translational modifications. • 200 amino-acid protein could have 20 to the 200th power possible sequences. • Structurally versatile • Polypeptide backbone can adopt a variety of conformations • Many conformers of side chains • Secondary structural elements can pack together in a wide variety of orientations • Various states of homo- and hetero- oligomerization • Proteins can bind prosthetic groups or cofactors (non-protein) • Heme • Metal ions • flavins • Structurally dynamic • Allosteric activation • Active and inactive forms 88 The structure of a protein is determined by the linear sequence of amino acids (1º structure) Ribonuclease An unfolded protein can be refolded in vitro. This demonstrates that the information needed to specify the tertiary structure is fully contained in the primary sequence. • The classic work of Christian Anfinsen in the 1950s on the enzyme ribonuclease revealed the relation between the amino acid sequence of a protein and its conformation. For this work he was awarded the Nobel Prize in Chemistry in 1972. Anfinsen discovered that: • Ribonuclease is a single polypeptide chain consisting of 124 amino acid residues cross-linked by four disulfide bonds. • Agents such as urea or guanidinium chloride effectively disrupt the noncovalent bonds., • The disulfide bonds can be cleaved reversibly by reducing them with a reagent such as β-mercaptoethanol. • When ribonuclease was treated with β-mercaptoethanol in 8 M urea, the product was a fully reduced, randomly coiled polypeptide chain devoid of enzymatic activity. In other words, ribonuclease was denatured by this treatment. • Anfinsen then made the critical observation that the denatured ribonuclease, freed of urea and β-mercaptoethanol by dialysis, slowly regained enzymatic activity. All the measured physical and chemical properties of the refolded enzyme were virtually identical with those of the native enzyme. • These experiments showed that the information needed to specify the catalytically active structure of ribonuclease is contained in its amino acid sequence. • For a good discussion of these classic experiments: anfinsen-experiment-in-protein-folding.html 89 The 20 common amino acids Ala showing L- stereochemistry l 1 _ t a / / e w t h • 20 different common amino acids only differing in side chain • Note that stereochemistry at Cα has not been indicated in this figure. • All natural a.a.’s are in L-configuration • A more general system of stereochemical designation is the R/S system. The L- configuration nearly always corresponds to S in the R/S system. The exception is L- cysteine which is R. • You might want to keep this sheet handy as a reference. • I will often used the one letter codes and you should learn these. • Most are easy, but I find D, E, N and Q the most tricky to remember • Q: Do we need to memorize the structure and names of amino acids on the test? • A: Yes. You should know the structure, name, 3 letter abbreviation, and 1 letter code of all the common amino acids. 90 Amino acid classification by property • Various simple textbook classifications for a.a.’s • e.g. small, nucleophilic, hydrophobic, aromatic, acidic, amide, basic • e.g. aliphatic, non-polar, aromatic, polar, charged -ve, charged +ve • However, no simple classification can properly capture the diversity of a.a. interactions and properties. • the same amino acid in different charge states can go from polar to nonpolar (H or K for example) • Different portions of the same amino acid can have different properties (aliphatic chain vs. guanidinium of arginine) • Generally find aliphatic/hydrophobic residues inside proteins and polar/charged on the surface of proteins • Notes: • Cysteine is special because it is best nucleophile, is most easily oxidized, and can form disulphide bonds. • Proline has a tertiary as opposed to secondary amide nitrogen and induces bend in polypeptide chain. • Theonine and Isoleucine have chiral carbons in side chain 91 Free amino acids are almost always zwitterions Commentary on the topic of zwitterions: • Amino acids in solution at neutral pH exist as dipolar ions (zwitterions). • In the dipolar form, the amino group is protonated (-NH3+) and the carboxyl group is deprotonated (-CO2-). • Under almost any conceivable physiologically relevant conditions, the amino and carboxylate group of a free amino acid will be in its charged state. • This is also true of a polypeptide chain: the N-terminus and the C-terminus will be in the charged states • Possible exceptions • Groups buried in the interior of proteins or lipid bilayers • Proteins in the stomach 92 pKa values of protein functional groups • Seven of the 20 amino acids have readily ionizable side chains. These 7 amino acids are able to donate or accept protons to facilitate reactions as well as to form ionic bonds. • The above table gives equilibria and typical pKa values for ionization of the side chains of tyrosine, cysteine, arginine, lysine, histidine, and aspartic and glutamic acids in proteins. • Two other groups in proteins—the terminal α-amino group and the terminal α-carboxyl group—can be ionized. • You should know the approximate values for all of these ionizable groups. It is safe to say that all carboxylic acids in proteins have a pKa of about 3-4. • Q: What is so special about Histidine? It has a pka of ~6, but did you mention that it does not react with anything much? • A: Histidine is very good at donating and accepting protons at physiological pH. This is a very important part of many enzyme mechanisms. I may have mentioned that histidine is not such a good nucleophile. For enzyme mechanisms that involve a nucleophilic attack on the substrate, cysteine would be the best amino acid, followed by lysine. • Q. Proteins buried in lipid bilayers are charged on one terminal end or not at all? if its charged on part which one is it? • A. The N-terminus is always positively charged and the C-terminus is always negatively charged under normal pH conditions (near neutral).  Under some circumstances, such as when the N- or C-terminus is buried in a very hydrophobic environment, I suppose they could be uncharged. The pKa of an ionizable group is going to depend on its environment. • Q. Proteins in stomach are charged on their N terminals, am i right? • A. I believe that the stomach is very low pH, like 2-3. At such low pH, practically every group in proteins will be protonated. It is close to the pKa for the C-terminus, so it might be partially protonated. • Q. Are the pKa values of AAs will be given in the test or not? • A. They won't be provided. You should know which residues are positively and negatively charged at neutral pH. • 93 An oligopeptide • Oligopeptide: A compound made up of the condensation of a small number (typically less than 20) of amino acids • Polypeptide: A compound made up of the condensation of more than ~20 amino acids • Each type of protein differs in its sequence and number of amino acids. It is the particular sequence of the various side chains that makes each protein distinct. • The two ends of a polypeptide chain are chemically different: the end carrying the free amino group (NH3+, sometimes incorrectly written as NH2) is the amino, or N-terminus, and that carrying the free carboxyl group (CO2-, sometimes incorrectly written as CO2H) is the carboxyl, or C-terminus. • The amino acid sequence of a protein is always presented in the N to C direction, reading from left to right. This corresponds to the 5’ to 3’ direction in which genes are read. 94 The peptide bond is planar and almost always trans All amino acids except proline Proline • Note that the C-N bond length of the peptide is 10% shorter than that found in usual C-N amine bonds. This is because the peptide bond has some double bond character (40%) due to resonance which occurs with amides. As a consequence of this resonance all peptide bonds in protein structures are found to be almost planar. This rigidity of the peptide bond reduces the degrees of freedom of the polypeptide during folding. • The planarity of the peptide bond is described using the angle ‘omega’. This is the dihedral angle between the C alphacarbonyl bond and the N-C alphaond. The omega (ω) angle is almost always 180º (trans) though sometimes (extremely rarely) it is 0º (cis). • Of the cis-peptide bonds found in proteins, almost all involve proline residues . The overall atom geometry in cis proline is very similar to the trans-proline case. Energetically, the trans proline structure is not markedly more favorable than its cis-proline counterpart since much the same spatial conflicts are present in both cases. Approximately 1% of prolines in proteins are cis. • A cis-peptide bond induces a very sharp kink in the polypeptide chain. • Q. It is stated that "Approximately 1% of prolines in proteins are cis." Does it mean 99% of prolines in proteins are trans? So, trans-proline is still more favourable than cis-proline (Slide 87)? Also, do you mean that proline is the only amino acid that can exist in cis while 19 other amino acids cannot. • A. Correct. 99% of all prolines are trans and trans is more favourable than cis. The difference in energy for cis vs. trans is smaller than it is for any of the other amino acids, and this is why we occasionally see cis prolines. It is extremely rare to find any of the other 19 amino acids in a cis conformation. 95 Certain combinations of φ and ψ angles are preferred Scans downloaded from: • Linus Pauling and Robert Corey analyzed the geometry and dimensions of the peptide bonds in the crystal structures of molecules containing one or a few peptide bonds. This analysis led Pauling to correctly predict the existence and structure of the alpha helix and beta sheets (for which he was awarded the 1954 Nobel Prize in Chemistry) • The take home message is that the secondary structure elements of proteins can be predicted by looking at the structure of an individual amino acid. That is, an amino acid in an alpha helical or beta sheet conformer is also in a minimal energy conformer because its bonds are staggered and the peptide bond is planar. • A polypeptide can be thought of as a series of planar units (peptide bonds) joined by flexible hinges (Cα-atoms). • Each Cα-atom has two rotatable bonds, the C-N bond (φ, phi) and the C-C bond (ψ, psi) • Only certain combinations of φ and ψ angles are allowed due to steric clashes between the adjacent residues. 96 The Ramachandran Plot (φ vs. ψ) β-strand conformation α-helical conformation • A graph of φ angle vs. ψ angle vs. occurrence in proteins is called a Ramachandran plot. • There are actually only a few conformations that are strongly preferred and these give rise to the common elements of secondary structure. 97 The Ramachandran Plot of a typical protein (as output by the program PROCHECK) • The Ramachandran plot for a particular protein shows the phi-psi torsion angles for all residues in the structure • By looking at how well the angles match up with expected distribution, the quality of a structure can be assessed. • Glycine residues are separately identified by triangles as these are not restricted to the regions of the plot appropriate to the other sidechain types. • The coloring/shading on the plot represents the various levels of favorability: the darkest areas (here shown in red) correspond to the "core" regions representing the most favorable combinations of phi-psi values. • A properly folded protein will have over 90% of the residues in these "core" regions. • The percentage of residues in the "core" regions is one of the better guides to stereochemical quality for assessing experimental protein structures. • An ideal Ramachandran plot can be generated computationally using known atomic radii and bond distances. 98 alpha-helices: an ‘island’ of preferred conformation • As mentioned earlier, Pauling and Corey twisted models of polypeptides around to find ways of getting the backbone into regular conformations which would agree with experimental diffraction data (much like the way the structure of DNA was determined). The most simple and elegant arrangement is a right-handed spiral conformation known as the 'alpha-helix'. • The structure repeats itself every 5.4 Angstroms along the helix axis, i.e. we say that the alpha-helix has a pitch of 5.4 Angstroms. Alpha-helices have 3.6 amino acid residues per turn, i.e. a helix 36 amino acids long would form 10 turns. The separation of residues along the helix axis is 5.4/3.6 or 1.5 Angstroms, i.e. the alpha-helix has a rise per residue of 1.5 Angstroms. • Every mainchain C=O and N-H group is hydrogen-bonded to a peptide bond 4 residues away (O(i) to N(i+4)). This gives a very regular, stable arrangement. • The peptide planes are roughly parallel with the helix axis and the dipoles within the helix are aligned. That is, all C=O groups point in the same direction and all N-H groups point the other way. This alignment of C=O and N-H bonds gives the alpha-helix a permanent dipole with a partial positive charge at the amino-terminus and a partial negative charge at the carboy-terminus. • Side chains point outward from helix axis and are generally oriented towards its amino- terminal end. • All the amino acids have negative phi and psi angles, typical values being -60 degrees and -50 degrees, respectively 99 beta-strands: another ‘island’ of preferred conformation • In addition to the alpha helix, Pauling and Corey discovered another periodic structural motif which they named the β-pleated sheet (β because it was the second structure that they elucidated, the α helix being the first). • The β-sheet differs markedly from the rodlike α-helix. A polypeptide chain, called a β- strand, in a β-sheet is almost fully extended rather than being tightly coiled as in the α- helix. A range of extended structures are sterically allowed. The side chains of adjacent amino acids point in opposite directions. • A β-sheet is formed by linking two or more β-strands by hydrogen bonds. Adjacent chains in a β-sheet can run in opposite directions (antiparallel β-sheet) or in the same direction (parallel β-sheet). • In the antiparallel arrangement, the NH group and the CO group of each amino acid are respectively hydrogen bonded to the CO group and the NH group of a partner on the adjacent chain. • In the parallel arrangement, for each amino acid, the NH group is hydrogen bonded to the CO group of one amino acid on the adjacent strand, whereas the CO group is hydrogen bonded to the NH group on the amino acid two residues farther along the chain. • Many strands, typically 4 or 5 but as many as 10 or more, can come together in β-sheets. Such β-sheets can be purely antiparallel, purely parallel, or mixed. • β-sheets can be relatively flat but most adopt a somewhat twisted shape. 100 Turns and loops connect strands and helices • Most proteins have compact, globular shapes, requiring reversals in the direction of their polypeptide chains. Many of these reversals are accomplished by reverse turns (not shown) and hairpins (shown). • The residues forming these two-residue turns have torsion angles in characteristic regions of the Ramachandran plot. • For type I' turns, residue 2 is always glycine whereas for type II' turns residue 1 is always Gly. This is because amino acids other than glycine would cause steric hindrance involving the residue's side chain and the main chain. • In other cases, more elaborate structures are responsible for chain reversals. These structures are called loops or sometimes Ω loops (omega loops) to suggest their overall shape. Unlike α-helices and β-strands, loops do not have regular, periodic structures. Nonetheless, loop structures are often rigid and well defined. Turns and loops invariably lie on the surfaces of proteins and thus often participate in interactions between proteins and other molecules. • For example, a part of an antibody molecule has surface loops (shown in red) that mediate interactions with other molecules. • Q. Do beta-hairpins only exists among beta-sheet? • A. As the name implies, the beta hairpin is most commonly found as a connector between strands of an antiparallel beta sheet. The reverse turn is a a bit more general and can be found in loops that connect both helices and strands. • Q. What are the differences/relationship between reverse turns, beta-hairpin turns and omega loops? • A. Reverse turns and beta turns do look very similar when you look at the structures on the slide. However, there are key differences in the conformations of amino acids that define each of these two types of turns. I don't expect you to know the details of these differences. One thing you should remember is that beta turns are typically used to connect two strands of anti- parallel beta sheet. An omega loop is a larger structure that is supposed to look something like the omega character (Ω). That is, the ends are very close together but the loop itself is large and extends out into space. The variable regions of an antibody can be described as omega loops. Experimental measurement of 101 β-sheet propensities • Kim and Minor mutated position 53 of protein GB1 to all 20 possible amino acids, and then measured the melting temperature for all of the resulting proteins • This analysis revealed that certain amino acids are more favorable in β-sheets than others. • Specifically, residues that are branched at the β-carbon of the amino acid tend to stabilize a β-sheet structure. • This observation is consistent with the concept of amino acid propensities. That is, every amino acid has a certain preference for being in a β-sheet, α-helix, or turn region. • For β-sheet vs. α-helix propensity are opposed to each other. That is, the reason a residue has a high β-sheet propensity, is because it has a low α-helix propensity, and vice versa. • image from: Proteins are generally composed of α-helices 102 and/or β-sheets connected by turns and loops • The α-helical content of proteins ranges widely, from nearly none to almost 100%. For example, about 75% of the residues in ferritin, a protein that helps store iron, are in α- helices. Single α-helices are usually less than 45 Å long. However, two or more α-helices can entwine to form a very stable ‘coiled coil’ structure, which can have a length of 1000 Å (100 nm, or 0.1 μm) or more. Such α-helical ‘coiled coils’ are found in myosin and tropomyosin in muscle, in fibrin in blood clots, and in keratin in hair. The helical cables in these proteins serve a mechanical role in forming stiff bundles of fibers, as in porcupine quills. The cytoskeleton (internal scaffolding) of cells is rich in so-called intermediate filaments, which also are two-stranded α-helical coiled coils. Many proteins that span biological membranes also contain α-helices. • The β-sheet is an important structural element in many proteins. For example, fatty acid- binding proteins, important for lipid metabolism, are built almost entirely from β-sheets. 103 Protein folding is largely driven by hydrophobic interactions Myoglobin Hydrophobic Hydrophilic surface cross section • Myoglobin, the oxygen carrier in muscle, is a single polypeptide chain of 153 amino acids. The capacity of myoglobin to bind oxygen depends on the presence of heme, a prosthetic (helper) group consisting of protoporphyrin IX and a central iron atom. • The folding of the main chain of myoglobin, like that of most other proteins, is complex and devoid of symmetry. A unifying principle emerges from the distribution of side chains. The striking fact is that the interior consists almost entirely of nonpolar residues such as leucine, valine, methionine, and phenylalanine. Charged residues such as aspartate, glutamate, lysine, and arginine are absent from the inside of myoglobin. The only polar residues inside are two histidine residues, which play critical roles in binding iron and oxygen. • The outside of myoglobin, on the other hand, consists of both polar and nonpolar residues. This contrasting distribution of polar and nonpolar residues reveals a key facet of protein architecture. In an aqueous environment, protein folding is driven by the strong tendency of hydrophobic residues to be excluded from water. • The polypeptide chain therefore folds so that its hydrophobic side chains are buried and its polar, charged chains are on the surface. • The secret of burying a segment of main chain in a hydrophobic environment is pairing all the NH and CO groups by hydrogen bonding. This pairing is neatly accomplished in an α- helix or β-sheet. • The ability to predict whether or not a given polypeptide sequence will fold into a given tertiary structure remains one of the ‘grand challenges’ of science. • In nature, protein fold either independently or with the help of other proteins known as chaperones. 104 Membrane proteins have grease on the outside K -channel lipid bilayer Three views of the same structure • Some proteins that span biological membranes are “the exceptions that prove the rule” regarding the distribution of hydrophobic and hydrophilic amino acids throughout three-dimensional structures. For example, ion channels are covered on the outside largely with hydrophobic residues that interact with the neighbouring alkane chains. The inner channel is quite polar and there are many specific interactions with the ion being transported. • David S. Goodsell: The Molecule of the Month appearing at the PDB • Potassium ions move through this channel from inside the cell to the outside. The driving force for this movement is simply the concentration gradient. Cells concentrate potassium ions inside, and then these ions are released when the membrane depolarizes (for example, during transmission of signals through the nervous system). The selectivity filter is the part with the backbone carbonyls oriented towards the ion in the centre of the channel. Only potassium (not sodium) is perfectly coordinated by these carbonyl oxygen atoms, and so only it can pass through the channel. It is my understanding that potassium ions are normally surrounded by 8 water molecules, whereas sodium is normally surrounded by 6. • The 2003 Nobel Prize in Chemistry was awarded for work in the area of channels • Roderick Mackinnon pioneered x-ray crystallography of ion channels. • Peter Agre discovered water channels. • Water channels facilitate the rapid transport of water across cell membranes in response to osmotic gradients. These channels are believed to be involved in many physiological processes that include renal water conservation, neuro-homeostasis, digestion, regulation of body temperature and reproduction. Members of the water channel superfamily have been found in a range of cell types from bacteria to human. 105 Chaperone assisted protein folding • Folding of proteins in vitro tends to be an inefficient process, with only a minority of unfolded molecules undergoing complete folding within a few minutes. • More than 95 percent of the proteins present in cells are in their native conformation. • The explanation for the cell’s remarkable efficiency in promoting protein folding probably lies in chaperones, a family of proteins found in all organisms from bacteria to humans. • There are two general families of chaperones: molecular chaperones, which bind and stabilize unfolded or partially folded proteins, thereby preventing these proteins from being degraded; and chaperonins, which directly facilitate their folding. • Chaperonins are probably used for a specific and relatively small selection of proteins, whereas molecular chaperones are used for most, if not all, proteins. • All chaperones have ATPase activity, and their ability to bind and stabilize their target proteins is specific and dependent on ATP hydrolysis. • Molecular chaperones include the Hsp70 family of proteins. When bound to ATP, Hsp70 assumes an open form in which an exposed hydrophobic pocket transiently binds to exposed hydrophobic regions of the unfolded target protein. Hydrolysis of the bound ATP causes Hsp70 to assume a closed form, releasing the target protein. Molecular chaperones are thought to bind all nascent polypeptide chains as they are being synthesized on ribosomes. More on GroEL 106 Hydrophobic stripe ATP-binding site Large cavity David S. Goodsell: The Molecule of the Month appearing at the PDB • Proper folding of a small proportion of proteins (e.g., the cytoskeletal proteins actin and tubulin) requires additional assistance, which is provided by chaperonins. • Shown on this slide is the bacterial chaperonin, GroEL, which contains 14 identical subunits stacked in two concentric rings (green). GroES is shown at the bottom in pink. • The large GroEL-GroES complex is available in PDB entry 1aon. In this picture, three of the subunits in each GroEL ring have been removed to show the interior, leaving four subunits in each ring. On the two in back, the hydrophobic amino acids, LEU, ILE, VAL, MET, PHE, TYR and TRP, are coloured blue. • Notice the stripe of hydrophobic amino acids around the entry at the top. This will interact strongly with unfolded proteins by coaxing them into the upper cavity. Once the unfolded protein is bound, ATP and GroES bind to GroEL. This causes a conformational change that forces the protein into the larger lower cavity that is much more hydrophilic than the upper cavity. • Now that the protein is in a hydrophilic environment, it will be forced to fold in order to minimize they unfavourable interactions between its hydrophobic portions and its hydrophilic environment. • After the protein has folded, ATP is hydrolyzed and GroES (the lid on the cavity) is released along with the newly folded protein. • Q: When use chaperonin to help proteins to fold, the GroES will bind to GroEL to the large cavity side or hydrophobic stripe side? • A: I believe it can bind to both sides. Don't worry about the details. 107 Proteins often consist of multiple independent domains and have 4 structure o CD4 Cro hemoglobin Rhinovirus (antibody) • Some polypeptide chains fold into two or more compact regions that may be connected by a flexible segment of polypeptide chain, rather like pearls on a string. • These compact globular units, called domains, range in size from about 30 to 400 amino acid residues. • For example, the extracellular part of CD4 (shown at top), the cell-surface protein on certain cells of the immune system to which the human immunodeficiency virus (HIV) attaches itself, comprises four similar domains of approximately 100 amino acids each. Often, proteins are found to have domains in common even if their overall tertiary structures are different. • Antibodies (immunoglobins) have a distinct domain structure in addition to quaternary structure. We will be taking a much closer look at antibody structure in the next section. • Quaternary structure refers to the spatial arrangement of subunits and the nature of their interactions. • The simplest sort of quaternary structure is a dimer, consisting of two identical subunits. This organization is present in the DNA-binding protein Cro found in a bacterial virus called λ. • More complicated quaternary structures also are common. More than one type of subunit can be present, often in variable numbers. For example, human hemoglobin, the oxygen-carrying protein in blood, consists of two subunits of one type (designated α) and two subunits of another type (designated β). Thus, the hemoglobin molecule exists as an α2β2 tetramer. • Viruses make the most of a limited amount of genetic information by forming coats that use the same kind of subunit repetitively in a symmetric array. The coat of rhinovirus, the virus that causes the common cold, includes 60 copies each of four subunits. The subunits come together to form a nearly spherical shell that encloses the viral genome. • Q: It mentioned that the coat of rhinovirus includes 60 copies each of four subunits. But from the picture I only see three coloured subunits. What's wrong in this. • A: There is a 4th protein that is inside and not visible from the outside. 108 Post-translational modifications of proteins N-link glycosylation O-link glycosylation S-link glycosylation C-Mannosylation (Asn) (Ser, Thr) (Cys) (Trp) O HN HO OH OH HO O O O HOO NH O HO HHO HO O H R NH HN HN HO N O S HO O H H HO HO O N N H H H N N O O H O H O H O H O H O N N N N N N N N H H H H O O N-MethylatiN+ N-AcylatiHN O phosphoryla-O P O sulfatio-O S O (Lys) (Lys, N-term) (Ser, Thr, Tyr)- (Tyr) O O O S S n H H prenylation/farnesylatiNn N N N S-Acylation (Cys) H H (Cys) O O • Many proteins are covalently modified, through the attachment of groups other than amino acids, to augment their functions. Many proteins, especially those that are present on the surfaces of cells or are secreted, acquire carbohydrate units on specific asparagine residues. The addition of sugars makes the proteins more hydrophilic and able to participate in interactions with other proteins. Conversely, the addition of a fatty acid to an α-amino group or a cysteine sulfhydryl group produces a more hydrophobic protein that will be tightly associated with the membrane. • Glycosylation is the most common modification in mammalian cells. • Proteins can also be reversibly modified to regulate their activity. Perhaps the most important modification for signaling pathways is phosphorylation and dephosphorylation of serine, threonine, and tyrosine residues. Regulation of protein activity by phosphorylation is basis for intracellular signalling. The enzymes that catalyze the addition of phosphate groups (from ATP donors) are called kinases (why kinases?). Enzymes that remove phosphate groups are called phosphatases. • Histones—proteins that assist in the packaging of DNA into chromosomes as well as in gene regulation—are rapidly acetylated and deacetylated on specific lysine residues in vivo. More heavily acetylated histones are associated with genes that are being actively transcribed. A more permanent modification of lysines in histone proteins is methylation. • The attachment of ubiquitin, a protein comprising 72 amino acids, is a signal that a protein is to be destroyed, the ultimate means of regulation. • This slides shows only a few of the common examples. A number of additional post-translational modifications are known. Lysine H C=O2 2 ε-N-monomethyllysine HP1 chromodomain recognize short peptide motifs that are embedded in 2 target proteins, but do not bind stably until the pep- Acetyl O 109 tide has acquired an appropriate PTM c Acetylation domains usually have a conserved binding pocket for + CoA CoA Post-translational modifications are catalyzed NH3 HN the modified residue and a more variable surface that selectivelyengagestheflankingaminoacids,andthereby HAT
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