amino acids, peptides, and proteins Flashcards
(124 cards)
1
Q
amino acids
A
Amino acids are building blocks of peptides and proteins. Each amino acid is made of a C atom, an amino group, a carboxyl group, and a side chain R group. Amino acid is a dipolar ion at physiological pH, with its amino group carrying a positive charge, while its carboxyl group carries a negative charge. The side chain group gives each amino acid unique properties. Each amino acid has a three-letter code and a one-letter code. These are the basic language of any biochemist used to describe amino acids and proteins.
2
Q
non-polar/aliphatic amino acids
A
are (from less hydrophobic) glycine, alanine, valine, leucine, methionine and isoleucine (to more hydrophobic). The R groups of these amino acids have either aliphatic or aromatic groups. This makes them hydrophobic (“water fearing”). In aqueous solutions, globular proteins will fold into a three-dimensional shape to bury these hydrophobic side chains in the protein interior. (GAV LIM)
3
Q
aromatic amino acids
A
A side chain is aromatic when it contains an aromatic ring system. The strict definition has to do with the number of electrons contained within the ring. Generally, aromatic ring systems are planar, and electons are shared over the whole ring structure. Includes (from less hydrophobic) to tyrosine, tryptophan, and phenylalanine (to more hydrophobic) (Try Trippin on Phenylalanine)
4
Q
Polar and uncharged amino acids
A
are proline, serine, cysteine, threonine, asparagine, and glutamine. The side chains in this group possess a spectrum of functional groups. However, most have at least one atom (nitrogen, oxygen, or sulfur) with electron pairs available for hydrogen bonding to water and other molecules. (Come Take Poison GAS)
5
Q
Basic amino acids
A
are polar and positively charged at pH values below their pKa’s, and are very hydrophilic. Even though the basic amino acids are almost always in contact with the solvent, the side chain of lysine has a marked hydrocarbon character, so it is often found NEAR the surface, with the amino group of the side chain in contact with solvent. Includes Histidine (pKa 6.5), lysine (pKa 10), and arginine (pKa 12) (HAL)
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Q
polar and negatively charged amino acids
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are acidic and include glutamate (pKa 4.4) and aspartate (pKa 4.4) (Guess what Mate, your an Asshole)
7
Q
Cysteine (C)
A
can form disulfide bonds and the importance of disulfide bonds in protein stability and structure. Disulphide bridges can link amino acid chains together and can make inter and intra-chain linkages: Human insulin
8
Q
L isomer
A
The groups: COOH, R, NH2 and H (where R is the side-chain) are arranged around the chiral center carbon atom. With the hydrogen atom away from the viewer, if the arrangement of the CO→R→N groups around the carbon atom as center is counter-clockwise, then it is the l form. All proteins are L configuration
9
Q
Henderson-Hasselbalch equation
A
pKa = pH + log [Acid]/[Base] If the pH of a solution = the pKa, then the acid is in equilibrium – it is half dissociated. if pH is less than pKa then it’s mainly protonated acid; if pH is more than pKa it’s mainly deprotonated.
10
Q
zwitterions
A
a neutral molecule with a positive and a negative electrical charge, though multiple positive and negative charges can be present. Zwitterions are distinct from dipoles, at different locations within that molecule. Amino acids are the best-known examples of zwitterions. These compounds contain an amino group (pKa 9.6) and a carboxylate group (pKa 2.34), and can be viewed as arising via a kind of intramolecular acid–base reaction: The amine group deprotonates the carboxylic acid. NH2RCHCO2H is in equilibrium with NH3+RCHCO2−
11
Q
Absorbance Assay (280 nm)
A
Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 nm. Secondary, tertiary, and quaternary structure all affect absorbance, therefore factors such as pH, ionic strength, etc. can alter the absorbance spectrum.
12
Q
Lambert-Beer Law
A
relates the attenuation of light to the properties of the material through which the light is traveling. A = log I0/I = ε c l Absorbance is proportional to extinction coefficient, concentration, and path length
13
Q
Isoelectric point (pI)
A
the pH at which a particular molecule carries no net electrical charge. Amino acids that make up proteins may be positive, negative, neutral, or polar in nature, and together give a protein its overall charge. At a pH below their pI, proteins carry a net positive charge; above their pI they carry a net negative charge. Proteins can, thus, be separated according to their isoelectric point. It is like a pKa of protein
14
Q
essential amino acids
A
an amino acid that cannot be synthesized de novo (from scratch) by the organism being considered, and therefore must be supplied in its diet. The nine amino acids humans cannot synthesize are arginine, phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine. (P.V.T T.I.M. H.A.L- private tim hall)
15
Q
Phenylketonuria
A
an autosomal recessive metabolic genetic disorder characterized by mutations in the gene for the hepatic enzyme phenylalanine hydroxylase (PAH), rendering it nonfunctional. This enzyme is necessary to metabolize the amino acid phenylalanine (Phe) to the amino acid tyrosine (Tyr). When PAH activity is reduced, phenylalanine accumulates and is converted into phenylpyruvate (also known as phenylketone), which can be detected in the urine
16
Q
Cystinuria
A
an inherited autosomal recessive disease that is characterized by the formation of cystine stones in the kidneys, ureter, and bladder. Cystinuria is caused by mutations in genes that encode parts of a transporter protein that is made primarily in the kidneys. These defects prevent proper reabsorption of basic, or positively charged, amino acids: lysine, ornithine, arginine.
17
Q
Selenocysteine
A
is the 21st proteinogenic amino acid. It exists naturally in all kingdoms of life as a building block of selenoproteins. Selenocysteine is a cysteine analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group.
18
Q
4-hydroxyproline and 5-hydroxylysine
A
major component in collagen. Proline hydroxylation requires ascorbic acid (vitamin C). The most obvious, first effects (gingival and hair problems) of absence of ascorbic acid in humans come from the resulting defect in hydroxylation of proline residues of collagen, with reduced stability of the collagen molecule, causing scurvy. created in post translational modification
19
Q
Methyllysine
A
found in myosin
20
Q
g-carboxyglutamate
A
Carboxylation in biochemistry is a posttranslational modification of glutamate residues, to γ-carboxyglutamate, in proteins. It occurs primarily in proteins involved in the blood clotting cascade, specifically factors II, VII, IX, and X, protein C, and protein S, and also in some bone proteins. This modification is required for these proteins to function. in prothrombin. In the blood coagulation cascade, Vitamin K is required to introduce gamma-carboxylation of clotting factors II, VII, IX, X and protein Z.
21
Q
Demosine
A
(lysine derivative), found in elastin
22
Q
Functions of amino acids other than constituents of proteins
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Intermediates of amino acid synthesis. Eg histidine-> histamine; L-Thyroxin (the thyroid hormone)
23
Q
warfarin
A
Warfarin works by blocking recycling of vitamin K, so that the body and tissues have lower levels of active vitamin K, and thus a deficiency of the active vitamin. Vitamin K is needed to incorporate gamma-carboxyglutamate into coagulation factors
24
Q
Vitamin K
A
a group of structurally similar, fat-soluble vitamins the human body needs for complete synthesis of certain proteins that are required for blood coagulation, and also certain proteins that the body uses to manipulate binding of calcium in bone and other tissues.
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Glycosylation
Many secreted and cell surface proteins are glycosylated. O-linked glycans attached to the hydroxyl oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains, or to oxygens on lipids such as ceramide. N-linked glycans attached to a nitrogen of asparagine or arginine side-chains. N-linked glycosylation requires participation of a special lipid called dolichol phosphate. This is the basis of ABO blood type. can make the protein more stable, more soluble and are important for cell to cell interactions
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congenital disorder of glycosylation
one of several rare inborn errors of metabolism in which glycosylation of a variety of tissue proteins and/or lipids is deficient or defective. Affected infants may have weak muscle tone (hypotonia), retracted (inverted) nipples, an abnormal distribution of fat, eyes that do not look in the same direction (strabismus), developmental delay, and a failure to gain weight and grow at the expected rate (failure to thrive). Mutations in the PMM2 gene cause CDG-Ia. The PMM enzyme is involved in a process called glycosylation, which attaches groups of sugar molecules (oligosaccharides) to proteins.
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(4) Modification of Side Chains by Acetylation and Methylation
eg. Acetyl lysine, di-methyl lysine, and di-methyl arginine; could change its charge and effect its interaction with DNA or other proteins; plays important roles for all things related to DNA
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Vorinostat
inhibit histone deacetylases (HDAC) for the treatment of cutaneous T cell lymphoma (CTCL)
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gleevec
bcr-abl tyrosine kinase inhibitor, in chronic myelogenous leukemia (CML) The BCR-ABL transcript is continuously active and does not require activation by other cellular messaging proteins. Gleevec competively binds to kinase active site on bcr-abl kinase, preventing it from phosphorylating and activating other enzymes
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ubiquitination
a post-translational modification (an addition to a protein after it has been made) where ubiquitin is attached to a substrate protein. The addition of ubiquitin can affect proteins in many ways: It can signal for their degradation via the proteasome, alter their cellular location, affect their activity, and promote or prevent protein interactions.
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Velcade
therapeutic proteasome inhibitor for treating relapsed multiple myeloma; proteasome inhibition may prevent degradation of pro-apoptotic factors, permitting activation of programmed cell death
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Multiple Myeloma
a cancer of plasma cells, a type of white blood cell normally responsible for producing antibodies. Has elevated proteosome activity.
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primary structure of a protein
refers to the amino acid sequence of a protein when amino acids are joined together to form the linear protein chain (also referred to as the polypeptide chain). Amino acids are joined together by the peptide bond formed between the carboxyl group of the first amino acid and the amino group of the second amino acid. Multiple amino acids are joined together to form a linear polypeptide chain and we often refer to each amino acid in the chain as a residue.
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secondary structures
The protein chain folds itself to form local secondary structures. The two major types of secondary structures are the alpha helix and the beta pleated sheet (or simply called the beta sheet). The turns and loops connect alpha helix or beta sheet to form tertiary structures. Left handed triple helix is a unique secondary structure present in collagens and is important for the function of collagen.
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tertiary structures
The secondary structures interact with each other to form three-dimensional tertiary structures. There are two major classes of tertiary structures: fibrous and globular structures.
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quaternary structures
Sometimes more than one protein chain comes together to form quaternary structures. For example, four polypeptide chains come together to form a functional hemoglobin. Using the protein structure, we can understand how protein functions. We can also design drugs that modulate these protein functions.
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polypeptide backbone
There are three kinds of covalent bonds in a polypeptide backbone: the bond between Ca and the carbonyl carbon within the first amino acid; the peptide bond between the carbonyl carbon of the first amino acid and the amide nitrogen of the second amino acid; the bond between the amide nitrogen and Ca of the second amino acid. The peptide bond has partial double bond property and cannot be rotated, but the other two covalent bonds can be rotated.
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Ramachandran plot
a way to visualize backbone dihedral angles ψ against φ of amino acid residues in protein structure. shows the common secondary structure elements. Not all phi and psi angles are possible because some rotations cause steric crowding of the backbone atoms. The possible phi and psi angles are clustered in small regions of a Ramachandran plot. In a fully extended polypeptide, phi and psi are 180°
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peptide bond
is synthesized when the carboxyl group of one amino acid molecule reacts with the amino group of the other amino acid molecule, causing the release of a molecule of water (H2O) has partial double bond character There is no rotation around it, C, O, N, H, and Ca are in a plane
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f (phi
angle around the Ca—amide nitrogen bond
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y (psi)
angle around the Ca—carbonyl carbon bond
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steric crowding
Some f and y combinations are unfavorable because of
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H-bonding interactions
Some f and y combinations are favorable because of H-bonding interactions along the backbone
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polymorphic proteins
about 20-30% of human proteins are polymorphic, meaning that these proteins have slightly different amino acid sequences at non-essential positions in each individual. On the other hand, Many genetic diseases are caused by proteins with one amino acid mutated.
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proteases
The peptide bond in a polypeptide chain can also be broken down by enzymes called proteases. There are proteases that digest any peptide bond, such as trypsin, chymotrypsin, and pepsin, which are important proteases that break down protein in food in the digestive system. There are other proteases that digest specific peptide bonds. These specific proteases often serve to activate a particular enzyme through peptide bond cleavage.
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blood clotting factors
many blood clotting factors are proteases normally existing in their inactive form. Upon trauma or surface damage, the most upstream clotting factor is cleaved and converted into active protease that triggers further cleavage of downstream clotting factors.
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Higher order protein structures
are driven by a number of forces, including hydrogen bond, ionic interaction, hydrophobic interaction, and Van der Waal’s interaction.
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Hydrogen bond
Hydrogen bond is an attractive force formed between a hydrogen donor and a hydrogen acceptor. Hydrogen bonds between the protein backbone amide nitrogen and carbonyl oxygen are the driving force for the formation of protein secondary structures.
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alpha helix
hydrogen bonds are formed between the carbonyl oxygen of the nth amino acid and the amide nitrogen of the n+4th amino acid within the same polypeptide chain. These hydrogen bonds force the polypeptide chain to form a right-handed screw-like helical structure. All side chains of amino acids in an alpha helix point outward from the helix. The first and eighth residues in a helix are aligned nicely on top of each other. Some amino acids (for example Ala and Leu) have a higher tendency to form alpha helices but other amino acids (Pro and Gly) cannot form secondary structures such as alpha helices.
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hemoglobin
An example of proteins that is mainly alpha helical is hemoglobin.
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Thalassemia
a genetic disease where mutations often cause the instability of alpha helices in hemoglobin.
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beta sheet
hydrogen bonds are formed between two polypeptide chains. The two chains can be arranged in parallel or anti-parallel fashions. An example of protein which is mainly composed of beta sheets is immunoglobulin or antibody. Circular Dichroism is a method to monitor the secondary structure content of a protein.
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Diverse functions of peptide
some functions include hormones and pheromones (insulin and ghrelin), neuropeptides, antibiotics, and protection (toxins)
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Vasopressin-AntiDiuretic Hormone
a peptide hormone that increases water permeability of the kidney's collecting duct and distal convoluted tubule, which in turn increases arterial pressure
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substance p
neuropeptides, pain mediator
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Angiotensin II
a peptide hormone that causes vasoconstriction and a subsequent increase in blood pressure. Angiotensin I is converted to angiotensin II (AII) through removal of two C-terminal residues by the enzyme angiotensin-converting enzyme (ACE
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holoenzyme
a catalytically active enzyme consisting of an apoenzyme combined with its cofactor
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Cofactor
functional non-amino acid component. Prosthetic groups are covalently attached cofactors (heme in myoglobin). Vitamins are often cofactors
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Coenzyme
complex organic or metalloorganic cofactors ( NAD+ in lactate dehydrogenase)
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Amino acid sequence and function
Amino acid sequence determines the function of each protein. Each different protein in our body has a unique amino acid sequence. Proteins with similar sequences have similar functions. Many genetic diseases are caused by proteins with one amino acid mutated. On the other hand, about 20-30% of human proteins are polymorphic, meaning that these proteins have slightly different amino acid sequences at non-essential positions in each individual.
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sickle cell anemia
a known mutation of a single nucleotide (see single-nucleotide polymorphism - SNP) (A to T) of the β-globin gene, which results in glutamic acid being substituted by valine. The sickle-cell disease occurs when the sixth amino acid, glutamic acid, is replaced by valine to change its structure and function; as such, sickle-cell anemia is also known as E6V. Valine is hydrophobic, causing the haemoglobin to collapse on itself occasionally.
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Proteases
hydrolyse peptide bonds at particular amino acid residues in a protein. Some are specific and some are general
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peptidases
hydrolyse peptide bonds at particular amino acid residues in a peptide
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trypsin
a more general protease,a serine protease from the PA clan superfamily, found in the digestive system of many vertebrates
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HIV protease
a more specific protease, inhibitor is crixivan. a retroviral aspartyl protease (retropepsin) that is essential for the life-cycle of HIV, the retrovirus that causes AIDS. HIV protease cleaves newly synthesized polyproteins at the appropriate places to create the mature protein components of an infectious HIV virion. Without effective HIV protease, HIV virions remain uninfectious. Can use the structure of this protein to develope drugs that target it
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HIV and CD4
a co-receptor that assists the T cell receptor (TCR) in communicating with an antigen-presenting cell. HIV-1 uses CD4 to gain entry into host T-cells. By investigating this protein interaction, we could create ways to interfere with such binding
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Antibody as drugs
could be used to inhibit protein-protein interactions that are harmful. Examples: Bevacizumab (Avastin): Anti-VEGF; Treatment of tumors. Infliximab (Remicade): Anti-TNFa; Treatment of rheumatoid arthritis, interstitial bowel disease. Rituximab (Rituxan):Anti-CD20,Treat B cell lymphoma, lupus and other autoimmune diseases.
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angiotensin converting enzyme ACE
indirectly increases blood pressure by causing blood vessels to constrict. It does that by converting angiotensin I to angiotensin II, which constricts the vessels. a more specific protease, inhibitor is captopril
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examples of proteins activated by protease cleavage
Activation of digestive enzymes such as trypsin, chymotrypsin. Activation of insulin. Activation of complement enzymes. Activation of blood clotting. Activation of some transcription factors eg Notch. Activation of enzymes involved in programmed cell death.
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blood clotting cascade
tissue damage activates clotting factor 7, which cleaves factor 10, which cleave prothrombin to create thrombin, which creates fibrin through cleavege to create blood clotts
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van der waals interactions
the sum of the attractive or repulsive forces between molecules (or between parts of the same molecule) other than those due to covalent bonds
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hydrogen donor
backbone NH, His N, Asn N, Gln N, Arg N, Lys N, Trp N, Ser O, Thr O, Tyr O
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hydrogen acceptor
backbone O, Ser O, Thr O, Tyr O, Asp O, Glu O, Gln O
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Leucine Zipper
Luecine is hydrophobic causing them to form a dimer in many TF
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Secondary structur in fibrous proteins
Proteins composed mostly of a-helices (eg Keratin-hair, nails, horns; Myosin; Tropomyosin; fibrinogen) and proteins composed of beta sheets include fibroin silk and spider webs
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Secondary structure in globular proteins
Proteins composed mostly of a-helices; (eg. Hemoglobin Myoglobin) and proteins composed mostly of beta-pleated sheets (eg Ig, fibroblast growth factor, pepsin, HIV protease)
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immunoglobulin structure
composed completely of antiparrallel beta sheets
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Circular Dichroism (CD) Analysis
CD measures the absorption difference (De) of left- and right- circularly polarized light. CD signals depend on the chain conformation
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beta Turns
b-turns occur frequently whenever strands in b sheets change the direction. The 180° turn is accomplished over four amino acids. The turn is stabilized by a hydrogen bond from the carbonyl oxygen of the 1st amino acid to amide proton of the 4th amino acid. Proline in position 2 or glycine in position 3 are common in b-turns.
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Proline Isomers
Most peptide bonds are in the trans configuration. For peptide bonds involving proline, ~6% are in the cis configuration. Proline isomerization is catalyzed by prolyl isomerases
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Fibrous proteins
tend to be long and have one kind of secondary structure (alpha helix or beta sheet). These proteins are often insoluble and perform a structural or protective role.
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a-Keratin
a family of fibrous structural proteins. Keratin is the key structural material making up the outer layer of human skin. It is also the key structural component of hair and nails. Fibrous keratin molecules supercoil to form a very stable, left-handed triple-helical motif to multimerise, forming filaments consisting of multiple copies of the keratin monomer.
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Collagen
Collagen is an important constituent of connective tissue: tendons, cartilage, bones, cornea of the eye. Each collagen chain is a long Gly- and Pro-rich left-handed helix. Three collagen chains intertwine into a right-handed superhelical triple helix. The triple helix has higher tensile strength than a steel wire of equal cross section. The proteins are often hydroxylated (Hydroxyproline) in order to create more hydrogen bonding and increase stability. G-X-X patteren allow proteins to pack closer together (glycine is small).
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Globular proteins
Most proteins in the body are globular proteins which are water or lipid soluble and carry out diverse functions. Turns and loops play important roles in the structure of globular proteins. These turns or loops enable the polypeptide chain to form particular structures instead of staying as extended linear chains. Sometimes loops and turns play important roles in interacting with other proteins such as the variable loops in an immunoglobulin molecule. The most frequently found amino acids in turns and loops are Glycine and Proline amino acids.
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Why is proline and glycine prevalent in Beta turns?
Proline is an unusually shaped amino acid, meaning it can provide the "kink" necessary to make beta-sheets turn the correct way. Glycine is a very flexible amino acid, and it has a very small side chain, so it will not interfere with the directions.
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special properties of proline
The distinctive cyclic structure of proline's side chain gives proline an exceptional conformational rigidity compared to other amino acids. It also affects the rate of peptide bond formation between proline and other amino acids. When proline is bound as an amide in a peptide bond, its nitrogen is not bound to any hydrogen, meaning it cannot act as a hydrogen bond donor, but can be a hydrogen bond acceptor. Proline acts as a structural disruptor in the middle of regular secondary structure elements such as alpha helices and beta sheets. Proline is also commonly found in turns (another kind of secondary structure), and aids in the formation of beta turns. This may account for the curious fact that proline is usually solvent-exposed, despite having a completely aliphatic side chain. Proline is a helix breaker because the side chain is basically jammed into the space that should be occupied by the backbone of the alpha helix -- a methylene group is in the space that would normally be occupied by a hydrogen-bonding amide proton, thus disrupting the H-bond network and sterics of the helix.
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structural motifs
The most frequently found amino acids in turns and loops are Glycine and Proline amino acids. Different proteins sometimes use common structural motifs which are specific arrangements of secondary structure elements. Common structural motifs often carry out similar functions. Many proteins are composed of domains which are often independent folding and functional units.
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dissociation constant
commonly used to describe the affinity between a ligand (L) (such as a drug) and a protein (P) i.e. how tightly a ligand binds to a particular protein. Ligand-protein (C) affinities are influenced by non-covalent intermolecular interactions between the two molecules such as hydrogen bonding, electrostatic interactions, hydrophobic and Van der Waals forces. They can also be affected by high concentrations of other macromolecules, which causes macromolecular crowding. Kd=(P)(L)/(C) The binding specificity is achieved through the lock and key complementary model or the induced fit model. the lower the Kd the tighter the binding (you need less enzyme to bind 50% of substrate)
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lock and key complementary model
At the active sites, the enzyme has a specific geometric shape and orientation that a complementary substrate fits into perfectly. According to this theory, the enzyme and substrate shape do not influence each other because they are already in a predetermined perfectly complementary shape. As a result, the substrate will be stabilized. This theory was replaced by the induced fit model which takes into account the flexibility of enzymes and the influence the substrate has on the shape of the enzyme in order to form a good fit.
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induced fit model
the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding.
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myoglobin
an iron- and oxygen-binding protein found in the muscle tissue of vertebrates in general and in almost all mammals. It is related to hemoglobin, which is the iron- and oxygen-binding protein in blood, specifically in the red blood cells. Myoglobin is only found in the bloodstream after muscle injury. It is an abnormal finding, and can be diagnostically relevant when found in blood. Myoglobin can only store but cannot transport oxygen because while it binds well to O2, it does not release it well. consists of eight alpha helices connected by loops.
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hemoglobin
the iron-containing oxygen-transport metalloprotein in the red blood cells. has a quaternary structure composed of four poly peptide chains (alpha helix). The heme binding sites in these four chains interact with each other through hydrogen bonding which enables hemoglobin to have a Tense and Relaxed state with different oxygen binding affinities. This is how binding at one site is sensed but another. These unique structure and binding properties enable hemoglobin to bind oxygen tightly in the lung and release the oxygen in tissues. Carbon monoxide binds heme much tighter than oxygen, leading to CO poisoning. it can also transport H and CO2
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allostery
also called cooperativity is the regulation of a protein by binding an effector molecule at a site other than the protein's active site. positive cooperativity: first binding event increases affinity at remaining sites. negative cooperativity: first binding event reduces affinity at remaining sites. Positive cooperativity can be recognized by sigmoidal binding curves
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tense and relaxed state of hemoglobin
Hemoglobin exists in two forms, a taut (tense) form (T) and a relaxed form (R). Various factors such as low pH, high CO2 and high 2,3 BPG at the level of the tissues favor the taut form, which has low oxygen affinity and releases oxygen in the tissues. Conversely, a high pH, low CO2, or low 2,3 BPG favors the relaxed form, which can better bind oxygen. The partial pressure of the system also affects O2 affinity where, at high partial pressures of oxygen (such as those present in the alveoli), the relaxed (high affinity, R) state is favoured. Inversely, at low partial pressures (such as those present in respiring tissues), the (low affinity, T) tense state is favoured. Additionally, the binding of oxygen to the Iron-II heme pulls the iron into the plane of the porphyrin ring (electron donation Fe2+ -> Fe3+ causes Fe to shrink), causing a slight conformational shift. The shift encourages oxygen to bind to the three remaining hemes within hemoglobin (thus, oxygen binding is cooperative).
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heme
a chemical compound of a type known as a prosthetic group consisting of an Fe2+ (ferrous) ion contained in the centre of a large heterocyclic organic ring called a porphyrin, made up of four pyrrolic groups joined together by methine bridges.
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Bohr effect
hemoglobin's oxygen binding affinity (see Oxygen–haemoglobin dissociation curve) is inversely related both to acidity and to the concentration of carbon dioxide. That is to say, an increase in blood CO2 concentration which leads to a decrease in blood pH will result in hemoglobin proteins releasing their load of oxygen. Conversely, a decrease in carbon dioxide provokes an increase in pH, which results in hemoglobin picking up more oxygen. Since carbon dioxide reacts with water to form carbonic acid, an increase in CO2 results in a decrease in blood pH.
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CO poisoning
carbon monoxide toxicity arises from the formation of carboxyhemoglobin, which decreases the oxygen-carrying capacity of the blood and inhibits the transport, delivery, and utilization of oxygen by the body. The affinity between hemoglobin and carbon monoxide is approximately 230 times stronger than the affinity between hemoglobin and oxygen so hemoglobin binds to carbon monoxide in preference to oxygen. The binding of carbon monoxide at one of these sites increases the oxygen affinity of the remaining three sites, which causes the hemoglobin molecule to retain oxygen that would otherwise be delivered to the tissue. This situation is described as carbon monoxide shifting the oxygen dissociation curve to the left. Because of the increased affinity between hemoglobin and oxygen during carbon monoxide poisoning, little oxygen will actually be released in the tissues. Carbon monoxide also binds to the hemeprotein myoglobin.
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crystallography method
a tool used for identifying the atomic and molecular structure of a crystal, in which the crystalline atoms cause a beam of incident X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. analyses of X-ray diffraction pattern generated by protein crystals lead to the determination of the protein structure.
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NMR determination of protein structure
analyses of magnetic resonance signal generated by high concentration of protein in solution within a magnetic field lead to the determination of the protein structure. NMR involves the quantum mechanical properties of the central core ("nucleus") of the atom. These properties depend on the local molecular environment, and their measurement provides a map of how the atoms are linked chemically, how close they are in space, and how rapidly they move with respect to each other. This is very difficult with large proteins.
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protein denaturing
Proteins can be denatured by heat and chemicals. denatured ribonuclease A can completely refold to its native state on its own, suggesting that all the information required to fold a protein into its native state is embedded in the primary amino acid sequences of the protein. The temperature at which half of the proteins are denautured is Tm
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Levinthal’s paradox
Proteins fold to the lowest-energy fold in the microsecond to second time scales. Assuming each aa has 10 conformations, a 100aa protein will have 10100 conformations. If sampling each in 10-13 sec, will take 1087 years. Therefore the search for the minimum energy level is not random
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Two models of folding pathways
1. Secondary structures first and then loops and tertiary structures. This is supported by the fact that The globular structure of myoglobin and many other proteins is stabilized by interactions between the side chains of amino acids on one a helix and those on another.
2. Hydrophobic amino acids condense to form a molten globule and then other secondary and tertiary structure features. This is supported by the distribution of hydrophobic and hydrophilic residues in proteins. 3. Or some combination of the two…
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Why don’t all proteins refold into their native structures after denaturation?
1. In the cell the protein folds as it is synthesized. 2. The protein may have been irreversibly insolubilised by the denaturation process. 3. Proper folding of some proteins in the cell is aided by various
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chaperones
not all proteins can refold on their own. proteins that assist the non-covalent folding or unfolding and the assembly or disassembly of other macromolecular structures.
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Hsp70
As newly synthesized proteins emerge from the ribosomes, the substrate binding domain of Hsp70 recognizes sequences of hydrophobic amino acid residues, and interacts with them. This spontaneous interaction is reversible, and may relatively freely bind and release peptides. By binding to partially synthesized peptide sequences, Hsp70 prevents them from aggregating and being rendered nonfunctional. Hsp70 proteins can act to protect cells from thermal or oxidative stress. They are induced at elevated temperatures and bind to hydrophobic region of unfolded protein and prevent aggregation. They can also help transport some proteins cross membranes in unfolded states. prokaryotic homologs: DnaK and DnaJ
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GroEL
the chaperonin family of molecular chaperones, and is found in a large number of bacteria. Unfolded substrate proteins bind to a hydrophobic binding patch on the interior rim of the open cavity of GroEL, forming a binary complex with the chaperonin. Binding of substrate protein in this manner, in addition to binding of ATP, induces a conformational change
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Macromolecular crowding
alters the properties of molecules in a solution when high concentrations of macromolecules such as proteins are present. These high concentrations of macromolecules occupy a large proportion of the volume of the cell, which reduces the volume of solvent that is available for other macromolecules. This excluded volume effect increases the effective concentration of macromolecules (increasing their chemical activity), which in turn alters the rates and equilibrium constants of their reactions. In particular this effect alters dissociation constants by favoring the association of macromolecules, such as when multiple proteins come together to form protein complexes, or when DNA-binding proteins bind to their targets in the genome
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Protein disulfide isomerase
n enzyme in the endoplasmic reticulum in eukaryotes that catalyzes the formation and breakage of disulfide bonds between cysteine residues within proteins as they fold. This allows proteins to quickly find the correct arrangement of disulfide bonds in their fully folded state, and therefore the enzyme acts to catalyze protein folding.
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Prolyl isomerase
interconverts the cis and trans isomers of peptide bonds with the amino acid proline.[1] Proline has an unusually conformationally restrained peptide bond due to its cyclic structure with its side chain bonded to its secondary amine nitrogen. Most amino acids have a strong energetic preference for the trans peptide bond conformation due to steric hindrance, but proline's unusual structure stabilizes the cis form so that both isomers are populated under biologically relevant conditions.
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Cyclophilins
also known as peptidylprolyl isomerase. The cyclosporin-cyclophilin A complex inhibits a calcium/calmodulin-dependent phosphatase, calcineurin, the inhibition of which is thought to suppress organ rejection by halting the production of the pro-inflammatory molecules TNF alpha and interleukin 2.
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cystic fibrosis
defects in cystic fibrosis transmembrane conductance regulator (CFTR), most common mutation is the deletion of F508, which causes protein misfolding
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prion disease
The normal prion protein which is rich in alpha helix structure. Prions are not considered living organisms but are misfolded protein molecules which may propagate by transmitting a misfolded protein state. If a prion enters a healthy organism, it induces existing, properly folded proteins to convert into the disease-associated, misfolded prion form; the prion acts as a template to guide the misfolding of more proteins into prion form. These newly formed prions can then go on to convert more proteins themselves; this triggers a chain reaction that produces large amounts of the prion form. All known prions induce the formation of an amyloid fold, in which the protein polymerises into an aggregate consisting of tightly packed beta sheets. Amyloid aggregates are fibrils, growing at their ends, and replicating when breakage causes two growing ends to become four growing ends. Creutzfeldt Jacob disease is an example of prion disease in humans
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Alzheimer’s disease
Most of autosomal dominant familial AD can be attributed to mutations in one of three genes: those encoding amyloid precursor protein (APP) and presenilins 1 and 2. Most mutations in the APP and presenilin genes increase the production of a small protein called Aβ42, which is the main component of senile plaques. Some of the mutations merely alter the ratio between Aβ42 and the other major forms—e.g., Aβ40—without increasing Aβ42 levels. This suggests that presenilin mutations can cause disease even if they lower the total amount of Aβ produced and may point to other roles of presenilin or a role for alterations in the function of APP and/or its fragments other than Aβ. There exist variants of the APP gene which are protective.
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Amyloid precursor protein (APP)
an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. Its primary function is not known, though it has been implicated as a regulator of synapse formation, neural plasticity and iron export. APP is best known as the precursor molecule whose proteolysis generates beta amyloid (Aβ), a 37 to 49 amino acid peptide whose amyloid fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients.
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Presenilins
Most cases of Alzheimer's disease are not hereditary. However, there is a small subset of cases that have an earlier age of onset and have a strong genetic element. In patients suffering from Alzheimer's disease (autosomal dominant hereditary), mutations in the presenilin proteins (PSEN1; PSEN2) or the amyloid precursor protein (APP) can be found. The majority of these cases carry mutant presenilin genes. To form Aβ, APP must be cut by two enzymes, beta secretases and gamma secretase. Presenilin is the sub-component of gamma secretase that is responsible for the cutting of APP.
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Parkinson’s disease
a degenerative disorder of the central nervous system. The motor symptoms of Parkinson's disease result from the death of dopamine-generating cells in the substantia nigra, a region of the midbrain; the cause of this cell death is unknown. Mutations in specific genes have been conclusively shown to cause PD. The role of the SNCA gene is important in PD because the alpha-synuclein protein is the main component of Lewy bodies (abnormal aggregates of a protein).
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amyloidosis
a non-specific term that refers to a number of different diseases collectively called amyloidoses. Amyloids are proteins whose secondary structure changes, causing the proteins to fold in a characteristic form, the beta-pleated sheet.[1] When the normally soluble proteins fold to become amyloids, they become insoluble and deposit in organs or tissues, disrupting normal function.[2][3] Different types of amyloidoses have different signs and symptoms depending on where and in which organs the amyloid proteins aggregate. Amyloidoses can be inherited or acquired.
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gel filtration chromatography
separates proteins based on their size differences. a small molecule that can penetrate every region of the stationary phase pore system "sees" a total volume equal to the sum of the entire pore volume and the interparticle volume. This small molecule will elute late due to the pore- and interparticle interactions). On the other extreme, a very large molecule that cannot penetrate any region of the pore system and interacts only the interparticle volume and will elute earlier when this volume of mobile phase has passed through the column.
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ion-exchange chromatography
Proteins have numerous functional groups that can have both positive and negative charges. Ion exchange chromatography separates proteins according to their net charge, which is dependent on the composition of the mobile phase. By adjusting the pH or the ionic concentration of the mobile phase, various protein molecules can be separated. For example, if a protein has a net positive charge at pH 7, then it will bind to a column of negatively charged beads, whereas a negatively charged protein would not. By changing the pH so that the net charge on the protein is negative, it too will be eluted.
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affinity chromatography
a method of separating biochemical mixtures based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.
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gel electrophoresis
Proteins, unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the polyacrylamide gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are usually denatured in the presence of a detergent such as sodium dodecyl sulfate (SDS) that coats the proteins with a negative charge. Generally, the amount of SDS bound is relative to the size of the protein, so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape.
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mass spectrometry
by bombarding it with electrons, the sample's molecules break into charged fragments. These ions are then separated according to their mass-to-charge ratio, typically by accelerating them and subjecting them to an electric or magnetic field: ions of the same mass-to-charge ratio will undergo the same amount of deflection. The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier. Results are displayed as spectra of the relative abundance of detected ions as a function of the mass-to-charge ratio. The atoms or molecules in the sample can be identified by correlating known masses to the identified masses or through a characteristic fragmentation pattern.
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Edman degradation
a method of sequencing amino acids in a peptide.Phenylisothiocyanate is reacted with an uncharged terminal amino group, under mildly alkaline conditions, to form a cyclical phenylthiocarbamoyl derivative. Then, under acidic conditions, this derivative of the terminal amino acid is cleaved as a thiazolinone derivative. The thiazolinone amino acid is then selectively extracted into an organic solvent and treated with acid to form the more stable phenylthiohydantoin (PTH)- amino acid derivative that can be identified by using chromatography or electrophoresis. This procedure can then be repeated again to identify the next amino acid.
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Western blot
It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane, where they are stained with antibodies specific to the target protein. The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample.
