TBT4135 - Biopolymerkjemi
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Fakta høst 2009
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Øvingsopplegg høst 200+
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Lab høst 2009
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Innføring i biologiske polymerer (polysakkarider, proteiner), med laboratorieøvinger i anvendte teknikker.
Innhold
Oppsummering
DNA
Deoxyribose (2-deoxy D-ribose), attached to a phosphate group on 3', this is 3' end), and next phosphate group on 3' (3' end). <math>\beta</math>-linked to pyrimidine (cytosin and guanine) or pyrine (adenine or thymin/uracil) at C-1.
PCR
Melt DNA (double -> singlestranded). Add small primers of known sequence near region of interest, bases and DNA polymerase. Rinse and repeat.
Sequencing
Maxam-Gilbert: Base specific cleavage of DNA after marking 5' end by radioactive phosphate, and seperated in gel electrophoresis. Can sequence up to 200 bases. Dideoxy: Synthesize DNA by biological methods, but add small amounts of a type of dideoxy base, which stops synthesis at certain places. Seperate by gel electrophoresis and put together sequence.
Proteins
Amino acids
All proteins formed of L-amino acids.
20 essential amino acids: Non-polar amino acids Alanine (Ala, A): methyl Valine (Val, V): isopropyl Leucine (Leu, L): isobuthyl Isoleucine (Ile, I): 1-methyl propane Proline (Pro, P): Propyl linked to amine in main Phenylalanine (Phe, F): Alanine with phenylfunction Tryptophan (Trp, W): Alanine with indole group Methionine (Met, M): CH3(2)-S-CH3, can be synthesized from cystein.
Polar amino acids Glycine (Gly, G): H Serine (Ser, S): methanol Threonine (Thr, T): tert-propanol Cysteine (Cys, C): methanethiol Tyrosine (Tyr, Y): Phenylalanine with hydroxy in para. Aspargine (Asp, N): Aspartic acid with amino instead of hydroxy Glutamine (Gln, Q): Glutamic acid with amino instead of hydroxy
Acidic amino acids Aspartic acid (Asp, D): Acetic acid Glutamic acid (Glu, E): Propylic acid
Basic amino acids Lysine (Lys, K): amino-buthane Arginine (Arg, R): propyl-guanidinium Histidine (His, H): methyl-imidazole
In general the <math>\alpha</math>-carboxyl group has a pKa of about 2 and the <math>\alpha</math> amino group has a pKa of around 9.5. Asp and Glu have pKa around 4, Cys, Thr and Lys have around 10, Arg has around 12.5 while histidine is special at around 6. Calculate pI by testing what net charge the protein has at a given pH and then try again.
Sequencing
Sanger's method: Attach dinitrofluorobenzene to N-terminal, degrade protein completely and then identify amino acid that is attached to reagent. Can do similar to carboxyl end. This can be used to sequence di- or tripeptides (middle amino acids identified by chromatography). Use mild degradation to obtain mixture, put together puzzle. Not used anymore.
Edman's method: Disconnect only amino-terminal amino acid, identify, and repeat. Can be done automatically in parallell.
Gene coding: Find the first few amino acids (7-10), use this to make a DNA probe (primer in PCR), amplify gene and sequence. Only works on prokaryotes, due to introns in eukaryotes.
Structure
Partial double bond in peptide bond hinders rotation in peptide chain except on each side of the <math>\alpha</math>-carbon (with R-group) although limited to certain angles. R-groups alternating side of chain.
Arnfinsens experiment: Disrupt disulfide bonds with mercaptoethanol and denature with 8M urea, reverse and regain most of activity - folding is native low energy state.
<math>\alpha</math>-helixes
Left-handed helix, full turn every 3.6 amino acids, with a rise of about 1,5 Å and pitch 5,4 Å. R-groups facing out of helix. Stabilized by uncharged, medium-sized amino acids: Ala, Leu, Phe, Tyr, Trp, Cys, Met, His and Asn. Small or large R-groups, or charged amino acids, de-stabilize the helix: Gly, Ile, Glu, Asp, Lys, Arg, Ser, Thr. Proline and hydroxyproline break the helix, due to hindered rotation in cyclobuthanol-ring. Threonine and serine have intramolecular hydrogen bonds that compete with intermolecular hydrogen bonds. There are other types of <math>\alpha</math>-helixes, such as <math>\alpha_10</math> or pi helixes, which are similar but with less or more amino acid residues per turn. In the standard helix amino acid i and i+3 hydrogen bond. Keratine is rich in <math>\alpha</math>-helixes and <math>\beta</math>-sheets (see below).
Collagen has a triple helical structure that is right-handed with about 20 amino acids per turn, i.e. a much loser structure. Typical sequence is Gly-X-Y where X is often proline and Y is often hydroxyproline. These lock the bond angles to favour this type of helix. Gelatin is denatured collagen that partially reforms the helixes upon gelation. Collagen triple helixes form intermolecular hydrogen bonds to other helixes to make strong filaments, but does not form intramolecular hydrogen bonds.
Collagen is the most abundant protein in mammals, 25-35% of total protein content. Collagen type I, II and III are fibrillar collagen and are found in most connective tissues and bone, cartilage and vitreous humor, and extensible connective tissues respectively. Collagen type IV is part of the basal laminae. Together they account for >90% of the collagen in the body. There are different chain types in the different forms of collagen, type I has two <math>\alpha</math>1 and one <math>\alpha</math>2 chain, type II has three <math>\alpha</math>1 chains, type III has three <math>\alpha</math>3 chains, while type IV has a mixture.
Collagen is built by first forming tropocollagen (three helixes bound together). In the ECM the ends are cleaved and the tropocollagen assembles into fiber bundles. The bundles have a striated appearance. Allysine and lysine residues form Schiff base covalent crosslinks.
Elastin is another ECM protein with a random coil shape, rich in glycine, valine, alanine and proline. Gives flexability to the ECM.
There are many diseases associated with ECM disorders. Marfan syndrome (long arms, legs, extra stretchy) caused by mutation in fibrillin, an important structural protein holding elastin in place. Ehler-Danlos syndrome (stretchy skin, lesions, bruises, bendable limbs) caused by mutation in collagen III.
<math>\beta</math>-sheets
Stretched <math>\alpha</math>-keratins, <math>\beta</math>-keratins and silk fibroin have a common protein structure called a <math>\beta</math>-sheets. Hydrogen bonds are formed between the backbone amide groups, while the R-groups stick up and down in the plane, forming intra-layer bonds in addition.
From the amino acid sequence the secondary structures above can sometimes be estimated. Hydropathy plots can also be made to map regions heavy in hydrophilic or hydrophobic regions, to see where they are most likely to be found in a tertiary structure. Tertiary structures can be assembled into quaternary structures, which are stabilized by weak interactions or disulfide bonds between segments.
Polysaccharides
Polysaccharides are the most abundant biopolymer. The basic building blocks are monosaccharides.
Monosaccharides
They are designated D or L depending on the orientation of the highest numbered chiral carbon atom. If the hydroxy group is pointing right in the Fischer structure it is a D-sugar. This corresponds to the non-ring carbon to be pointing up in the Haworth projection. Opposite for L sugars. L sugars are mirror images of R sugars with the corresponding name, i.e. all groups are mirrored. If only one group is mirrored the sugars are C-X epimers. If the hydroxygroup on C-1 is cis with the non-ring carbon group the sugar is <math>\beta</math>, or <math>\alpha</math> if trans. Pentoses are ribose (RR), arabinose (LR), xylose (RL) and lyxose (LL). Hexoses are allose (RRR), altrose (LRR), glucose (RLR), mannose (LLR), gulose (RRL), idose (LRL), galactose (RLL) and talose (LLL).
