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.
Alpha-helix: 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, Ph
