BioChem: Proteins and Peptides

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What are amino acids (in terms of functional groups)?: -an amino derivative of carboxylic acids (both are linked to the alpha carbon)
What are Zeitterions?: -at physiological pH, the amino group os protonated and the carboxyl is ionized
What are the ploar uncharge amino acids?: -glycine
-serine
-asparagine
-threonine
-cysteine
-glutamine
-tyrosine
What are thh hydrophobic amino acids?: -leucine
-proline
-alanine
-valine
-methionine
-tryptophan
-phenylalanine
-isoleucine
What are the basic amino acids?: -lysine
-histidine
-arginine
What are the acidic amino acids?: -aspartic acid
-glutamic acid
How many of the 20 AA are chiral?: -19 out of 20!
(exist as stereoisomers)
What are stereoisomers?: -they have same molecular formula but different arrangements in space
There are about 200 naturally occurring AA. How can there be so many?: -exist as L-AA's in biochemical pathways
-D-AA's in microbe cell walls and antibiotics
-Post-translationally modified AA
What is the primary sequence of proteins?: -all peptide bonds are the same
-only sequence of side chains is different
-vary widely in size and complexity
Do cells contain lots of protein?: Yes, Rat liver has a lot. E-coli is a close second. Even spinach has protein!
What are the essential amino acids?: -arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tyrptophan, valine
What are the nonessential amino acids?: -alanine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, tyrosine
Which two essential amino acids are actually only needed in juveniles of rats and humans?: -arginine
-histidine
How does a Ramachandran plot work?: -only white spots are possible bond angles.
What is amino acid analysis?: -finding what amino acids are present and in what amounts in a protein
What is the sequence of amino acids (not analysis): -finding the unique sequence of amino acids in a protein
How do you perform acid hydrolysis of peptides?: 1) make up in 6 N HCL
2) heat at 110 C for 24 hours
What are different ways of separating out amino acids?: -peptide hydrolysis
-chromatography
-chromatogram
-the Ninhydrin reaction
Is the peptide bond flat or bent? Why?: -bond lies flat due to double bond character (DBC).
*Double bond moves between N=C and C=O
For the 20 amino acids we learned, what conformation are they even though they're not drawn that way?: -'L' acids
What are two amino acids that are rarely found in proteins?: -tryptophan
-thyamine
Which amino acid usually has disulfide bonds?: -cystein
what two groups will be destroyed if they are put in an acid hydrolysis?: -glutamine
-asparage
(amide groups get hydrolized)
*tryptophan can't stand it either
What is the point of Ninhydrin in a chromatogram?: everything turns purple except proline which is yellow
What does the height indicate of the peaks in a chromatogram?: -amount of amino acid present
What are 4 chromatographic methods?: 1) adsorption
2) ion exchange
3) partition
4) gel permeation
How does ion exchange work?: -AA's interact with a stationary charge
How does partition work?: -use of a liquid that separates objects
What does gel permeation do?: -acts as a molecular sieve by separating large sizes first
What is the problem with gel permeation?: -it's hard to find a medium that won't cause adsoption or ion exchange
Once again (and then some), what are the problems with acid hydrolysis?: -tryptophan destroyed
-glutamine and asparagine hydrolysed
-serine and thronine lowered (desaturation)
Why do scientists still use this method?: -testing facilities insist on it!
How is the procedure for Ion exchange chromatography?: 1)add cation exchange bead before sample
2) add mixture of Asp, Ser, Lys
3) add Na+ (such as NaCl): Asp lets go first due to least positive charge
4) increase Na+: Ser elutes next
5) increase Na+ more: Lys will elute last since it has highest charge
What protein is sometimes able to denature and renature?: -RNAase
What are two major protein sequencing reagents?: -Sangers Reagent (DNFB)
-Dansylchloride
What are 4 major protein cleaving reagents?: -Chymotrypsin
-Trypsin
-Endoprotease V8
-Cyanogenbromide
Where do cleaving reagents cleave?: -on carboxy side of group
How does Chymotrypsin work?: -cleaves on carboxy side of: Phe, Tyr, and Trp
-works on the aromatics
How does Trypsin work?: -cleaves on carboxy side of: Lys and Arg.
-works on basic groups
How does Endoprotease V8 work?: -cleaves on carboxy side of: Glu or Asp
-works on strong acidic groups
How does Cyanogenbromide work? What is different with this reagant from the others?: Converts Met into homoserine
*Cyanogenbromide is a chemical reagant NOT an enzyme, nor does it cleave.
What is the major difference between a twisted fiber and a pleated fiber?: -twisted can stretch to twice the length and back again; pleats barely increase before returning to form
Where do the characteristics of fibrous proteins come from?: -a result of their sequence and repeated bond angles
What amino acids make up silk fibroin and what is their ratio?: -gly, ala, ser
-3:2:1
ex; g-s-g-a-g-a-g-s-g-a-g-a-g-s
What shape is silk fibroin?: -found in beta pleated sheets
How does silk stretch in terms of wool?: -very poorly
-doesn't bounce back
What makes up alpha keratins?: -alpha helices (this is how you would make string into a rope)
What component of wool makes it so stretchy?: -sulfur content: it has cystein which has disulfide bonds
Is wool an alpha keratin?: Yes
What item makes up 25-50% of the protein in vertebrates?: -collagen
What are the key characteristics to collagen?: -very insoluble
-strongly resists stretching
-has lots of gly and pro (or hydroxypro which is more stable)
What is the sequence for collagen?: -mostly repeating Gly-X-Pro
Why is there so much Gly found in collagen?: -it is woven tightly and only Gly can fit in the center where the twists meet
What are the fours levels of protein stucture?: -primary
-secondary
-tertiary
-quartenary
How many levels of protein structure does hemoglobin show?: -all four levels
Describe the first level of protein structure: primary: -primary structure is the order of AA's in the polypeptide
Describe secondary structure.: -secondary structure is regularities in local conformations
Describe tertiary structure.: -tertiary structure is the completely folded protein, interactions may be between distant parts of the sequence
Describe quarternary structure.: Quarternary structure subunit interactions in an oligomeric protein
What causes Sickle-Cell anemia?: -An alteration in the sequence of the hemoglobin beta-chain at position 6 from Glu to Val
What affect does the change in hemoglobin have on shape of RBC's?: Deoxyhemoglobin is more hydrophobic in one area and this leads to the formation of long rigid fibers
What is denaturation?: -disruption of native conformation of a protein, with loss of biological activity
-can be caused by heating or chemicals (very small energy)
How can denaturation be monitored?: -monitor changes in ultraviolet (blue), viscosity (red), optical rotation (green).
How does 'in vivo' (inside cell) protein folding occur?: -folded proteins occupy a very low-energy well which makes it very stable
-proteins fold spontaneously into this conformation
-folding continued by previous folds through interactions
-folding is extremely rapid <1s
-
As a protein is folding, what happens to its shape and why?: -the polypeptide collapses in upon itself due to the hydrophobic effect
-an intermediate "molten globule" forms with elements fo secondary structure
-backbone rearranged to acheive a stable native conformation
What is RNase?: -Anfinsen denatured it using 8 M urea and beta mercaptoethanol (ME)
-then he removed them to allow refolding (it didn't work)
-he added catalytic amounts of beta ME and RNase reformed to 100% of its activity and function! He won the nobel.
What does the hydrophobic effect contribute in detail to protein folding?: -nonpolar side chains associate causing a polypeptide chain to collapse to the molten globule
-nonpolar chains inside while polar and charged side chains remain on surface facing water
What is the driving force for protein folding?: -the LARGE increase in entropy from water released to bulk solvent!
*hydrophobic collapse of a polypeptide occurs at the same time as formation of secondary structures
How does hydrogen bonding contribute to protein folding?: -contributes to cooperativity of folding
-helps stabilize secondary structures and native conformation
How do Van der Waals interactions contribute to protein folding?: -VDW contacts occur between nonpolar side chains and contribute to stability
-Charge-Charge interactions between oppositely charged side chains in protein interior also may stabalize
What is Anfinson's Flicker hypothesis?: I don't know!
-prepared antiserum to native Staph Nuclease or to a peptide fragment of aminoacids I-99
How did Anfinson go about his Flicker Hypothesis?: -purified antibodies to peptides through affinity chromatography
-Introduced antibodies into native conformation over the peptide column
-had same effect on native form as it did from the peptide

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