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45 Cards in this Set
- Front
- Back
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Properties of collagen (4)
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1) insoluble fibrous protein
2) important in bones, cartilage, dentin, vasculature, skin, cornea, tendons, etc. 3) composed of alpha chains (NOT helix) that form trimers called tropocollagen 4) most abundant protein |
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type of helix of a-chain
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L-handed helix
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tropocollagen definition
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3 a-chains wrapped around each other forming a r-handed triple helix
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why is collagen such a good structural protein?
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strong and insoluble in water (which is what the body is made up of)
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properties of collagen alpha-chain (4)
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1) composed of mainly Gly-X-Y where X = proline and Y = hydroxyproline or hydroxylysine
2) helical structure is stabilized by H-bonding BETWEEN chains (unlike a-helix)- if you unwind the tropocollagen, each a-chain will lose it's L-helix structure 3) ~1000 amino acids 4) lots of prolines |
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how do a-chains hydrogen bond?
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between a-chains, carbonyl groups on prolyl rsidues project perpendicular to axis of helix and bind with OH groups on hydroxyproline/lysines.
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a-chain vs. a-helix
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a-helix stabilized by H-bonds between AAs located 4 residues apart from each other.
a-chain is stabilized by carbonyl groups H-bonding with OH groups on separate chains. |
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structure of tropocollagen and it's aggregates (3)
-types of a-chains -possible structures |
1) may contain 3 identical or non-identical a-chains
2) some types of tropocollagen spontaneously aggregate to form microfibrils (superstructure) 3) tropocollagen molecules that constitute microfibrils eventually become covalently cross-linked to each other (meshwork) |
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proper alignment of tropocollagen in microfibril formation requires...
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glycosylation of certain hydroxylysyl residues in order to stack the right way (staggered)
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overlap zone
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where 2 tropocollagen overlap
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hole zone
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space between 2 tropocollagens in a line
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Tropocollagen crosslinking
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lysyl group (or hydroxylysyl) undergoes deamination by peptidyl lysyl oxidase, Cu++ and B6 coenzymes--> Becomes (hydroxy)lysyl semialdehyde-->crosslinks reversibly with (hydroxy)lysyl residue on another tropocollagen-->releases H2O-->schiff base-->irreversibly reduced by 2H to have a stable secondary amine (NH bonded to 2 carbons on 2 separate tropocollagen units within a microfibril)
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Schiff base
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intermediate of tropocollagen crosslinking
A Schiff base (or azomethine), named after Hugo Schiff, is a functional group that contains a carbon-nitrogen double bond with the nitrogen atom connected to an aryl or alkyl group—but not hydrogen |
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Would additional OH to lysyl residue increase or decrease formation of schiff base? why?
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increase, because OH group increases partial positive charge on the carbon
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deficiency of peptidyl lysyl oxidase (PLO)
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lathyrism- hyperflexible joints, soft stretchy skin, predisposition to aneurysms
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Other causes of lathyrism
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PLO deficiency
copper deficiency (cofactor for PLO) excessive consumption of sweet pease which contains aminopropionylnitrile- potent inhibitor of PLO |
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aminopropionylnitrile
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found in sweet peas
inhibitor of PLO |
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why doesn't b6 deficiency cause lathyrism?
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it does, but the neurological symptoms (seizure, depression) are more pressing so it often goes unnoticed
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Vitamin C deficiency causes defective collagen synthesis because...
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1) needed for hydroxylation of prolyl residues
2) hydroxylation of lysyl residues |
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importance of prolyl hydroxylation in collagen
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OH group of hydroxyprolyl residues needed for interchain H-bonding betwen a-chains within tropocollagen trimer
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importance of lysyl hydroxylation in collagen (2)
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1) oh groups on hydroxylysyl residues serve as glycosylation sites needed for proper alignment of tropocollagen units within microfibrils
2) OH groups on hydroxylysyl semialdehydes promote formation of schiff base for crosslinking |
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hydroxylysyl semialdehyde
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product of PLO/Cu+= interaction with hydroxylysyl
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collagen microfibril
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aggregates of glycosylated Tropocollagen
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Types of electrophoresis separation of proteins (3)
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1) net charge of native protein
2) isoelectric point of protein 3) molecular weight of proteins |
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Process of electrophoresis based on net charge
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1) run gel at slightly basic pH (~8.5)
2) protein band will travel to + end, distance will be based on how negative the R groups are 3) heterozygous will show up as 2 bands |
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net charge of proteins are generally...
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negative
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Isoelectric focusing process
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1) apply proteins in middle
2) gel has pH gradient in substrate surface 3) protein migrates to corresponding direction (+ or -) based on R groups 4) at low or high pH, protein will stop migrating (or focus) based on it's pI (becomes deprotonated or protonated and loses it's charge) |
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electrophoresis by size process
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1) pretreat proteins with SDS
2) load protein onto gel on (+) side 3) proteins will move through 3D polyacrylamide gel matrix at a rate based on size |
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SDS and it's purpose
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sodium dodecasulfate
binds every other AA in a protein, linearizing it and applying it's negative charge (eliminates net charge and shape differences) |
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Emphysema
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destruction of the alveolar walls in lungs by action of enzyme elastase on elastin (structural protein)
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elastin
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structural protein in walls of alveoli
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purpose of elastic alveoli
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assists exhalation of inspired air
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elastin stain
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dark staining
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elastase (what does it do, where is it normally found, and what class of protein is it)
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serine protease that cleaves by hydrolysis, peptide bonds.
produced in body- leaks out of dead neutrophils into blood |
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alpha1-antitrypsin (AT)- what it does and where does it come from
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a1-trypsin inhibitor that also inhibits elastase and many other serine proteases (serpin)
secreted by liver into blood |
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Serpin
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serine protease inhibitor
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serine protease (indicative AA residues)
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key amino acid residue: Ser-173
His-41 and Asp-88 also important |
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Key amino acid residue of serpins (specifically AT)
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Met-358 (Ser-359 also important)
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2 common causes of emphysema (and why)
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1) smoking:
-contains ROS which oxidizes key Met residue on AT (deactivates it) -particulate matter from smoke is foreign and stimulates recruitment of neutrophils 2) mutation AT gene (defective, absence) --> deficiency in AT |
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where is the protein mutation located in AT gene mutation?
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position 342 on AT protein
Glu (-) changes to Lys (+) which results in mutant AT having higher pI (because to neutralize the new + charge, you need to raise the pH) |
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cause of multiple bands on a protein subject to isoelectric focusing
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post transitional modification results in subpopulations with different pI
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instances of post translational modification that affect isoelectric focusing (4) Indicate which are more common.
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1) partial proteolysis ("trimming"). Very common.
2) Carboxylation (rare) 3) phosphorylation (rare) 4) glycosylation- some sugar groups have (-) charges; also common (more negative charges = lower pI than normal due to needing to lower the pH to provide H+ for neutralization) |
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Why would an emphysema patient have fewer isoforms of AT and different pI than a normal person?
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mutation can change R group charges, and prevent some of the normal post-translational modifications (in case of AT mutation, lack of glycosylation)
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what is a marker enzyme and how is it used
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enzyme that is abundant in a certain organ (and less abundant in most other organs)
you measure serum levels of the marker enzyme to assess damage (damaged cells will leak marker enzyme into blood)- if a panel comes back with one particular organ's marker enzymes consistently elevated, then that organ is probably damaged |
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connection between mutant AT and liver damage
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AT mutation (produced in liver) --> lack of glycosylation--> no "destination tag" -->accumulates in liver-->damage
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