Exam 2 Flashcards
Insulin: synthesis
In β-cells of the pancreas, proinsulin is cleaved into insulin and connecting peptide (C-peptide). Both are
stored in vesicles and secreted into the blood circulation
Insulin: structure and storage
Insulin: primary structure (51 a.a)
2nd: alpha and beta helices
quaternary: zinc-insulin hexamer (6 insulin molecules stabilized by zinc ions)
In β-cells, insulin is first stored in secretory vesicles
What determines a proteins function
its structure
Formation of a peptide bond
Peptide bond formation: condensation reaction (removal of water) costs energy (GTP, ribosome)
C (w/ =O) binds to N of other a.a
> carboxylic group binds to amine group
Polypeptide chain
Peptide chain is amino acids linked through peptide bonds, also called amide bonds
> The properties of a protein is determined by the side chains
N terminus is amine end
C terminus is carboxylic end
alpha-helix
Backbone: Formed by polypeptide chain
R-groups (side chains): These stick outwards from the helix, away from the helical axis
Intra-chain Hydrogen Bonds: They form within the same chain between the backbone atoms.
Specifically:
The carbonyl oxygen (C=O) of an amino acid forms a hydrogen bond with the amide hydrogen (N-H) of an amino acid.
These bonds run parallel to the helix axis, holding the coil tightly together
a-helix can anchor proteins in a membrane: pore proteins (transport) and hormone receptors (signal transduction)
helix breakers: Chemical (oxidative) changes in amino acid side chains that disturb structure (loss in structure = loss in function)
Collagen
Rigid structure: 3 intertwined a-helices (triple helix)
Primary structure, more than 1000 amino acids is composed of the repeat: Gly-X-Y, with Y often proline or 4-hydroxyproline
Multiple tropocollagen units pack together to form fibrils
In collagen, side chains of prolines and lysines are often hydroxylated
* 4-Hydroxy-proline forms hydrogen bonds to stabilize the triple helix
* 5-Hydroxy-lysine is attachment site for disaccharides, galactose−glucose
Collagen suprastructure
In the ER:
1) Synthesis of Pre-procollagen
begins on ribosomes
pre-procollagen includes:
> A signal peptide (helps it enter the ER)
> The actual collagen sequence (with Gly-X-Y repeats)
The signal peptide is cleaved once inside the ER → now it’s procollagen.
2) hydroxylation of proline and lysine residues (via Prolyl hydroxylase and lysyl hydroxylase, with vitamin C as a cofactor) (pro & lys react with alpha-ketoglutarate which turns into succinate once reaction takes place)
3) glycosylation (w/ Galactose or glucose–galactose disaccharides) of selected 5-hydroxylysine residues
In the Golgi Apparatus:
Triple-Helix Formation
> Procollagen is still flanked by propeptides on both N- and C-termini to prevent premature fibril formation.
Secretion into the Extracellular Space
After secretion, procollagen peptidases cleave N- and C-terminal propeptides
This creates tropocollagen, the mature form capable of self-assembly (fibrils into fibers)
Synovial joints
Surrounded by ligament (capsule), a fibrous connective tissue with collagen fibers arranged in orderly parallel manner
End of bones at the joint have cartilage, consists of collagen and proteoglycan
Synovial cavity contains synovial fluid, with hyaluronan (also called hyaluronate), to increase the viscosity and elasticity
Ehlers–Danlos syndrome
Hyperflexible joints
Mutations in the following can cause EDS:
* Fibrous proteins: COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, and TNXB
* Enzymes: ADAMTS2, PLOD1
PLOD1 = Lysyl hydroxylase (or procollagen-lysine 5-dioxygenase)
Helix-breakers
> Some natural occurring amino acids do not fit in an a-helix, they disturb the structure
> A mutation that introduces a helix breaker can have dramatic effects on protein function
* Skin (COL5A1 and COL5A2)
* Blood vessels (COL3A1)
Layers of the arteries and veins
Walls of both are made up of three layers:
tunica intima
tunica media
tunica adventitia
Elastic arteries
Have vasa vasorum (networks of small blood vessels that supply the walls of arteries with oxygen and nutrients)
Right under endothelium:
Internal elastic lamina (layer of elastic tissue)
Tunica media (smooth muscle cells
and elastic tissue)
Tunica adventitia (mainly composed of collagen)
Collagen versus elastin
Both are structural proteins
Blood vessel walls contain an extracellular matrix with a specific mix of proteins for the right amount of rigidity (collagen) and elasticity (elastins)
Disulfide bonds
Extracellular proteins have disulfide bonds to covalently fix the three-dimensional structure made by hydrogen bonds
(SH groups interacting covalently)
Cysteine thiol is a redox group
Oxidation: 2 -SH + O2 <> -S−S- (disulfide bond) + H2O2
> i.e Disulfide-bond formation is an oxidation reaction, reduced to SH thiol groups
Vitamin in collagen hydroxylases
Vitamin C (ascorbate) is a reducing cofactor in collagen synthesis
> H of proline or lysine side chain turns into OH
Vitamin C dietary deficiency
Results in scurvy
> vitamin C (ascorbic acid) is required for the synthesis of collagen in humans.
> Scurvy leads to the formation of spots on the skin, spongy bleeding gums, and bleeding from the mucous membranes. > The spots are most abundant on the thighs and legs, and a person with the ailment looks pale, feels depressed, and is partially immobilized.
> advanced scurvy: open, suppurating wounds and loss of teeth.
Scurvy does not occur in most animals as most animals can synthesize their own vitamin C
Vitamin C: antioxidant
Vitamin C (ascorbate) is the reduced form, which can donate electrons (e − )
to reduce radicals, such as some ROS
The oxidized form dehydroascorbic acid can be reduced back to ascorbate
by glutathione transferases present in most cells.
beta-Strands and beta-sheets
Multiple b-stands can form a b-sheet
A β-strand is a single, extended stretch of polypeptide chain
The backbone is zigzagged
> Side chains alternate above and below the strand, creating a pleated sheet appearance when multiple strands are aligned.
beta-sheet
> Formed when two or more β-strands run alongside each other and are linked via hydrogen bonds between the carbonyl oxygen (C=O) of one strand and the amide hydrogen (N-H) of another.
Sheets can be:
Parallel (strands run in the same N → C direction)
Antiparallel (strands run in opposite directions)
Protein folding and surface
A polypeptide chain folds into its thermodynamically most stable form.
> polar side chains point outward, can form H-bonds with water
> Hydrophobic core region: apolar side chains pointing inward, H-bonds between different peptide bonds
Charged amino acids at the protein surface, hydrophobic amino acids in the interior
Lysozyme: structural domains
a single polypeptide chain that has 2 structural domains:
alpha-helical domain and beta-sheet domain
Prion diseases
a group of rare, fatal neurodegenerative disorders caused by the misfolding of a normal protein into a pathogenic form
The normal form of the prion protein: PrPc
The disease-causing form: PrPsc
> change in structure, from a-helices to b-sheet
> has a higher β-sheet content
> results in protein precipitates in brain
Domain structure of transcription factor GAL4
has a DNA-binding regions on either end and a regulatory domain in the middle (a regulation RNA-polymerase)
> A protein domain is a structural and functional unit within a single
continuous amino acid chain (usually encoded by single exon)
Quaternary structure of proteins
> Many functional proteins consist of more than 1 peptide chain
The subunits can be identical or different (different genes)
They often contain co-enzymes or help-molecules (metal ions)
Hemoglobin archetypal example
- has 4 subunits interacting w/ eachother, 2alpha and 2beta
- each subunit contains a heme group w/ an iron molecule that binds oxygen
Hemoglobin quaternary structure
quaternary structure enables cooperativity
When one subunit binds O₂, the entire tetramer changes shape, making it easier for the next subunit to bind O₂.
> This cooperative effect gives hemoglobin its sigmoidal (S-shaped) oxygen-binding curve—perfect for loading oxygen in the lungs and unloading it in tissues.
whereas myoglobin only has a single chain = no cooperative binding
A human disorder of hyaluronan metabolism (& example in dogs)
Serum hyaluronan levels are elevated at birth and then decrease to the normal range
> This form of cutaneous mucinosis is caused by skin fibroblasts producing abnormally large amounts of acid mucopolysaccharides, usually hyaluronan.
> folding and thickening of the skin
The thickened and wrinkled skin in Shar-pei dogs is caused by the excessive production of hyaluronan by the hyaluronan synthase 2 (HAS2) gene. > Hyaluronan lubricates the animals’ joints.
> Shar-pei dogs are born with wrinkles and lose them as they get older because they literally grow into their skin
Proteoglycans
long, highly charged, and repetitive polysaccharides (such as hyaluronan, chondroitin sulfate, and keratan sulfate) attached to core proteins, forming highly hydrated compressible gels
Hyaluronan (Hyaluronate)
Disaccharide unit = Glucuronic acid + N-acetylglucosamine
Linked by β glycosidic bonds
Chondroitin 6-sulfate
Disaccharide unit = Glucuronic acid + N-acetylgalactosamine-6-sulfate
Proteoglycans contain much larger, repetitive sugar polymers than glycoproteins
Hyaluronic acid (hyaluronan)
proteoglycan aggregate
Hyaluronic acid serves as a scaffold: long, unbranched polysaccharide made up of repeating disaccharide units (Glucuronic acid & N-acetylglucosamine)
Core proteins attached to hyaluronan via link proteins.
Chondroitin sulfate & Keratan sulfate are covalently attached to the core protein
major component of the extracellular matrix
found in connective tissue, skin, eyes, and joints
cosmetic application:
Hylauronic acid fillers
Vitamin E
It is an anti-oxidant: Protects amino acids from ROS-damage
Affects Maillard (browning) reaction on collagen
> Maillard reaction: causes browning and makes collagen stiffer and more prone to degradation = aging
> Vitamin E neutralizes free radicals that accelerate Maillard reactions, and reduces oxidative steps that lead to the browning
How does selenium neutralize free radicals and other skin-damaging compounds before they can lead to wrinkles?
Glutathione peroxidase reduces hydrogen peroxidase (H2O2) to water
> Glutathione peroxidase transfers e- from glutathione (GSH) to hydrogen peroxide
Selenium is present in the active site of Glutathione Peroxidase as the amino acid selenocysteine
States of selenocysteine
1) Selenolate (E-Se-, active)
Attacks hydrogen peroxide (H₂O₂)
Selenolate is oxidized to selenenic acid
2) Selenenic acid (E–SeOH)
Further oxidized, reacts with GSH
3) Selenosulfide (E–Se–S–G)
reduced back to selenolate form by glutathione (GSH)
- GSH > GSSG, selenosulfide gives the S & G
Protein structure summary
Primairy structure: sequence of linked amino acids
Secondary structure: a-helix, b-sheets and loops
Tertiary structure: total three-dimensional shape
> same sequence gives same shape = conformation
Quaternary structure: When a functional protein contains several polypeptide chains (subunits, such as hemoglobin)
Large proteins often consist of protein domains (each built from a-helices, b-sheets), linked through unstructured loops that execute distinct parts of function, often encoded by single exons in genes.
A single amino acid substitution can lead to serious disease when it disturbs the structure of the protein (such as a helix breaker in collagen, sickle cell anemia Glu>Val).
Connective tissue
main function is to support, bind, and protect other tissues and organs
Integrins (transmembrane receptor proteins) bind extracellular proteins (fibronectin and collagen)
Collagen provides the tensile strength
Fibronectin: a large glycoprotein that
has binding domains for integrins, collagen etc
> Adhesive molecule: connects cells (via integrins) to the collagen
Proteoglycans: Provides gel structure, resists compression
what can UDP-glucose synthesize?
glycogen, proteoglycans, glycoproteins, glycolipids
UDP-glucuronate, UDP-galactose
UDP-glucose is activated glucose
Glycoproteins
short carbohydrate chains covalently linked to serine or asparagine residues in the protein
> most secreted from cells
e.g of a branched glycoprotein: fucose
contain unique sugar structures
> O-glycosylation (serine residues) and N-glycosylation (asparagine residues) of proteins
Synthesis reaction:
UDP-glucose > glycosylated protein
Enzyme: glycosyltransferase
Cofactor: Protein-OH > UDP
Act as sugar flags outside the cell
> Often recognized as “self” by immune system
> different ‘flags’ in diff blood types
Conveyer-belt synthesis of N-glycans
1) Initiation on cytosolic side:
Sugars like (e.g N-acetylglucosamine) are added one-by-one to dolichol-Phosphate
2) partially assembled glycan flips across the ER membrane (to the lumen side)
> More mannose and glucose residues are added
3) N-glycosylation of Secreted Proteins
> glycan is transferred from dolichol to the nascent protein
> happens whilst the protein in being made (translated)
4) Complete N-glycans are Attached Post-translationally
> Although the core oligosaccharide is transferred co-translationally, it undergoes processing and maturation afterward:
> In the ER: Glucose and some mannose residues are trimmed.
> In the Golgi: Further modification
Processing of N-linked glycan
Processing of N-glycans occurs in the endoplasmic reticulum and Golgi apparatus
Endo H-sensitive N-glycans are high-mannose glycans or hybrid glycans with at least 5 mannose residues, two of which must be terminal mannose residues
What are mucins?
transmembrane glycoproteins
> transmembrane mucins are glycoproteins expressed in the GI tract, forming the extracellular glycocalyx of the enterocytes and the inner mucus layer in the colon
> have a beta chain and an alpha chain
healthy colon mucus layer:
inner mucus layer is relatively sterile
inner mucus layer limits invasion of enteropathogens, such as Salmonella enterica and Campylobacter jejuni
