Biochem Exam 2

• Intermediate filament
• Hair, nails, claws, skin
• Disulfide crosslinking defines strength

• The most abundant protein (up to 30% total content)
• Found in extracellular matrix of connective tissues/skin
• Ropelike fibers enforced by chemical crosslinking provide support and elasticity to animal tissues

up to 30%

• Tendons
• Ligaments and skin
• Cartilage, bones
• Blood vessels
• Intervertebral discs

Higher level of collagen crosslinking and its overall lower levels contribute to skin aging

• Polymeric gels, which can be reversibly disrupted at high temps
• Property is widely used in cooking (jello)

Every third residue is Gly which allows tight packing into this helix

• Intermolecular H-bonds between amide and carbonyl groups of Gly
• Bonds are between the strands!!
• Alignment of fibers generates characteristic striated pattern

• Gly-Pro-X and/or Gly-X-Hyp (hydroxyproline) which form left-handed coils- every third aa is Gly 
• The coils coil around each other to form a right-handed triple helix (type II; not alpha helices)

• They are resistant to compression and assure high tensile strength
• As tough as copper wire

• Hydroxyproline
• Hydroxylysines

• Uses Proly-4-hydroxylaze (P4H)
• Stabilizes the triple helix by H-bonds

• Oxidation to highly reactive aldehydes by Lysyl Oxidase (LOX)
• Stabalized by intra- and inter- molecular crosslinking
• Substrate for glycosylation (covalent addition of oligosaccharides); attachment sites for carbs

• Aldehydes spontaneously react with each other

• Ascorbic acid (vitamin C) is required by the enzymes to convert proline to hydroxyproline and lysine to hydroxylysine. 
• Without Vitamin C, collagen cannot be processed correctly and this occurs
• Can be reversed by dietary vitamin C

• Bone pain
• Skin and gum disease
• Loosening of teeth
• Poor wound healing
• Emotional changes
• Death

Ascorbic Acid (vitamin C)

Required for a second reaction which reduces  Fe3+ back to Fe2+ reactivating the enzyme which converts pro to hyp

• Essentially made of Gly-Ala repeats in ~extended beta sheet structure
• Doesn't stretch easily, since structure is fully extended

• Doesn't stretch but is very flexible: non-covalent weak interactions between layers
• H bonds between strands
• Van der Waals interactions between sheets
• Anti-parallel strands

• Intermediate filaments (keratins, lamins)
• Microtubules (tubulin)
• Microfilaments (actin)

• Cell shape: determined by cytoskeletal proteins and signaling cascades
• Cell motility: cellular and intracellular
• Cell integrity: connection and integration of extracellular matrix (ECM), cytoplasmic membrane, and organelles

• Assembles into double helical homopolymers 
• 5-9 nm in diamater
• Forms microfilaments
• Polar (+/- ends); all units are formed in the same direction and has directionality

• Stable dimers (2 subunits) of alpha and beta tubulin self-assemble into hollow hetero-polymers of tubular shape 
• 25 nm
• Polar with directionality

• A large family of proteins (65 genes in humans)
• Keratins (at least 19 types)
• Lamins A and B (nuclear); neurofilaments (axons)
• 10-14 nm in diameter
• Apolar (opposite attachment so no directionality); not used in tracks because there is no directionality

• Coliled-coils
• Hydrophobic a,d (1,4) positions are buried; twist of helices due to 3.6 residues per turn
• Heptad is ~two full turns

• Coiled-coils align to form oligomers
• Terminal globular domains help align fibers
• Fibers can be enforced by disulfide bonds in extracellular spaces (hair/nails)
• Disassembly is mediated by phosphorylation (regulates dynamics and assemblies- posttranslational modification)

• Highly resistant to cold, high-salt, and detergent solutions
• Can be dissolved by guanidinium- HCl or Urea

E to G mutation in Keratin 5

• Lamin's mutation prevents the protein to maintain its shape

Actin Polymerization

Hexokinase (metabolic enzyme) and actin belong in the same family of proteins

• Catalyzes the first step of glycolysis
• Binds both ATP and glucose
• Catalyzes transfer of the terminal Pi from ATP to glucose

• Hydrolyzes ATP upon polymerization
• ATP hydrolysis is translated to conformational changes that work as internal clock of actin filament aging

ATP hydrolysis is not required for polymerization, only for sensing age of F-actin to temporally control its depolymerization

• Nucleation: formation of actin trimer; dimers are unstable; rate limiting step
• Elongation: "+" end polymerizes faster than the "-" end; when (G-Actin) is high, both ends are growing
• Steady state: "-" end disassemble, while the "+" end polymerizes= treadmilling

• ATP-bound (freshly polymerized)
• ADP-Pi bound (central, older actin)
• ADP-bound (aged actin, the least stable)

• Unstable ADP region (at the "-" end) predominantly depolymerizes
• ADP-monomers exchange nucleotide from ADP to ATP
• Recycled ATP-monomers are added to the "+" end of the filament
• ATP is needed

• Cell migration: directed actin polymerization pushes the membrane and causes the cell to migrate
• Pathogens can hijack actin: intracellular pathogenic bacterium Listeria (and others) initiates the same general mechanism to move inside human cells; the put actin inside of them and use them to push the pathogen along

• Hydrolysis by myosin catalytic (head) domains causes large conformational changes in the molecule
• Since myosin grabs actin in one conformation and releases in another, two molecules move with respect to each other Myosins convert chemical energy into mechanical movement

• Reaction begins with myosin head bound to an actin subunit of the thin filament. ATP binding alters the configuration of the myosin head so that it releases actin
• Rapid hydrolysis of ATP to ADP+Pi which rotates the myosin lever
• Binding to actin causes Pi and then ADP to be released
• ATP then replaces the lost ADP

• Alpha and beta tubulin dimers assemble in microtubules- 25nm in diameter
• Cell shape
• Cellular motility (cilia)
• Intracellular motility
• Chromosome segregation in cell division

• GTP hydrolysis at the beta subunit destabilizes the filament
• Can lead to catastrophe

• When microtubule enters the stage of fast disassembly
• If GTP is hydrolyzed before the new tubulin dimer is added (if GTP-dimer is low), the microtubule + end will contain GDP and become unstable

• Essential to chromosome segregation
• Daughter chromosomes bind to the +ends of MT and get separated between the divided cells powered by controlled depolymerization of MTs

Cell division since cancer cells are among the most actively dividing cells in the organism

• Binds to beta-tubulin subunit and stabilizes microtubules
• Blocks cell division 
• Used for cancer chemotherapy

• Any molecule that binds to a protein
• Typically reversible, transient and specific
• When the protein is an enzyme, often this is a substrate

• A protein with an "empty" binding site
• Without ligand

Ligand-bound form of protein

• Molecules that assist enzyme in its activity
• Inorganic (metals)
• Co-enzymes (complex organic molecules, vitamin derivatives)

• Co-substrates: (soluble cofactors, nor firmly associated); reversible
• Prosthetic groups: tightly or even permanently associated with their enzymes

• H bonds
• Hydrophobic effect
• Electrostatic interactions
• Dipole-dipole interactions
• Rarely: covalent interactions

• Lock and key hypothesis: binding site of protein is a perfect match for the substrate; barely the case
• Induced fit hypothesis: binding site of protein is similar to the substrate but when bound, changes occur in the structure of both species; more common case

• Specificity of a protein for its ligand arises from complementary shapes
• Geometric complementarity: size and shape of cleft and ligand
• Electronic complementarity: recognizes hydrophobicity, charge, hydrogen bonding

When 50% of binding sites are occupied (half saturation)

If [L]>Kd, more is bound; [L]<Kd, less is bound

Ligand concentration

• Stronger interactions

• Muscle oxygen carrier
• Oxygen allows oxidation of organic molecules to CO2 and releasing ~16 times more energy

a prosthetic group that is a porphyrin derivative that chelates iron

• Coordinating
• Sensing oxygen binding

• Heterotetramer:
• 2 alpha subunits
• 2 beta subunits
• 4 different O2 binding sites

No O2

Bound O2

• It ineeds to exchange oxygen
• Myoglobin is a binder bc it needs to steal oxygen from hemoglobin (neither are inefficient)

• Regulation of a protein by ligand binding at a distant site other than the protein's active site
• Long-range allostery is especially important in cell signaling and metabolism
• Allosteric effects mediated by long range conformational changes in proteins

Ligands that enhance the protein's activity

Ligands that decrease the protein's activity

• R: High affinity state; binds oxygen readily with flat heme and shifted helix
• T: Low affinity state; binds oxygen poorly

• Iron is off of the heme plane in the oxygen-free state due to coordination to a His residues in underlying helix
• Binding of oxygen and its bonding to His64 repositions iron "in plane" with the heme ring
• As a result, an adjacent helix is also repositioned

• Competes and interferes with O2 release
• It binds to Hb~300x more efficiently than O2 -> fewer sites available for O2 binding
• Additionally, binding of CO induces transition to the high affinity R state, inhibiting O2 release to tissues
• CO binding is nearly irreversible and strongly delays release of O2

Reduces it from 7 hours to ~10-20 minutes

• Normal CO-Hb is ~1% 
• Heavy smokers might have up to 15% CO-Hb
• 25% CO-Hb causes severe poisoning
• 50% CO-Hb in blood causes death

• pH effects on respiration
• Low pH: protonation of hemoglobin (at several His sites) decreases affinity for oxygen stimulating release of O2

Deprotonates hemoglobin and allows for the reformation of CO2 from bicarbonate in the blood and its release to atmosphere

• Low pH= high [H+]= high [HbH+]
• This then stabilizes the T-state and releases O2 into tissues

• CO2 + Hb
• Carries CO2 to lungs
• Facilitates O2 release

• CO2 covalently binds to amino terminus of hemoglobin subunits
• Resultant carbamates form salt bridges that stabilize the low-affinity T state of hemoglobin
• Transition to T state leads to release of O2 through allosteric regulation
• Released protons further the Bohr effect

• Tissues: high pCO2, low pO2
• Favors T-state
• Release of O2

• Low pCO2, high pO2
• R-state is favored
• O2 binding, CO2 release

2,3-Bisphosphoglycerate (BPG)

• Produced by red blood cells
• Binds to a basic region (His) at the interface of all 4 subunits that is only present in the low affinity T state
• Therefore, it stabilizes the T state and promotes the release of O2

• Doesn't bind BPG 
• Steals O2 from mother

• At a low pH, the capacity of Hb for oxygen is reduced as well as the affinity to oxygen. Bound protons break cooperatively of O2 binding to Hb
• This allows fish to fill the swim bladder with O2 against high gradient of concentrations
• O2 doesn't bind to Hb at low pH even at high pO2

• Disease of protein structure
• Single mutation: surface Glu is mutated to Val in beta subunit
• The mutated hemoglobin is insoluble when deoxygenated (T state)

• Hydrophobic surface patch leads to protein  polymerization (insolubility)
• it is sufficient to induce aggregation (polymerization)
• Aggregation involves deoxyHb and develops under low oxygen pressure

• Homozygote: carriers of two copies will die
• Heterozygotes: resist malaria (advantageous mutation)

• Heterozygotes develop anemia as sickle cells are fragile and don't transit well through capillaries
• Sickle cells are lysed readily

• Not consistent- half lives of proteins vary from minutes to "infinity"
• Most proteins: 100-200 hours

• Regulatory proteins
• Enzymes that catalyze committed steps
• Transcription factors

Special cases (dentin, crytallins)

• On average, protein's half-life correlates with its N-terminal residue 
• Depends on post translation modification

Proteins with Met, Ser, Ala, Thr, Val, or Gly

Proteins with Phe, Leu, Asp, Lys, or Arg (charged or large hydrophobic)

• Pulse-chase analysis with a radioactive probe
• Protein synthesis inhibition with cycloheximide

Better method to determine protein half life bc you can distinguish the difference from old and new proteins

• Incubate with radioactive aa
• Remove the isotope
• Purify the protein of interest by antibody at designated time points
• Analyze by SDS-gel/western blot
• Check radioactivity and total protein (western blot)

• A way to measure protein half life
• Cycloheximide:a drug that blocks the elongation step of protein translation
• Analyze by Western Blot after incubation

• 80-90%
• Most intracellular proteins

• 10-20%
• Extracellular proteins
• Cell organelles
• Some intracellular proteins
• Protein aggregates

• Phagocytosis
• Autophagy
• Receptor-mediated endocytosis

• Digests: ingested materials, obsolete cell components
• Degrades: macromolecules of all types i.e proteins, nucleic acids, carbohydrates, lipids

• Acidic pH of lysosomes maintained by a proton pump in the lysosomal membrane (requires ATP)
• Acid hydrolases: active at pH <5 (inside the lysosome), inactive if released into cytosol (pH 7.2)

• Cell "eating" of material
• Bigger sizes

• Cell "drinking"
• Smaller sizes

Particles are tagged for destruction

• "self eating" of old worn out organelles
• Important in cell degradation during apoptosis
• Important under starvation and to recycle entire organelles/aggregates

• Activated under nutrient deficit; stimulated autophagy with nutrient and energy supply
•  
• Inhibited under conditions of food abundance

• Prevents accumulation of aggregation-prone proteins
• Eliminates misfolded proteins: reduction of ER stress
• Removes damaged organelles" limits production of ROS

Ubiquitin

• Small protein destined for degradation
• Reaction is catalyzed by ubiquitin ligases
• Ubiquitination is N-end rule dependent

C-terminal glycine (carboxyl group donor) of ubiquitin forms this with the epsilon-amino group of lysine residues on the substrate

• Other ubiquitin molecules can be attached to ubiquitin lysines leading to this
• Proteins destined to be degraded are this
• Polyubiquitinated proteins are recognized and degraded by 26S proteasome

• Cylindrical complex consisting of four stacked, seven membered rings
• Two outer rings are alpha subunits (inactive)
• Two inner rings are beta subunits; these are proteolytically active

• Cleaves only poly-ubiquitinated proteins 
• This degradation requires hydrolysis of ATP
• Energy of ATP hydrolysis is used to unfold poly-ubiquitinated targeted proteins

• Recognizes unfolded sequences
• "consumes" and digest them
• Does not require ATP

• Recognizes poly-U
• Remoces ubiquitin
• Unfolds (requires ATP)
• Translocates to 20S

Heat shock proteins (i.e HSP70)

• Stress causes high level of misfolded proteins and HSP70 is produced
• HSP70 binds to 19S and dissociates it from 20S
• Unfolded/disordered proteins are recognized by 20S an degraded
• ATP is not required

May be different in solution bc of the pKa of the individual amino acids