Nonenzymatic protein function and protein analysis Flashcards
Motif
Repetitive organization of secondary structural elements
Collagen
- Extracellular of connective tissue
- trihelical fiber
- strength and fiber
Elastin
-Extracellular of connective tissue
Stretch and recoil like a spring
Keratin
- Intermediate filaments in epithelial cells
- mechanical integrity of cells
- regulatory proteins
- hair and nails
Actin
- Subunit of microfilaments
- Have + and - ends which allow for unidirectional travel for motor proteins
Tubulin
- Subunit of microtubules
- Intracellular transport with kinesin and dynesin
- end is near nucleus and + end is near periphery of cell
- also involved in structure and chromosome seperation
Myosin
Interacts with actin in myofibrils, has one head and one neck
Kinesin and dyneins have how many heads?
2 of which one is atleast attached to tubulin
Kinesins
- move to + end
- align chromosomes in metaphase and depolymerizing microtubules during anaphase of mitosis
- In neurons, bring besicles to synaptic terminal
Dyneins
- move to - end
- in neurons bring waste or recycled nuerotransmitters to soma (retrograde transport)
Binding proteins
Bind to a specific substrate, either to sequester it in body or hold it at steady concentrations
Cadherins
- Glycoproteins that are calcium dependent
- hold similar tissues together
- specific tissues have specific cadherins
Integrins
- Bind and communicate with extracellular matrix
- have a role in cell signaling, cell division, apoptosis, and other processes
- have 2 membrane-spanning chains alpha and beta
Selectins
- Bind to carbohydrate molecules outside of other cells
- Found on white blood cells and endothelial cells of blood vessels
- Host defense, inflammation, white blood cell migration
Antibodies
Immunoglobins (Ig) made by B cells
Shape and structure of anitbodies
Y shaped with two heavy and two light chains held together by disulfide and noncovalent bonds
Antigen-binding site
At tips of “Y”, specific polypeptide sequence that binds to one antigenic sequence
Constant region
Recruits and binds immune cells eg macrophages
Antibodies neutralize antigen –> effect?
Antigens unable to exert effect on body
Opsonization
Antibodies mark antigen for destruction by white blood cells
Agglutinating
Antibodies clump antigen and antibody into insoluble complex to be phagocytosed and digested by macrophages
Facilitated diffusion
Passive transport where diffusion is down a gradient through a pore created by transmembrane protein
Are ungated channels regulated?
No
Voltage-gated channels
Gated by membrane potential near channel
Ligand gated channels
- Binding of specific substance/ligand can open or close channels
- Can use Michealis-Menten and Lineweaver Burke plots for this as well
G protein coupled receptors
Heterotrimeric G protein. A ligand binds to a receptor and that receptor binds to G protein to activate it
Gs
Stimulates adenylate cyclase to increase cAMP
Gi
Inhibits adenylate cyclase to decreases cAMP
Gq
Activates phospholipase C to increase intercellular Ca2+ concentration
Phospholipase C
It cleaves a phospholipid to form PIP2 which is then cleaved to DAG and IP3. IP3 opens up calcium channels in ER
IP3
Opens up calcium channels in ER
Inactive GPCR
Has α, β,γ subunits together and α is bound to GDP
What happens to GPCR when a ligand binds to a receptor that binds to GPCR?
GDP is replaced with GTP and α subunit dissociates. α subunit alters activity of adenylyl cyclase.
How is the GCPR signal cascade turned off?
GTP is hydrolyzed to GDP and the α subunit binds to beta and gamma subunits to make G protein inactive.
Electrophoresis
Subject molecules to electric field and move them accordingly to net charge and size
Polyacrylamide gel
It is a slightly pourous mixture. Larger and electrically neutral move slower than smaller and charged.
Native PAGE
- Analyze proteins in native charge
- can compare similar size molecules based on size or charge
- Stain
What happens to protein if you stain the native PAGE?
It denatures and cannot be recovered.
SDS-PAGE
- Seperates by molecular mass
- SDS (detergent) disrupts noncovalent interactions and surrounds proteins with negative charge
- Stain to visualize bands afterwards
Isoelectric focusing
A mixture of proteins is placed in a gel with pH graident and then electric field placed. Positively charged proteins move to cathode and negative charged proteins move to anode. They will stop at their pI and become neutral.
Dalton
g/mol
Chromatography
- Place sample on stationary phase/adsorbent
- Run mobile phase for sample to run through stationary phase or elute
- Components with high affinity for stationary move slower
Retention time
Time spent in stationary phase
Column chromatography
Adsorbent is polar silica or alumina beads. Size and polarity determine speed. Each fraction’s solvent can be evaporated to keep compound of interest.
Ion-exchange chromatography
Beads have charged substances and they bind to attractive charges. You elute with salt gradient.
Size-exclusion chromatography
Beads are porous which capture smaller molecules and slow them down. Larger molecules move faster.
Nickel has high affinity for ?
Histidine tags
Affinity chromatography
Beads are coated with a receptor. Eluent can be free receptor but it will be harder to seperate protein from eluent later on. It can also have varying pH pr salinity level to disrupt protein-bead interactions.
X-ray crystallography
Protein is isolated, crystallized, and measured for electron density. Patterns are interpreted for structure
Hydrolysis to study protein structure
Can tell you about amino acid composition but nothing about sequence
Edman degradation
Sequential digestion of proteins (50-70 AA). It selectively and sequentially removes the N-terminal AA, which can be analyzed via mass spectroscopy
Can you find location of disulfide bonds through electrophoresis?
No, because they are broken down.
How do you determine protein activity?
Looking at how it affects a known reaction, often accompanied by color change
How can you analyze aromatic amino acids?
UV spectroscopy
Bradford Protein Assay
It is initially green-brown and is protonated. It gives up protons to amino acids in proteins and turns blue. If there is more than one protein, this test is not accurate.
