Chapter 3- Nonenzymatic Protein Function and Protein Analysis Flashcards
motif
repetitive organization of secondary structural elements together
primary structural proteins in body
collagen, elastin, keratin, actin, and tubulin
collagen
trihelical fiber (3 a-helices woven together to form a secondary helix). provides strength and flexibility—ex: makes up most of extracellular matrix of connective tissue
elastin
extracellular matrix of connective tissue. stretch and recoil like a spring.
kertains
intermediate filament proteins found in epithelial cells. mechanical integrity of cell and also function as regulatory protein.—– primary protein in hair and nails
actin
protein that makes up microfilaments and thin filaments in myofibrils. MOST ABUNDANT PROTEIN IN EUKARYOTIC CELLS.
Tubulin
protein that makes up microtubules. provide structure, chromosome separation in mitosis, and intracellular transport with kinesin and dynein. has polarity.
kinesins and dyneins
motor proteins associated with microtubules. have 2 heads, one is always attached to tubulin at all times. important for vesicle transport in the cell.
kinesins
play key roles in aligning chromosomes during metaphase and depolymerizing microtubules during anaphase of mitosis. bring vesicles toward positive end of microtubule (ex: toward synaptic terminal in neurons)
dyneins
involved in sliding movement of cilia and flagella. bring vesicles toward negative end of microtubules (ex: back to soma in neurons).
examples of binding proteins
hemoglobin, Ca2+ binding proteins, DNA-binding proteins.
Cell adhesion molecules (CAMs)
proteins found on the surface of most cells that aid in the binding of the cell to the extracellular matrix. all integral membrane proteins.
3 types of adhesion molecules
- cadherins
- integrins
- selectins
cadherins
group of glycoproteins that mediate Ca2+ dependent cell adhesion. hold similar types of cells together (ex: epithelial cells)
integrins
group of proteins that all have two membrane-spanning chains called a and B. important for binding and communication w/ extracellular matrix. cellular signaling (promoting division or apoptosis). also stabilize clots
selectins
bind to carbohydrate molecules that project from other cell surfaces. weakest bonds formed by the CAMs . expressed on white blood cells and endothelial cells that line blood vessels.
3 ourcomes when antibodies bind to their targets (antigens)
- neutralize antigen
- opsonization (marking pathogen for destruction by other white blood cells immediately
- agglutinating (clumping together) the antigen and antibody into large insoluble protein complexes that can be phagocytized and digested by macrophages.
3 types of ion channels
- ungated (unregulated)
- voltage-gated (regulated by membrane potential change)
- ligand-gated (binding specific substance or ligand to channel for it to open or close)
enzyme-linked receptors
3 primary protein domains.
- membrane-spanning domain (anchors the receptor in the cell membrane)
- ligand-binding domain (stimulated by the appropriate ligand and induces a conformational change that activates the catalytic domain)
- catalytic domain (once activated, typically initiates a second messenger cascade)
G-protein coupled receptors
integral membrane proteins involved in signal transduction
heterotrimeric G protein
used by G-protein coupled receptors to transmit signals to an effector in the cell. 3 subunits (a, B, gamma)
3 main types of G proteins
- Gs stimulates adenylate cyclase- increases levels of cAMP
- Gi inhibits adenylate cyclase- decreases levels of cAMP
- Gq activates phospholipase C- cleaves phospholipid from the membrane to form PIP2. PIP2 is then cleaved into DAG and IP3. IP3 can open Ca2+ channels in ER increasing Ca2+ levels in the cell.
inactive form of G protein
a subunit binds to GDP in complex with B and gamma
active form of G protein
ligand binds to GPCR, receptor becomes active, GDP is replaced with GTP, a-subunit is able to dissociate from B and gamma subunits, a-sub alters activity of adenylate cyclase. then GTP becomes dephosphorylated to GDP then B and gamma-sub rebind to a-sub rendering G protein inactive.
how are proteins/ biomolecules isolated from body tissues or cell cultures
lysis and homogenization (crushing, grinding, blending) tissue of interest into an evenly mixed solution, then centrifugation will isolate proteins
most common isolation techniques
electrophores and chromatography
electrophoresis
subjecting compounds to an electric field, which moves according to their net charge and size.
PAGE
method for analyzing proteins in their native states. limited to mass-to-charge and mass-to-size ratios. most useful to compare the molecular size or charge of proteins known to be similar in size from other analytic methods.
SDS-PAGE
separates proteins based on mass alone. denatures the protein.
Isoelectric point (pI)
pH at which protein or amino acid is electrically neutral, with an equal number of positive and negative charges.
Isoelectric focusing
separates amino acids on basis of pI. mixture of proteins placed in a gel with a pH gradient (acidic at positive anode, basic at negative cathode, and neutral in the middle) protein stops moving when pH = pI
chromatography
variety of techniques that require the homogenized protein mixture to be fractionated through a porous matrix. isolated proteins are available immediately for identification and quantification. CONCEPT: the more similar the compound is to its surroundings the mre it will stick to and move slowly through its surroundings. (better for large amounts of proteins)
process of chromatography
- place sample onto solid medium called stationary phase/ adsorbent
- let sample run through mobile phase into the stationary phase (elute)
- retention time: amount of time compound spends in stationary phase
- partitioning: varying retention times for each compound in solution results in separation of components within the stationary phase
column chromatography
Used to separate and collect other macromolecules besides proteins such as nucleic acids. column filled with silica/ alumina beads and gravity moves solvent down. both size and polarity matter. less polar = faster it’ll go through.
ion-exchange chromatography
beads in column are coated with charged substances, so they attract or bind compounds that have an opposite charge. at the end a salt gradient is used to elute the charged molecules that have stuck to the column.
size-exclusion chromatography
beads used in the column contain tiny pores of varying size. this allows the larger ones to move faster around the beads and the smaller ones to move slower
affinity chromatography
customized columns to bind any protein of interest by creating a column with a high affinity for that protein or specific antibody to the protein. the protein of interest is retained in the column. protein can be removed with elution (may not come out or protein used to elute it may stick to the protein of interest)
2 ways to determine protein structure
first the protein must be isolated
- X-ray crystallography (after isolation it must be crystallized. most reliable and common method. measures electron density on extremely high resolution scale and can also be used for nucleic acids)
- nuclear magnetic resonance (NMR) spectroscopy
Edman degradation
to determine primary structure. uses cleavage to sequence proteins of up to 50 to 70 amino acids. selectively and sequentially removes N-terminal aa of the protein, which can be analyzed via mass spectroscopy.
4 ways to determine concentration
- UV spectroscopy
- bicinchoninic acid (BCA) assay
- Lowry reagent assay
- Bradford protein assay (most common b/c its simple and reliable)
Bradford protein assay
mixes a protein in solution with Coomassie Brilliant Blue dye.
protonated (acidic) dye- brown/green
deprotonated (basic) dye- blue — created by interactions with proteins in solution
what type of receptor are hormones likely to act on
enzyme-linked and G protein-coupled receptors
