Biochemistry Ch 3. Nonenzymatic Protein Function and Analysis Flashcards
Structural proteins
Compose the cytoskeleton, anchoring proteins, and much of the extracellular matrix, generally fibrous in nature
Common structural proteins
Collagen, elastin, keratin, actin, and tubulin
Motor proteins
Have one or more heads capable of force generation through a conformational change, have catalytic activity acting as ATPases to power movement, common applications include muscle contraction, vesicles movement within cells, and cell motility
Common motor proteins
Myosin, kinesin, dynein
Binding proteins
Bind a specific substrate, either to sequester it in the body or hold its concentration at steady state
Cell adhesion molecules (CAM)
Allow cells to bind to other cells or surfaces
Cadherins
Calcium dependent glycoproteins that hold similar cells together
Integrins
Have two membrane spanning chains and permit cells to adhere to proteins in the extracellular matrix, some have signaling capabilities
Selectins
Allow cells to adhere to carbohydrates on the surfaces of other cells and are most commonly used in the immune system
Antibodies
aka immunoglobulins (Ig), are used in the immune system to target a specific antigen, contain a constant region and a variable region (responsible for antigen binding), formed of two identical heavy chains and two identical light chains, held together by disulfide linkages and non covalent interactions
Antigen
Protein on the surface of a pathogen (invading organism) or a toxin
Ion channels
Can be used for regulating ion flow into or out of a cell, three main types of ion channels
Ungated channels
Always open
Voltage gated channels
Open within a range of membrane potentials
Ligand gated channels
Open in the presence of a specific binding substance, usually a hormone or neurotransmitter
Enzyme linked receptors
Participate in cell signaling through extracellular ligand binding and initiation of second messenger cascades
G protein coupled receptors
Have a membrane bound protein associated with a trimeric G protein, also initiate second messenger systems
G protein coupled receptor steps
Ligand binding engages the G protein, GDP is replaced with GTP, the Alpha subunit dissociates from the beta and gamma subunits, the activated alpha subunit alters the activity of adenylate cyclase or phospholipase C, GTP is dephosphorylated to GDP, the alpha subunit rebinds to the beta and gamma subunit
G protein
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Adenylate cyclase
=
Phosphorlipase C
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Electrophoresis
Uses a gel matrix to observe the migration of proteins in response to an electric field
Native PAGE
Maintains the proteins shape, but results are difficult to compare because the mass to charge ratio differs for each protein
SDS PAGE
Denatures the proteins and maksthe native charge so that the comparison of size is more accurate, but the functional protein cannot be recaptured from the gel
Isoelectric focusing
Separates proteins by their isoelectric point (pI), the protein migrations toward the electrode until it reaches a region of the gel where pH=pI of the protein
Chromatography
Separates protein mixtures on the bases of their affinity for a stationary phase or a mobile phase
Stationer phase
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Mobile phase
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Isoelectric point
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Column chromatography
Uses beads of a polar compound, like silica or alumina (stationary phase), with a nonpolar solvent (mobile phase)
Ion exchange chromatography
Uses a charge column and a variably saline eluent
Size exclusion chromatography
Relies on porous beads, larger molecules elute first because they are not trapped in the small pores
Affinity chromatography
Uses a bond receptor or ligand and an fluent with free ligand or a receptor for the protein of interest
X-ray crystallography
Can be used after the protein is isolated to determine protein structure
Edman degradation
Sequential degradation that allows amino acid sequencing
Analyzing protein structure
X-ray crystallography or NMR
Analyzing amino acid composition
Simple hydrolysis
Analyzing amino acid sequence
Edman degradation
Analyzing activity levels for enzymatic samples
Determined by following the process of a known reaction, often accompanied by a color change
Analyzing protein concentration
Can be determined colorimetrically, either by UV spectroscopy or through a color change reaction
BCA assay, Lowry reagent assay, Bradford protein assay
Each test for protein and have different advantages and disadvantages, Bradford protein assay uses a color change from brown-green to blue and is the most common
