Nonenzymatic Protein Function and Protein Analysis Flashcards
motif
a repetitive organization of secondary structural elements together
collagen
characteristic trihelical fiber , makes up most of the extracellular matrix of connective tissue, provides strength and flexibility
elastin
component of the extracellular matrix of connective tissue, stretches and recoils
keratins
intermediate proteins in epithelial cells, in hair and nails
actin
protein that makes up microfilaments and thin filaments in myofibrils
tubulin
makes up microtubules, microtubules are important for providing structure, chromosomes separation in mitosis and meiosis, and intracellular transport with kinesin and dynein
motor proteins
display enzymatic activity, acting as ATPases that power the conformation al change necessary for motor function
myosin
primary motor function that interacts with actin
kinesin and dyneins
motor proteins associated with microtubules
binding proteins
hemoglobin, transcription factors
cell adhesion molecules (CAM)
proteins found on the surface of most cell sand aid in binding, the cell to the extracellular matrix or other cells
different families: cadherins, integrins, and selectins
cadherins
glycoproteins that mediate calcium dependent cell adhesion
integrins
a group of proteins that all have two membrane-spanning chains called alpha and beta; play a role in cell signaling
selectins
bind to carbohydrate molecules that project from other cell surfaces
immunoglobulins
also known as antibodies, proteins produced by B cells that function to neutralize targets in the body, such as toxins and bacteria and then recruit other cells to help with the threat
each antibody has a ?
antigen-biding region, at the tip of the Y
What are the targets for antibodies?
antigens (pathogens)
What are the three outcomes of antibodies binding to an antigen?
- neutralizing the antigen, making the pathogen or toxin unable to exert its effect on the body
- marking the pathogen for destruction by other white blood cells immediately; this marking is called opsonization
- clumping together (agglutinating) the antigen and antibody into large insoluble protein complexes that can be phagocytized and digested by macrophages
Opsonization
marking the 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
Ion channels
proteins that create specific pathways for charged molecules
facilitated diffusion
a type of passive transport, the diffusion of molecules down a concentration gradient through a pore in the membrane created by this transmembrane protein
ex. ungated, voltage-gated, and ligand-gated
ungated channels
have no gates and are therefore unregulated, ex. potassium channels
voltage-gated channels
gate is regulated by the membrane potential change near the channel, ex. voltage-gated non-specific sodium-potassium channels are found in the cells of the sinoatrial node of the heart
ligand-gated channels
binding of a specific substance or ligand to the channel causes it to open or close…the inhibitory neurotransmitter GABA binds to a chloride channel and opens it
Km
refers to the solute concentration at which the transporter is functioning at half of its maximum activity
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–> often results in the initiation of a second messenger cascade
G Protein Coupled Receptors
integral membrane proteins involved in signal transduction, utilize a heterotrimeric G protein (GDP and GTP)
3 types
1. G_s stimulates adenylate cyclase, increases levels of cAMP in the cell
2. G_i inhibits adenylate cyclase, which decreases levels of cAMP in the cell
3. G_q activates phospholipase C, which cleaves a phospholipid from the membrane to form PIP2. PIP2 is then cleaved into DAG and IP3; IP3 can open calcium channels in the endoplasmic reticulum, increasing calcium levels in the cell
GPCR - what separates away from GPCR protein?
alpha subunit can dissociate from alpha, beta, and gamma joined together in GPCR .
homogenization
crushing, grinding, or blending the tissue of interest into an evenly mixed solution
(centrifugation can then isolate proteins from much smaller molecules before other isolation techniques must be employed)
electrophoresis
subjects compounds to an electric field, moves them according to their net charge and size
uses polyacrylamide gel—> standard medium for protein electrophoresis
Polyacrylamide gel electrophoresis (Native PAGE)
maintains the protein’s shape, but results are difficult to compare because the mass-to-charge ratio differs for each protein
SDS-PAGE
denatures the proteins and masks the native charge so that 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 migrates toward an electrode until it reaches a region of the gel where pH=pI of the protein
chromatography
separates protein mixtures on the basis of the their affinity for a stationary phase or a mobile phase
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 charged column and a variable 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 bound receptor or ligand and an eluent with free ligand or a receptor for the protein of interest
X-ray crystallography
protein structure can be determined by this after the protein is isolated (NMR can also be used)
Edman degradation
a technique that allows sequential degradation for amino acid sequencing
Bradford protein assay
uses a color change from brown-green to blue, most common way to test for a protein
could also use BCA assay or Lowry reagent assay
