Nonenzymatic Protein Function and Protein Analysis Flashcards
Primary Structural Proteins of the Body
Collagen Elastin Keratin Actin Tubulin
Motif
repetitive organization of secondary protein structural elements together
Collagen
three left handed helices woven in fiber
makes up extracellular matrix of connective tissue
strength and flexibility
Elastin
extracellular matrix of connective tissue
stretch and recoil like spring to regain original shape of tissue
Keratins
intermediate filament proteins in epithelial cells
mechanical integrity and regulatory proteins
Actin
microfilaments and thin filaments in myofibrils
most abundant protein in EK cells
polarity to have motor proteins move along actin filament
Tubulin
makes up microtubules which provide structure, intracelluar transport and chromosomal seperation
has polarity like actin : + towards periphery of cell and - towards the nucleus
Motor proteins display ____ acting as ____ that power the conformational change necessary for motor function. Have transient interactions with either ____ or _____
enzymatic activity
ATPases
actin
microtubules
Myosin
primary motor protein interacting with actin
thick filament in myofibril
cellular transport
neck causes power stroke
Kinesins/Dynesins
motor proteins with microtubules
two heads where one is attahced to tubule at all times
align chromosomes at metaphase and depolymerize microtubules during anaphase
Dyenins slide the movement of cilia and flagella
Kinesisns bring vesicles toward positive end of microtubule
Dyenins bring vesicles toward the negative end
Binding Proteins
transport/sequester molecules by binding to them
hemoglobin, calcium binding proteins, DNA binding proteins
has affinity curve for molecule of interest
Cell Adhesion Molecules (CAMS)
proteins found on the surface of most cells that aid in binding the cell to ECm or other cells
integral proteins
Cadherins, Integrins, Selectins
Cadherins
glycoproteins that conduct calcium dependent cell adhesion
hold similar type cells together
ither cells have type specific cadherins E for epithelial and N for nerve
Integrins
proteins with two membran spanning chains of alpha and beta
bind and communicate with ECM
cellular signaling and cell division and apoptosis
Selectins
bind to carbohydratte molecules from the surface
weakest bonds formed by CAMs
expressed on white blood cells and endothelial cells of blood vessls
host defense
Antibodies/Immunoglobulins
proteins made by B cells to neutralize targets in the body like toxins/bacteria and recruit other cells to destroy them
___ hold the heavy and light side chains of antibodies togehter
disulfide linakges
Antigen binding Region
Tips of the Y that bind to a specigic antigenic sequence
Constant Region
part of antibody thT RECRUITS AND BINDS CELLS OF IMMUNE SYSTEM LIKE MACROPHAGES
Three Outcomes of Antigen Binding
1) Neutralizing Antigen
2) Making pathogen for destruction by other blood cells immediately (Opsonization)
3) Agglutinating antigen and antibody into insoluble complex to phagocitize
Biosignaling
process in whcih cells receive and act on signals
EC ligands, transporters for facilitated diffusion, receptor proteins, second messengers
Facilitated Diffusion
passive transport
diffusion down a gradient through pore in transmembrane
allow molecules to avoid the fatty chains of membrane
Ungated Channels
unregulated
no gates
potassium channels
net efflux of ions unless at eq
Voltage-Gated Channels
gate is regulated by membrane potential
neruons with sodium channels
depolarization causes a change to let them open
non specific Na/K channels in sinoatrial node of heart as pacemaker current
Ligand-Gated Channels
binding to a specific substance or ligand to cause channel to open or close
neurotransmitters
Km and vmax of enzymes apply here too
Enzyme Linked Receptors
three primary protein domains
membrane spanning - anchors receptor
ligand binding - stimulated by ligand yo induce change
catlyitc activateed by ligand binding domain changing
leads to second messenger cascafe
G Protein Coupled Receptors
family of integral membrane proteins involved in signal transduction
characterized by 7 alpha helices
receptors differ in specificity of the ligand binding area on extracellular surface
Heterotrimeric G Protein
g proteins for GPCR with intracellular link GDP/GTP
Three Types G Protein
Gs - stimulate adenylate cyclase to increase cAMP
Gi - inhibit adenylate cyclase to decrease cAMP
Gq - activates phospholipase C to leave phospholipid from PIP 2 to cleave from DAG and IP3 which then opens calcium channels
alpha beta and gamma subunits of G protein
alpha binds to GDP and is complex with B and gamma
when ligand binds to G protein, receptor is activasted and engages corresponding G protein
GDp replaced with GTP and alpha dissociate from beta and gamma
alpha alters adenylate cyclase activity
alpha s activates
alpha i enzyme is inhibited
once GTP on alpha is dephosphorylated alpha will go back to B and hgamma and inactivate the protein
Centrifugation
isolate proteins from smaller molecules before other isolation techniques
Electrophoresis
moves protein according to net charge and size
velocity is migration velocity
v = Ez/ f
E = e;ectroc field strength f = frictional coefficient z = net charge
Poly acrylamide Gel
used in electrophoresis
small go fast, large go slow
Native PAGE
mass to charge and mass to size ratios
molecular size and charge of proteins
SDS-PAGE
sodium dodecyyl sulfate
proteins seperated on mass
SDS disrupts non covalent interactions and negative charges
only E affects velocity
Isoelectric Focusing
seperation by isoelectric point pI
pH when protein is neutal
for individual amino acids this is zwitterion phase
Chromatography
fractioned through a porous matrix
seperated by chemical and physical properties
Stationary Phase Adsorbant
sasmple on solid medium
mobile phase for sample to elute out of
Column Chromatography
size and polarity have to role with how quickly compound goes
less polar, the faster it goes
pH, salinity, polarity
Ion Exchange CHromatography
charged beads
attract/bind opposin charge
after all compounds are gone, salt is used to compete with charged protein to elute it out
Szie Exclusion Chromatography
beads have pores allowing small compounds to elute out slowly and large ones to elute out faster
different MW are key here
Affinity Chromatography
coating stationary beads with item that has affinity for target like nickel for histags, antibodies, enzyme substrate,
target is then washed off using elutant that competes for affinity of target letting it elute out
eluent can also be certain pH or salinity to rid of ligand interaction on beads
X Ray Crystallography
protein is isolated and crystalized
measures electron density and generates a pattern of dots to determine structure
Nuclear Magnetic Resonance
protein structure
Edman Degradation
small protein analysis of AMINO ACIDS
CLEAVAGE TO SEQ PROTEINS OF 50-70
protein hydrolysis
removes N terminal amino acid one at a time
Chymotrypsin, TrypsinCyanogen Bromide
selectively cleaves proteins at specific amino acid residues to create smaller fragment for Edman/Gel electrophoresis
positions cannot be determines as disulfide/salt bridges are broken
Bradford Protein Assay
mixes protein in solution with coomassie blue dye
dye gives protons to ionizable groups in protein to turn blue
increase protein concentrations have larger concentration of blue dye
standard curve created measuring absorbance and then unknown
only one protein is effective
