Ch. 3: Nonenzymatic Protein Function and Protein Analysis Flashcards
what are the two main functions of proteins within the cell?
- supporting cellular shape and organization
- acting as enzymes
where are structural and motor proteins found? (2)
- within individual cells
- the extracellular matrix
in general terms, how are proteins involved structurally intracellulary and extracellularly?
INTRACELLULAR: the cytoskeleton (a 3-D web or scaffolding system for the cell) is comprised of proteins that are anchored to the cell membrane by embedded protein complexes
EXTRACELLULAR: matrices composed of proteins also support the tissues of the body (tendons, ligaments, cartilage, and basement membranes)
what are the 5 primary structural proteins in the body?
- collagen
- elastin
- keratin
- actin
- tubulin
char (3): structural proteins
- have a highly repetitive secondary structure
- have a super-secondary structure/motif
- regularity gives many a fibrous nature
defn: motif/super-secondary structure
a repetitive organization of secondary structural elements together
char + func (2) + location: collagen
char: has a characteristic trihelical fiber (three left-handed helices woven together to form a secondary right-handed helix)
func: 1. makes up most of the extracellular matrix of connective tissue
2. important in providing strength and flexibility
location: throughout the body
func (2): elastin
- another important component of the extracellular matrix of connective tissue
- main role is to stretch and then recoil like a spring, restoring the original shape of the tissue
defn + func (3): keratin
defn: intermediate filament proteins found in epithelial cells
func: 1. contribute to the mechanical integrity of the cell
2. function as regulatory proteins
3. the primary protein that makes up hair and nails
char (2) + func: actin
func: makes up microfilaments and the thin filaments in myofibrils
char: 1. the most abundant protein in eukaryotic cells
2. have a positive and a negative side (polarity that allows motor proteins to travel unidirectionally along an actin filament like a one way street)
func + char: tubulin
func: makes up microtubules
char: has polarity (negative end of a microtubule is usually located adjacent to the nucleus, the positive end is usually in the cell periphery)
can structural proteins have motor functions?
yes, some can in the presence of motor proteins (e.g. cilia and flagella of bacteria and sperm)
explain how motor proteins can display enzymatic activity
they act as ATPases that power the conformational change necessary for motor function
what 2 things do motor proteins have transient interactions with?
- actin
- microtubules
defn + func (3) + char: myosin
defn: the primary motor protein that interacts with actin
func: 1. the thick filament in a myofibril
2. involved in cellular transport
3. movement of the neck is responsible for the power stroke of sarcomere contraction
char: each myosin subunit has a single head and neck
defn + char (2) + func of both: kinesin and dynein
defn: the motor proteins associated with microtubules
char: 1. have 2 heads, at least one of which remains attached to tubulin at all times
2. have opposite polarities
func: important for vesicle transport in the cell
func (2): kinesin
- key role in aligning chromosomes during metaphase
- key role in depolymerizing microtubules during anaphase of mitosis
func: dynein
involved in the sliding movement of cilia and flagella
explain what it means that kinesin and dynein have opposite polarities
KINESINS bring vesicles toward the positive end of the microtubule
DYNEINS bring vesicles toward the negative end of the microtubule
diagram: stepwise activity of kinesins
kinesins move along microtubules in a stepping motion such that one or both heads remain attached at all times
defn: binding proteins
proteins that have stabilizing functions in individual cells and the body that act to transport or sequester molecules by binding to them
what are 3 common binding proteins/groups?
- hemoglobin
- calcium-binding proteins
- DNA-binding proteins (often transcription factors)
each binding protein has an affinity curve for its molecule of interest, what makes this curve differ?
this curve differs depending on the goal of the binding protein
what is the affinity when sequestration of the molecule is the goal? why?
the binding protein usually has high affinity for its target across a large range of concentrations so it can keep it bound at nearly 100 percent
what is the affinity for a transport protein? why?
transport proteins must be able to bind or unbind its target to maintain steady-state concentrations, and is thus likely to have a varying affinity depending on the environmental conditions
defn: cell adhesion molecules (CAMs)
proteins found on the surface of most cells (all integral membrane proteins) and aid in binding the cell to the extracellular matrix or other cells
what are the 3 categories of CAMs?
- cadherins
- integrins
- selectins
defn + func + char: cadherins
a group of glycoproteins that mediate calcium-dependent cell adhesion
func: often hold similar cell types together (such as epithelial cells)
char: different cells usually have type-specific cadherins (i.e. epithelial cells use E-cadherin, nerve cells use N-cadherin)
defn + func (3): integrins
a group of proteins that all have 2 membrane-spanning chains called alpha and beta that are very important in binding to and communicating with the extracellular matrix
func: 1. play a very important role in cellular signaling
2. can greatly impact cellular function by promoting cell division, apoptosis, or other processes
3. important role in host defense
what are 3 examples of processes which integrins are involved in?
- allowing platelets to stick to fibrinogen, a clotting factor, which causes activation of platelets to stabilize the clot
- white blood cell migration
- stabilization of epithelium on its basement membrane
defn + char (2) + func: selectins
defn: bind to carbohydrate molecules that project from other cell surfaces
char: 1. the weakest formed bonds by CAMs
2. expressed on white blood cells and the endothelial cells that line blood vessels
func: important role in host defense, including inflammation and white blood cell migration
what is the common purpose of the cells and proteins involved in the immune system?
to rid the body of foreign invaders
defn (2) + aka + char (3) + diagram: antibodies
defn: 1. the most prominent type of protein found in the immune system
2. 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 eliminate the threat
aka: immunoglobulins (Ig)
char: 1. Y-shaped proteins made up of two identical heavy chains and two identical light chains
2. disulfide linkages and covalent interactions hold the heavy and light chains together
3. each has an antigen-binding region at the tips of the Y
func: antigen-binding region
within this region, there are specific polypeptide sequences that will bind one (and only one) specific antigenic sequence
outside of the antigen-binding region, what is the rest of the antibody molecule called and what is its function?
name: constant region
func: involved in recruitment and binding of other cells of the immune system, such as macrophages
defn: antigens
the targets of antibodies
what are the three outcomes of when antibodies bind to their targets (antigens)?
- 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 (opsonization)
- clumping together (agglutinating) the antigen and antibody into large insoluble protein complexes that can be phagocytized and digested by macrophages
defn: biosignaling
a process in which cells receive and act on signals
in what 4 capacities do proteins participate in biosignaling?
what 2 functions do proteins have to participate?
capacities: 1. extracellular ligans
2. transporters for facilitated diffusion
3. receptor proteins
4. second messengers
functions: 1. substrate binding
2. enzymatic activity
defn: ion channels
proteins that create specific pathways for charged molecules
what are the 3 main groups of ion channels?
what is the similarity and differences between the ion channel groups?
- ungated channels
- voltage-gated channels
- ligand-gated channels
have different mechanisms of opening, but all permit facilitated diffusion of charged particles
defn + func (2): 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
func: 1. used for molecules that are impermeable to the membrane (large, polar, or charged)
- allows integral membrane proteins to serve as channels for these substrates to avoid the hydrophobic fatty acid tails of the phospolipid bilayer
defn: ungated channels
have no gates and are thus unregulated
defn: voltage-gated channels
the gate is regulated by the membrane potential change near the channel
defn: ligand-gated channels
the binding of a specific substance or ligand to the channel causes it to open or close
where can we derive the kinetics of transporters such as ion channels in membranes?
the Km and vmax parameters that apply to enzymes are also applicable
we can derive them from the Michaelis-Menten and Lineweaver-Burk plot equations where Km refers to the solute concentration at which the transporter is functioning at half of its maximum activity
defn: enzyme-linked receptors
membrane receptors that display catalytic activity in response to ligand binding
what are the three primary protein domains of enzyme-linked receptors?
- membrane-spanning domain
- ligand-binding domain
- catalytic domain
func: membrane-spanning domain
anchors the receptor in the cell membrane
char + func: ligand-binding domain
stimulated by the appropriate ligand
induces a conformational change that activates the catalytic domain which often results in the initiation of a second messenger cascade
what are 3 common classes of enzyme-linked receptors?
- receptor tyrosine kinases
- serine/threonine-specific protein kinases
- receptor tyrosine phosphatases
defn: G protein-coupled receptors
a large family of integral membrane proteins involved in signal transduction that differ in specificity of the ligand-binding area found on the extracellular surface of the cell and are characterized by their 7 membrane-spanning alpha-helices
func: heterotrimeric G protein
used by GPCRs to transmit signals to an effector in the cell
what are G proteins named for?
their intracellular link to guanine nucleotides (GDP and GTP)
how do GPCRs and G proteins work together?
- the binding of a ligand increases the affinity of the receptor for the G protein
- the binding of the G protein represents a switch to the active state and affects the intracellular signaling pathway
what are the 3 main types of G proteins + their functions?
- Gs –> stimulates adenylate cyclase, which increases levels of cAMP in the cell
- Gi –> inhibits adenylate cyclase, which decreases levels of cAMP in the cell
- Gq –> activates phospholipase C, which cleaves a phospholipid from the membrane to form PIP2, which is then cleaved into DAG and IP3 which can open calcium channels in the ER, increasing calcium levels in the cell
mnemonic: functions of heterotrimeric G proteins
GS Stimulates
GI Inhibits
Mind your P’s and Q’s: GQ activates Phospholipase C
what are the three subunits of the G protein?
alpha, beta, gamma
what is the status of the subunits when the G protein is in its inactive form?
the alpha subunit binds GDP and is in a complex with the beta and gamma subunits
what happens to the subunits when a ligand binds to the GPCR, the receptor becomes activated, and engages the corresponding G protein? + diagram
- the alpha subunit is able to dissociate from the beta and gamma subunits
- the activated alpha subunit alters the activity of adenylate cyclase
- if the alpha subunit is alphas –> the enzyme is activated, if it is alphai –> the enzyme is inhibited
- once GTP on the activated alpha subunit is dephosphorylated to GDP, the alpha subunit will rebind to the beta and gamma subunits, rendering the G protein inactive
how are proteins and other biomolecules isolated from body tissues or cell cultures?
- cell lysis
- homogenization
defn: homogenization
crushing, grinding, or blending the tissue of interest into an evenly mixed solution
func: centrifugation
can isolate proteins from much smaller molecules before other isolation techniques must be employed
what are the 2 most common isolation techniques? can they be used on both native and denatured proteins?
- electrophoresis
- chromatography
yes, they can be used on both native and denatured proteins
how does electrophoresis work?
by subjecting compounds to an electric field, which moves them according to their net charge and size
so negatively charged compounds will migrate toward the positively charged anode and positively charged compounds will migrate toward the negatively charged cathode
defn: migration velocity
velocity of the migration of charged compounds toward the charged anodes in electrophoresis
what is migration velocity, v, directly proportional to (2) and inversely proportional to (1)? + equation
directly: 1. electric field strength E
2. the net charge on the molecule z
inversely: a frictional coefficient f
what does the frictional coefficient f depend on?
the mass and shape of the migrating molecules
what is the standard medium for protein electrophoresis?
polyacrylamide gel
char (3): polyacrylamide gel
- a slightly porous matrix mixture
- solidifies at room temperature
- multiple samples can be run simultaneously due to the gel’s size
how does polyacrylamide gel interact with proteins during protein electrophoresis?
- proteins travel through this matrix in relation to their size and charge
- the gel acts like a sieve, allowing smaller particles to pass through easily while retaining large particles
what 3 things would make a molecule move faster through the gel? what 3 things would make a molecule move slower through the gel? (or not at all)
faster: 1. small
2. highly charged
3. placed in a large electric field
slower: 1. bigger and more convoluted
2. electrically neutral
3. placed in a small electric field
defn: PAGE
polyacrylamide gel electrophoresis
a method for analyzing proteins in their native states
diagram: electrophoresis
what is PAGE limited by?
the varying mass-to-charge and mass-to-size ratios of cellular proteins because multiple different proteins may experience the same level of migration
can the functional native protein be recovered from the gel after electrophoresis in PAGE?
only if the gel has not been stained because most stains denature proteins
for what is PAGE most useful?
to compare the molecular size or the charge of proteins known to be similar in size from other analytic methods like SDS-PAGE or size-exclusion chromatography
defn: SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
separates proteins on the basis of relative molecular mass alone
how does SDS-PAGE work? (4)
- starts with the same premise as PAGE, but adds SDS, a detergent that disrupts all noncovalent interactions
- it binds to proteins and creates large chains with net negative charges, thereby neutralizing the protein’s original charge and denaturing the protein
- as the proteins move through the gel, the only variables affecting their velocity are E, the electric field strength, and f, the frictional coefficient, which depends on mass
- after separation, the gel can be stained so the protein bands can be visualized and the results recorded
what unit is protein atomic mass expressed in?
daltons (Da) = g/mol
defn: isoelectric point (pI)
the pH at which the protein or amino acid is electrically neutral, with an equal number of positive and negative charges
defn: zwitterion
the electrically neutral form of individual amino acids
for most amino acids, what groups are protonated or deprotonated for the zwitterion form?
amino group protonated
carboxyl group deprotonated
side chain electrically neutral
what are the 2 amino acids that are exceptions to the above zwitterion formation pattern? when does their zwitterion form?
arginine and lysine (basic side chains)
amino group deprotonated (thus electrically neutral)
nitrogenous side chain protonated
carboxyl group deprotonated
how is the pI determined for polypeptides?
primarily determined by the relative numbers of acidic and basic amino acids
defn: isoelectric focusing
exploits the acidic and basic properties of amino acids by separating on the basis of pI
how does isoelectric focusing work? (4)
- the mixture of proteins is placed in a gel with a pH gradient (acidic gel at the positive anode, basic gel at the negative cathode, and neutral in the middle)
- an electric field is generated across the gel
- positively charged proteins migrate toward the cathode, negatively charged proteins migrate toward the anode
- as the protein reaches the portion of the gel where the pH is equal to the protein’s pI , the protein takes on a neutral charge and will stop moving
mnemonic: remembering anodes and charges in isoelectric focusing
Anode in isoelectric focusing: A+
Anode has Acidic (H+-rich) gel and a (+) charge
defn + basic premise: chromatography
a variety of techniques that require the homogenized protein mixture to be fractionated through a porous matrix
the more similar the compound is to its surroundings (polarity, charge, etc.), the more it will stick to and move slowly through its surroundings
what is one of the main reasons why chromatography is so valuable?
the isolated proteins are immediately available for identification and quantification
when is chromatography preferred over electrophoresis?
when large amounts of protein are being separated
process (5): chromatography
- place the sample onto a solid medium (the stationary phase or adsorbent)
- run the mobile phase through the stationary phase which will allow the sample to run through the stationary phase (elute)
- depending on the relative affinity of the sample for the stationary and mobile phases, different substances migrate through at different speeds (those that have a high affinity for the stationary phase will barely migrate, those that have a high affinity for the mobile phase will migrate much more quickly)
- varying retention times of each compound in the solution results in separation of the components within the stationary phase (partitioning)
- each component can then be isolated individually for study
defn: retention time
the amount of time a compound spends in the stationary phase
process (5) + diagram: column chromatography
- a column is filled with silica or alumina beads as an adsorbent and gravity moves the solvent and compounds down the column
- as the solution flows through the column, both size and polarity determine how quickly the compound moves through the polar beads (the less polar the compound, the faster it can elute)
- the solvent drips out the column’s end, and different fractions that leave the column are collected over time
- each fraction contain bands that correspond to different compounds
- the solvent can then be evaporated and the compounds of interest kept
what 3 things can be easily changed in column chromatography to help elute the protein of interest?
solvent:
1. polarity
2. pH
3. salinity
process (2): ion-exchange chromatography
- the beads in the column are coated with charged substances, so they attract or bind compounds that have an opposite charge
- after all other compounds have moved through the column, a salt gradient is used to elute the charged molecules that have stuck to the column
process (3): size-exclusion chromatography
- the beads used in the column contain tiny pores of varying sizes which allow small compounds to enter the beads, thus slowing them down
- large compounds can’t fit into the pores, so they’ll move around them and travel through the column faster
- the size of the pores may be varied so that molecules of different molecular weights can be fractionated
can size-exclusion and ion-exchange chromatography be used together?
yes, a common approach in protein purification is to use an ion-exchange column followed by a size-exclusion column
main purpose + process (3): affinity chromatography
main purpose: we can customize beads to bind any protein of interest by creating a column with high affinity for that protein
- coat beads with a receptor that binds the portein or a specific antibody to the protein such that the protein is retained in the column
- once the protein is retained in the column, it can be eluted by washing the column with a free receptor (or target or antibody), which will compete with the bead-bound receptor and ultimately free the protein from the column
- eluents can be created with a specific pH or salinity level that disrupts the bonds between the ligand and the protein of interest
what are 3 common stationary phase molecules in affinity chromatography?
- Nickel (used in separation of genetically engineered proteins with histidine tags)
- antibodies or antigens
- enzyme substrate analogous (mimic the natural substrate for an enzyme of interest)
what is the only drawback to the elution step in affinity chromatography?
the recovered substance can be bound to the eluent
what are the 2 methods by which protein structure can be determined? which is the more reliable and common method?
- X-ray crystallography (more reliable and common)
- nuclear magnetic resonance (NMR) spectroscopy
how does X-ray crystallography work? (2)
- measures electron density on an extremely high-resolution scale and can also be used for nucleic acids
- an x-ray diffraction pattern is generated in this method and the small dots in the diffraction pattern can then be interpreted to determine the protein’s structure
how can the amino acids that compose a protein be determined?
by complete protein hydrolysis and subsequent chromatographic analysis
HOWEVER, the random nature of hydrolysis prevents amino acid sequencing
SO, sequential digestion of the protein with specific cleavage enzymes is used
defn + use + process: Edman degradation
use: used for small proteins
defn: uses cleavage to sequence proteins of up to 50 to 70 amino acids
process: selectively and sequentially removes the N-terminal amino acid of the protein (can be analyzed via mass spectroscopy)
how is protein activity generally determined? (3)
- by monitoring a known reaction with a given concentration of substrate and comparing it to a standard
- activity is correlated with concentration but is also affected by the purification methods and the conditions of the assay
- reactions with a color change have particular applicability because microarrays can rapidly identify the samples from a chromatographic analysis that contains the compound of interest
how is concentration determined?
almost exclusively through spectroscopy
why can proteins be analyzed with UV spectroscopy without any treatment?
because proteins contain aromatic side chains
what is a downside to UV spectroscopy?
it is particularly sensitive to sample contaminants
what are the bicinchoininic acid (BCA) assay, the Lowry reagent assay, and Bradford assay? which of these 3 is the most common and why?
colorimetric changes with specific reactions caused by proteins
Bradford assay is the most common because of its reliability and simplicity in basic analyses
how does the Bradford protein assay work? (6) + diagram
- mixes a protein in solution with Coomassie Brilliant Blue dye
- the dye is protonated and green-brown in color prior to mixing with proteins (acidic form)
- the dye gives up protons upon binding to amino acid groups, turning blue in the process (basic form)
- ionic attractions between the dye and the protein then stabilize this blue from of the dye (so increased protein concentrations correspond to a larger concentration of blue dye in solution)
- samples of known concentration are reacted with the Bradford reagent and then absorbance is measured to create a standard curve
- the unknown sample is then exposed to the same conditions and the concentration is determined based on the standard curve
when is a Bradford assay good to use? when not? why?
very accurate: when only one type of protein is present in solution
not good: when more than one protein is present because of variable binding of the Coomassie dye with different amino acids
what is the Bradford protein assay limited by?
the presence of detergent in the sample or by excessive buffer
