Primary structural proteins in the body are collagen, elastin, keratin, actin and tubulin
These proteins have highly repetitive organization (motif)
Organization gives most structural proteins a fibrous nature
Trihelical fiber: three left handed helices woven together to form a secondary right-handed helix)
Makes up most of the extracellular matrix of connective tissue
Important in providing strength and flexibility
Primary role is to stretch and recoil like a spring so that it restores the original shape of the tissue
Component of the extracellular matrix
Intermediate filament proteins found in epithelial cells
Contribute to mechanical integrity of the cell
Also function as regulatory proteins
Primary protein that makes up hair and nails
Makes up microfilaments and the thin filaments in myofibrils
Most abundant protein in eukaryotic cells
Polar proteins: having a positive and negative side allows motor proteins to travel in one direction along an Actin filament
Makes up microtubules
Microtubules provide: structure; chromosome separation; and intracellular transport with kinesin and dynein
Has polarity: negative end is usually located near the nucleus while the positive end is located in the periphery of the cell
Some structural proteins have motor function in the presence of proteins
E.g. – motile cilia in bacteria or the flagella in sperm
Enzymatic Activity: act as ATPases which powers the conformational change necessary for motor function
Motor proteins interact either with actin or microtubules
Primary motor proteins that interacts with actin
Thick filament in a myofibril and is also involved in cellular transport
Each subunit has a head and a neck
Movement of the neck is responsible for the power stroke of sarcomere contraction.
Kinesins and Dyneins
Motor proteins associated with microtubules
Have two heads, at least one stays attached to tubulin at all times
Kinesin: play a role in aligning chromosomes during metaphase and depolymerizing microtubules during anaphase of mitosis.
Dyneins: involved in the sliding movement of cilia and flagella
Both proteins are important for vesicle transport in the cell
Kinesins bring vesicles towards the positive end of the microtubule
Dyneins bring vesicles towards the negative end of the microtubule
Classified by proteins that have a stabilizing function in individual cells of the body. These proteins transport or sequester molecules by binding to them
E.g. – hemoglobin, calcium-binding proteins, DNA-binding proteins
Each binding protein has an affinity curve for its molecule of interest
If goal of protein is sequestration: binding protein will usually have a high affinity over a long range of conditions in order to keep the target molecule bound at nearly 100%
If goal of protein is transport: varying affinity depending on environmental conditions so that equilibrium concentrations can be maintained
Cell Adhesion Molecules
Proteins found on the surface of most cells
Aid in binding the cell to the extracellular matrix or other cells
Are all integral membrane proteins
Group of glycoproteins that mediate calcium-dependent cell adhesion
Hold similar cell types together, each cells usually have type-specific cadherins
Group of proteins that have two membrane-spanning chains called a & b.
Chains are important in binding to and communicating with the extracellular matrix
Play an important role in cellular signaling and can greatly impact cellular function by promoting cell division, apoptosis, or other processes
Bind to carbohydrate molecules that project from other cell surfaces
Weakest bonds formed by CAMs
Expressed on white blood cells and endothelial cells that line blood vessels
Play an important role in host defense: including inflammation and white blood cell mitigation
Most prominent type of protein found in the immune system is the antibody (or immunoglobulins (Ig))
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 eliminate the threat.
Y-shaped proteins that are made up of two identical heavy chains
Disulfide linkage and noncovalent interaction hold the heavy and light chains together
Each antibody has an antigen-binding region at the tips of the “Y”
Specific polypeptide sequences that will bind one specific antigenic sequence.
Remaining part of an antibody is the constant region
Involved in the recruitment and binding of other cells of the immune system.
When antibodies bind to their targets (antigens), can cause one of three outcomes:
Neutralize the antigen which makes the pathogen or toxin unable to exert its effect on the body
Opsonization: marking the pathogen for destruction by other white blood cells immediately
Agglutinating: clump together the antigen and antibody into a large insoluble protein complexes that can be phagocytized and digested by macrophages
Process by which cells receive and act on signals
Proteins act as extracellular ligand, transporters for facilitated diffusion, receptor proteins and second messengers
Can have functions involved in substrate binding or enzymatic activity
Proteins that create specific pathways for charged molecules
All permit facilitated diffusion of charged molecules
Facilitated Diffusion: diffusion of molecules down a concentration gradient through a pore in the membrane created by a transmembrane protein.
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 phospholipid bilayer
Km & vmaxparamters that apply to enzymes can also apply to transporters (ion channels)
Km refers to the solute concentration at which the transporter is functioning at half of its maximum capacity
Unregulated since they have no gates
E.g. – potassium ion channel.
Will always be movement unless specified ion is at equilibrium
Gate is regulated by the membrane potential change near the channel
E.g. – voltage-gated sodium channels
Channels are closed under resting conditions; depolarization of the cell membrane leads to a conformational change in the protein that allows them to quickly open.
Binding of a specific substrate or ligand to the channel causes it to open or close
E.g. – neurotransmitters act at the postsynaptic membrane. GABA (inhibitory N.T) binds to the chloride channel and opens it.
Membrane receptors that display catalytic binding in response to ligand binding
Have three primary protein domains
Membrane-Spanning: anchors the receptor in the cell membrane
Ligand-Binding: stimulated by the appropriate ligand and induces a conformation change that activates the catalytic domain.
Catalytic: Often results in the initiation of a second messenger cascade
E.g. – Receptor tyrosine kinases (RTK)
G Protein-Coupled Receptors (GPCR)
Family of integral membrane proteins involved in signal transduction
Seven membrane spanning alpha-helices
Receptors differ in specificity of the ligand-binding area found on the extracellular surface of the cell
Heterotrimeric G Proteins: how the GPCRs transmit signals to an effector in the cell
Named for their intracellular link to guanine nucleotides (GDP & GTP)
Binding of ligand increases the affinity of the receptor for the G protein
Binding of G-protein represents a switch to the active state and affects the intracellular signaling pathway
G-proteins can result in either stimulation or inhibition, three main types:
Gs: stimulated adenylate cyclase which increase levels of cAMP in the cell
Gi: inhibits adenylate cyclase which decreases levels of cAMP in the cell
Gq: activates phospholipase C – 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 which increases calcium levels in the cell
G-Proteins have three subunits: a, b, and g
In inactive form, the alpha-subunit binds GDP and is in a complex with the other two subunits
When a ligand binds to the GPCR, the receptor becomes activated and engages the corresponding G protein
Causes the conversion of GDP to GTP which allows the alpha subunit to dissociate from the other two subunits
Alpha subunit alters the activity of adenylate cyclase (either inhibitory or stimulating)
Once GTP is dephosphorylated to GDP, the alpha subunit binds to the other two and the G protein is made inactive again.
Proteins and other biomolecules are isolated from body tissue or cell cultures by cell lysis or homogenization: crushing, grinding or blending the tissue of interest into an evenly mixed solution.
Centrifugation: can isolate proteins from much smaller molecules after homogenization
Precursor to other isolation techniques: electrophoresis and chromatography
Works by subjecting compounds to an electric field which moves them according to their net charge and size.
Negatively charged compounds will migrate towards the positively charged anode, and positively charged compounds will migrate towards the negatively charged cathode
Migration Velocity: velocity of the migration and is directly proportional to the electric field strength (E) and to the net charge of the molecule (z), and is inversely proportional to a frictional coefficient, f.
v = (Ez)/f
Polyacrylamide gel is the standard medium for electrophoresis
Slightly porous matrix mixture that solidifies at room temperature
Proteins travel through this matrix in relation to their size and charge
Gel allows smaller molecules to pass through easily and retains large particles.
A molecule will move faster through the gel if it is small, highly charged or placed in a large electric field
Native PAGE (Polyacrylamide gel electrophoresis)
Method for analyzing proteins in their native states.
This is limited by the varying mass-to-charge and mass-to-size ratios of cellular proteins since different proteins may experience the same level of migration
Protein can be recovered from the gel if the sample hasn’t been stained
Most useful to compare molecular size or the charge of proteins known to be similar in size
(Sodium Dodecyl Sulfate) SDS-PAGE
Separates proteins on the basis of relative molecular mass alone.
SDS disrupts all noncovalent interactions
Binds to protein and creates large chains with net negative charges which neutralizes the protein’s original charge and denatures the protein
The only variable that affects the velocity is E and f
Proteins can be separated based on their isoelectric point (pI)
pI is the pH at which the protein or amino acid is electrically neutral
Zwitterion is the neutral form for single amino acids, calculation of this point was done in first chapter
Exploits the acidic and basic properties of amino acids by separating on the basis of pI.
Mixture is placed in a gel with a pH gradient where the anode has acidic gel and is positive, and the cathode has basic gel and is negative, middle is neutral
Electric field is then generated across the gel
Positively charged proteins will migrate towards the cathode and negatively charged proteins will migrate towards the anode
When the protein reaches a portion of the gel that has a pH equal to its pI, the protein takes on a neutral charge and will stop moving.
Require the homogenized protein mixture to be fractionated through a porous matrix
Allow for the protein to be immediately available for identification and quantification
Overarching concept: the more similar a compound is to its surroundings (by polarity, charge, etc.), the more it will stick to and move slowly through its surroundings.
Process begins by placing a sample onto a solid medium called the stationary phase or adsorbent
Mobile phase is then run through the stationary phase
Allows the sample to elute: run the sample through the stationary phase
Components that have high affinity for the stationary phase will barely migrate at all
Components that have high affinity for the mobile phase will migrate quickly
Retention Time: amount of time a compound spends in the stationary phase
Varying retention times of each compound results in the separation of components within the stationary phase (partitioning)
Column is filled with polar silica or alumina beads as an adsorbent, and gravity moves the solvent + compounds down the column
The less polar a compound, the faster it will elute (shorter retention time)
Solvent polarity can be easily changed by altering the pH or salinity
Eventually solvent drips out of the column and it can be collected at different intervals to get a specific compound of interest.
Beads of column are coated with charged substances so that they attract or bind compounds that have an opposite charge
After all other compounds have moved through the column, a salt gradient can be used to elute the charged molecules that have stuck to the column
Beads used in the column contain tiny pores of varying sizes which allow small compounds to enter the beads
This effectively slows down small molecules while allowing larger molecules to elute quickly
Columns can be customized to bind any protein of interest by creating beads with a high affinity for that protein.
Can be accomplished by coating the beads with a receptor that binds to the protein or a specific antibody to the protein.
Protein is retained in the column
Once the protein is retained, it can be eluted by washing the column with a free receptor which competes with the bead-bound receptor and ultimately frees the protein from the column
Alternatively, an eluent can be created with a specific pH or salinity to disrupt the ligand bonds
Drawback is that the recovered substance may be bound to the eluent
Can be determined through X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy
X-Ray Crystallography: most reliable and common method
Protein must be isolated and crystallized beforehand
Measures electron density on an extremely high-resolution scale
Generates an X-Ray diffraction pattern, small dots in pattern are then used to determine protein’s structure
NMR is discussed in organic chemistry and accounts for 25% of the protein structure determination.
Amino Acid Composition
Can be determined by complete protein hydrolysis and subsequent chromatographic analysis, but actual sequence of amino acids cannot be determined since hydrolysis is a random process.
If the sequence of amino acids is needed, the protein needs to be sequentially digested with specific cleavage enzymes.
Edman Degradation: uses cleavage to sequence proteins of up to 50-70 amino acids
Selectively and sequentially removes the N-terminal amino acid of the protein which is then analyzed by mass spectroscopy
Larger proteins are digested with chymotrypsin, trypsin and cyanogen bromide
Selectively cleaves proteins at specific amino acid residues, which creates smaller fragments that can be analyzed using electrophoresis or the Edman degradation
Location of disulfide links and salt bridges cannot be determined with this method since those connections are broken
Protein activity can be determined by monitoring a known reaction with a given concentration of substrate and then comparing it with a standard
Activity is correlated with concentration but is also affected by the purification methods used
Most applicable when reactions have a colour change associated with it since the can be quickly identified from a chromatographic analysis
Determined through spectroscopy. Can be analyzed with UV spectroscopy without any treatment since proteins contain aromatic side chains
This analysis is sensitive to sample contaminant
Another method is to take advantage of the fact that proteins cause colorimetric changes with specific reactions: bicinchoninic acid (BCA) assay, Lowry reagent assay, and Bradford protein assay.
Bradford Protein Assay
Most common since it is reliable and simple
Mixes a protein in solution with blue dye
Dye gives up protons when it binds to amino acid groups and turns blue in the process
The larger the concentration of blue dye, the higher the concentration of the protein
Due to ionic attraction between dye and protein which causes stabilization of the blue dye
Samples of known concentrations are reacted with the Bradford reagent and the absorbance is measured to create a standard curve
Unknown sample is exposed to same conditions and the concentration is determine based on the standard curve
Very accurate when only one type of protein is present in the solution
Limited by the presence of detergent in the sample or by excessive buffer.