Non-enzymatic Protein, Function and Protein Analysis

Cellular Functions

Structural Proteins

• 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

Collagen

• 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

Elastin

• 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

Keratins

• 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

Actin

• 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

Tubulin

• 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

Motor Proteins

• 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

Myosin

• 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

Binding Proteins

• 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

Cadherins

• Group of glycoproteins that mediate calcium-dependent cell adhesion
• Hold similar cell types together, each cells usually have type-specific cadherins

Integrins

• 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

Selectins

• 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

Immunoglobulins

• 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

Biosignaling

• 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

Ion Channels

• 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
• K_{m &} v_max paramters that apply to enzymes can also apply to transporters (ion channels)
  • K_m refers to the solute concentration at which the transporter is functioning at half of its maximum capacity

Ungated Channels

• Unregulated since they have no gates
  • E.g. – potassium ion channel.
• Will always be movement unless specified ion is at equilibrium

Voltage-Gated Channels

• 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.

Ligand-Gated Channels

• 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.

Enzyme-Linked Receptors

• 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:
    • G_s: stimulated adenylate cyclase which increase levels of cAMP in the cell
    • G_i: inhibits adenylate cyclase which decreases levels of cAMP in the cell
    • G_q: 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.

Protein Isolation

• 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

Electrophoresis

• 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

Isoelectric Focusing

• 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.

Chromatography

• 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 Chromatography

• 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.

Ion-Exchange Chromatography

• 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

Size-Exclusion Chromatography

• 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

Affinity Chromatography

• 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

Protein Analysis

Protein Structure

• 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

Activity Analysis

• 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

Concentration Determination

• 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.