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

  • 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

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

  • 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

  • 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

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
  • Km & vmax paramters 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

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:
      • 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.

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

  • 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.

  • 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.