Nonenzymatic Protein Function and Protein Analysis

Structural proteins

• Cytoskeleton = 3D web/scaffolding system for the cell
• Comprised of proteins anchored to membrane by complexes
• Extracellular matrices also support tissues of body
  • Tendons, ligaments, cartilage, basement membranes are proteinaceous
• Primary structural proteins: collagen, elastin, keratin, actin, tubulin
• High repetitive organization of secondary structural elements (motif)

Collagen

• Trihelical fiber: 3 left-handed helices woven together → secondary right-handed helix
• Makes up most of ECM of connective tissue
• Found throughout body
• Provides strength and flexibility
• Major component of bone
• Structure relies on glycine interactions
  • Replacement of glycine → bone fragility
• Extracellular protein

Elastin

• ECM component of connective tissue
• Stretches and recoils like a spring → restores original shape
• Extracellular protein

Keratins

• Intermediate filament proteins in epithelial cells
• Contribute to mechanical integrity of cell
• Also function as regulatory proteins
• Primary protein in nails
• Extracellular protein

Actins

• Microfilaments + thin filaments in myofibrils
• Most abundant protein in eukaryotic cells
• Has positive and negative side (polarity)
• Lets motor proteins travel unidirectionally (one-way street)
• Cytoskeleton protein

Tubulin

• Makes microtubules: structure, chromosome separation, intracellular transport
• Has polarity:
  • Negative end by nucleus
  • Positive end by periphery
• Cytoskeleton protein

Motor proteins

• Cilia and flagella of bacteria and sperm
• Have ATPase activity
• Transient interactions with actin or tubules
• Myosin:
  • Primary motor protein with actin
  • Also involved in cellular transport
• Kinesins and dyneins:
  • Motor proteins with microtubules
  • Have 2 heads, one attached to tubulin
  • Kinesins align during metaphase, depolymerize microtubules during anaphase
  • Dyneins involved in sliding movement
  • Both important for vesicle transport:
    • Kinesin brings vesicles toward positive end
    • Dynein brings waste/recycled NT back to negative end
• Actin not involved in cell migration

Binding proteins

• Hb, calcium-binding proteins, DNA-binding proteins (TFs)
• Each binding protein has an affinity curve for its molecule
  • OxyHb dissociation curve is one of interest
• Affinity curve varies based on goal
• If protein sequesters molecule, it can keep stuff going at 100%
• Transport proteins bind and unbind
• If enough binding protein present, can have all receptors bound despite low affinity

Cell adhesion molecules (CAMs)

• Proteins on surface of most cells
• Help bind cells to ECM
• All are integral membrane proteins
• 3 main families: cadherins, integrins, selectins

Cadherins

• Glycoproteins
• Mediate Ca-dependent cell adhesion
• Often hold similar cell types together (ex: epithelial cells)
• Different cells have different cadherins:
  • Epithelial = E-cadherin
  • Nerve = N-cadherin

Integrins

• Two membrane-spanning chains: α and β
• Bind + communicate with ECM
• Also involved in signaling → impacts cellular processes
• Integrin αIIbβ3 allows platelets to stick to fibrinogen
  • Activates platelets to stabilize clot
• Also used for WBC migration and stabilization of epithelium on basement membrane

Selectins

• Bind to carbohydrate molecules projecting from other surfaces
• Weakest bonds formed by CAMs
• Expressed on WBCs and endothelial cells lining blood vessels
• Important for host defense
• Similar to integrins
• Some meds target selectins and integrins
• Cancer metastasis associated with unique CAM expression

Immunoglobulins (antibodies)

• Proteins produced by B cells
• Neutralize targets and recruit other cells to eliminate them
• Y-shaped:
  • 2 identical heavy chains
  • 2 identical light chains
• Held together by disulfide linkages + noncovalent interactions
• Antigen-binding region at tips of Y
  • Specific polypeptide sequences bind one (and only one) antigenic sequence
• Rest of antibody = constant region
  • Recruitment/binding of other immune cells (ex: macrophages)
• When antigens bind → 1 of 3 outcomes:
  • Neutralization (pathogen/toxin can’t exert effect)
  • Mark pathogen for destruction by WBCs immediately (opsonization)
  • Agglutination (clumping antigen–antibody into large insoluble complexes → phagocytized)

• Biosignaling = process by which cells receive and act on signals

Ion channels

• Create pathways for ions
• 3 types:
  • Ungated/unregulated (ex: K channels)
  • Voltage-gated (need depolarization)
  • Ligand-gated (need ligand binding)
• Transport kinetics have $K_m$ and $V_{max}$ values
• Cooperative
• Transporters do not have analogous $K_{eq}$ values

Enzyme-linked receptors

• 3 domains:
  • Membrane-spanning domain (anchors receptor)
  • Ligand-binding domain (stimulated by ligand)
  • Catalytic domain (activated after ligand binding)
• Can initiate second messenger cascades
• RTKs:
  • Monomers that dimerize upon ligand binding
  • Dimer = active form
  • Phosphorylates cellular enzymes

GPCRs

• Integral membrane receptor family
• Linked to heterotrimeric G protein (GDP/GTP)
• G proteins:
  • Gs increases AC → cAMP
  • Gi decreases AC → cAMP
  • Gq increases PLC → PIP₂ → DAG + IP₃
    • IP₃ opens Ca in ER → increases $[Ca^{2+}]$ in cell
• Subunits:
  • α, β, γ
  • α binds GDP and associates with β/γ
• Mechanism:
  • Ligand binds GPCR → GDP → GTP on α
  • α dissociates from β/γ
  • Alters AC activity
  • When GTP dephosphorylated, α rebinds β/γ
• Second messenger systems allow low concentration with high effect

Ions in cells

• Ca²⁺ usually protein-bound (muscle contraction, exocytosis, other stuff)
• Mg²⁺ also protein-bound
• Na⁺ and K⁺ exist in free state
• Cl⁻ excreted by kidneys → not bound to anything

• Proteins isolated by cell lysis + homogenization
  • Crushing, grinding, blending tissue into evenly mixed solution
• Centrifugation isolates proteins from smaller molecules

Electrophoresis

• Apply electrical field → proteins move by charge + size
• Opposites attract
• Migration velocity: $v = Ez/f$
  • $E$ = electric field strength
  • $z$ = charge
  • $f$ = frictional coefficient
• Polyacrylamide gel is standard
• Gel acts like sieve: smaller + highly charged move faster
• Can run multiple gels simultaneously

Native PAGE

• Polyacrylamide gel electrophoresis for native proteins
• Limited by mass/charge and mass/size ratios
• Functional native protein can be recovered only if gel not stained
  • Most stains denature proteins
• Most useful to compare size/charge when known to be similar

SDS-PAGE

• Sodium dodecyl sulfate PAGE
• Separates proteins by mass alone
• SDS disrupts noncovalent interactions
• SDS binds proteins → large chains with net negative charge
• Velocity depends mainly on $E$ and $f$

Isoelectric focusing

• Isoelectric point $pI$ = pH where protein is neutral (zwitterion)
• Separates amino acids based on $pI$
• Gel has pH gradient and electric field
• Positively charged proteins migrate toward anode
• Protein stops moving when pH = $pI$ (neutral charge)
• Can’t make gel equal to protein’s $pI$ if you want separation (won’t move + stays mixed with contaminants)
• Need pH that makes protein of interest negative while others positive/neutral
• Anode = positively charged, acidic
• Cathode = negatively charged, basic (OH⁻)

Chromatography

• Tool to separate and identify compounds
• Requires homogenized mixture fractionated through porous matrix
• Isolated proteins available for identification/quantification right away
• More similar compound is to surroundings, more it sticks + moves slowly
• Preferred over electrophoresis for large amounts of protein
• Sample placed on stationary phase (adsorbent)
• Mobile phase runs through to elute
• High affinity for stationary → barely moves
• High affinity for mobile → moves a lot
• Retention time = time compound spends in stationary phase
• Different retention times → separation (partitioning)
• Different media used

Column chromatography

• Column with silica or alumina beads
• Gravity moves solvent + compounds down
• Less polar elutes faster (shorter retention time)
• Can change solvent polarity, pH, or salinity
• Collect fractions over time
• Evaporate solvent, keep compounds
• Not limited to proteins

Ion exchange chromatography

• Beads coated with charged substances
• Bind opposite charge
• Positively charged column binds negatively charged protein
  • Can increase retention time or fully hold
• Salt gradient elutes molecules at end

Size exclusion chromatography

• Beads have pores: small compounds enter and slow down
• Large compounds travel faster around beads
• Pore size variation separates different molecules
• Often do ion exchange then size exclusion

Affinity chromatography

• Column designed with high affinity for target protein
• Beads coated with receptor or specific antibody
• Protein retained in column
• Example logic: if protein sticks in nonpolar column → protein is nonpolar (long retention time)
• Stationary phase molecules can be nickel, antibodies, enzyme substrate analogues
• Wash with free receptor to compete and free protein
• Recovered protein might be bound to eluent

Drawbacks

• Protein may have too high affinity and not elute
• Protein may be permanently bound to free receptor in eluent

Protein structure

• Determined by x-ray crystallography and NMR spectroscopy
• Must isolate and crystallize protein first
• Crystallography measures e⁻ density at high resolution
  • Also used for nucleic acids
• NMR determines minor amount (~25%) of structure

Amino acid composition

• Determined by complete protein hydrolysis + chromatographic analysis
• Random hydrolysis prevents sequencing

Edman degradation

• Sequences proteins ~50–70 amino acids (small proteins)
• Selectively/sequentially removes N-terminal amino acid
• For larger proteins:
  • Digest with chymotrypsin, trypsin, cyanogen bromide
  • Synthetic reagents cleave proteins at specific residues
  • Smaller fragments then sequenced with Edman degradation
• Disulfide links and salt bridges broken to get primary structure, so can’t be determined
• Cleavage for sequence determination must be selective

Activity analysis

• Monitor reaction at given $[substrate]$ and compare to standard
• Activity correlates with concentration
• Affected by purification methods + assay conditions
• Color-change reactions are useful

Concentration determination

• Spectroscopy-based
• UV spectroscopy works because proteins have aromatic side chains
  • Sensitive to contaminants
• Colorimetric assays:
  • BCA assay
  • Lowry reagent assay
  • Bradford protein assay

Bradford protein assay

• Mix protein solution with blue dye
• Dye is green-brown before mixing
• Dye gives up protons when binding to amino acids → turns blue
• More protein → more blue
• Measure absorbance to build curve
• Accurate for one type of protein
• Limitations:
  • Detergent or too much buffer can artificially increase measured protein level
  • Can lead to lower activity than expected
  • If protein eluted from affinity column by binding ligand → high concentration but low activity