MCAT Biochemistry - Nonenzymatic Protein Function and Protein Analysis

Structural Proteins

support the tissues of the body

tissues made of structural proteins

Tendons, ligaments, cartilage, basement membranes

primary structural proteins

collagen, elastin, keratin, actin, tubulin

motif

a repetitive organization of secondary structural elements; gives structural proetins a fibrous nature

Collagen

characteristic 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; found throughout the body and is important in providing strength and flexibility

osteogenesis imperfecta (brittle bone disease)

collagen cannot form its unique secondary helical structure in the abscence of glycine; replacement of glycine with other amino acids can cause improper folding of the collagen protein and cell death, leading to bone fragility

Elastin

important component of the extracellular matrix of connective tissue; primary role is to stretch and then recoil like a spring, which restores the original shape of the tissue

Keratins

intermediate filament proteins found in epithelial cells; contribute to the mechanical integrity of the cell and also function as regulatory proteins; primary protein that makes up hair and nails

Actin

protein that makes up microfilaments and the thin filaments in myofibrils, most abundant protein in eukaryotic cells, have a positive side and a negative side; polarity allows motor proteins to travel unidirectionally along an actin filament, like a one-way street

Tubulin

protein that makes up microtubules; important for providing structure, chromosome separation in mitosis and meiosis, and intracellular transport with kinesin and dynein; polarity; the negative end of a microtubule is usually located adjacent to the nucleus, whereas the positive end is usually in the periphery of a cell

Motor proteins

molecular motors that convert chemical energy into mechanical work by moving along the cytoskeleton of cells; also display enzymatic activity (ATPase); transient interactions with either actin or microtubules

ATPases

enzymes that break down ATP, esp. to do work

examples of motor proteins

motile cilia and flagella of bacteria and sperm; contraction of the sarcomere in muscle

Myosin

primary motor protein that interacts with actin; the thick filament in a myofibril; can be involved in cellular transport; each subunit has a single head and neck; movement at the neck is responsible for the power stroke of sarcomere contraction

Kinesins

play key roles in aligning chromosomes during metaphase and depolymerizing microtubules during anaphase of mitosis; two heads, one always attached to tubulin; important for vesicle transport towards positive end

ex. neurons - neurotransmitters towards synaptic terminal

dyneins

involved in the sliding movement of cilia and flagella; important for vesicle transport towards negative end

ex. neurons - vesicles of waste or recycled neurotransmitter back toward the soma

Binding proteins

have stabilizing functions in individual cells and the body; transport or sequester molecules by binding to them

examples of binding proteins

hemoglobin, calcium-binding proteins, DNA-binding proteins (transcription factors)

oxyhemoglobin dissociation curve

haemoglobin’s affinity curve for oxygen; varying affinity depending on the environmental conditions

Cell adhesion molecules (CAMs)

are proteins found on the surface of most cells and aid in binding the cell to the extracellular matrix or other cells

cadherins, integrins, selectins

Cadherins

are a group of glycoproteins that mediate calcium-dependent cell adhesion; similar cell types together (epithelial cells); type-specific (epithelial - E-cadherin; nerve - N-cadherin)

Integrins

group of proteins that all have two membrane-spanning chains called α and β; important in binding to and communicating with extracellular matrix; cellular signalling, cell division, apoptosis, white blood cell migration, stabilization of epithelium on its basement membrane, etc.

ex. integrin αIIbβ3 allows platelets to stick to fibrinogen, a clotting factor, which causes activation of platelets to stabilize the clot

Selectins

bind to carbohydrate molecules that project from other cell surfaces; weakest bonds; expressed on white blood cells and the endothelial cells that line blood vessels; host defense, inflammation, white blood cell migration, etc.

ex. cancer medication target unique CAMs that allow cancer to metastasise

Antibodies/immunoglobulins (Ig)

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 and two identical light chains held together by disulfide & noncovalent interactions

antigen-binding region

tips of the ‘Y’s of antibodies that contains pecific polypeptide sequences that will bind one, and only one, specific antigenic sequence

antigens

target of antibodies; a molecule, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor

constant region

remaining part of the antibody molecule which is involved in recruitment and binding of other cells of the immune system, such as macrophages

Antigen neutralisation

making 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

Clumping together the antigen and antibody into large insoluble protein complexes that can be phagocytized and digested by macrophages

Biosignaling

a process in which cells receive and act on signals

Ion channels

proteins that create specific pathways for charged molecules

ex. heart disease medication - calcium channel blockers; seizure medication - sodium channel blockers; IBS medication - acetylcholine receptor/cation channel blockers

Facilitated diffusion

a type of passive transport; the 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)

ungated channels

have no gates and are therefore unregulated

ex. all cells - potassium channels (net efflux of K+)

voltage-gated channels

gate is regulated by the membrane potential change near the channel

ex. neurons - sodium channels (depolarisation drives conformational change)

ligand-gated channels

the binding of a specific substance or ligand to the channel causes it to open or close

ex. neurons - chloride channel (inhibitory neurotransmitter γ-aminobutyric acid (GABA))

enzyme-linked receptors

display catalytic activity in response to ligand binding; three primary protein domains: a membrane-spanning domain, a ligand-binding domain, and a catalytic domain

ex. Receptor tyrosine kinases (RTK), serine/threonine-specific protein kinases and receptor tyrosine phosphatases

membrane-spanning domain

anchors the receptor in the cell membrane

ligand-binding domain

stimulated by the appropriate ligand and induces a conformational change that activates the catalytic domain

catalytic domain

initiates a second messenger cascade

second messenger cascade

other proteins cause further change in the cell

heterotrimeric G proteins

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

ex.

G_s stimulates adenylate cyclase, which increases levels of cAMP in the cell

G_i inhibits. adenylate cyclase, which decreases levels of cAMP in the cell

“Mind your p’s and q’s”: G_q activates phospholipase C → cleaves a phospholipid from the membrane → form PIP2 → cleaved into DAG and IP3 → IP3 opens calcium channels in the endoplasmic reticulum → increasing calcium levels in the cell

G protein-coupled receptors (GPCR)

a large family of integral membrane proteins involved in signal transduction; characterized by their seven membrane-spanning α-helices

α subunit

binds GDP and is in a complex with the β and γ subunits; can be s or i

adenylate cyclase

an enzyme that catalyzes the conversion of ATP to cAMP, a second messenger involved in signal transduction

homogenization

crushing, grinding, or blending the tissue of interest into an evenly mixed solution

Centrifugation

a mechanical process which involves the use of the centrifugal force to separate particles from a solution according to their size, shape, density, medium viscosity and rotor speed; isolate proteins from much smaller molecules

electrophoresis

subjecting compounds to an electric field, which moves them according to their net charge and size; Negatively charged compounds will migrate toward the positively charged anode, and positively charged compounds will migrate toward the negatively charged cathode; small/more charged/strong electric field molecules move faster; uses electrolytic cell

migration velocity (v)

velocity of electrophretic migration; proportional to the electric field strength, E, and to the net charge on the molecule, z, and is inversely proportional to a frictional coefficient, f

v = Ez/f

Polyacrylamide gel

standard medium for protein electrophoresis; a slightly porous matrix mixture that acts as sieve

Polyacrylamide gel electrophoresis (PAGE)

method for analyzing proteins in their native states; the functional native protein can be recovered from the gel after electrophoresis if unstained; 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

daltons (Da)

an alternative term for molar mass (g/mol)

average molar mass of one amino acid

~ 100 daltons, or 100 g/mol

Sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE)

separates proteins on the basis of relative molecular mass alone

SDS

a detergent that disrupts all noncovalent interactions; binds to proteins and creates large chains with net negative charges, thereby neutralizing the protein’s original charge and denaturing the protein

isoelectric point (pI)

the pH at which the protein or amino acid is electrically neutral, with an equal number of positive and negative charges

In isoelectric focusing, a protein stops moving when pH = pI.

zwitterion

a molecule that contains an equal number of positively and negatively charged functional groups

For most amino acids, occurs when the amino group is protonated, the carboxyl group is deprotonated, and any side chain is electrically neutral (except arginine and lysine; occurs when the amino group is deprotonated while the nitrogenous side chain is protonated)

Isoelectric focusing

separating proteins on the basis of isoelectric point (pI); 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); electric field is then generated across the gel; positively charged toward the cathode and negatively charged toward the anode, pH = pI → neutral charge → stop moving

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 (by polarity, charge, and so on), the more it will stick to and move slowly through its surroundings; preferred for large amounts

stationary phase/adsorbent.

solid medium in chromatography that does not move

mobile phase

liquid/gas medium in chromatography that moves with the sample

elute

run through the stationary phase

retention time

amount of time a compound spends in the stationary phase

partitioning

separation of the components within the stationary phase due to varying retention times

column chromatography

a column is filled with silica or alumina beads as an adsorbent, and gravity moves the solvent and compounds down the column; size and polarity; can be used to separate and collect other macromolecules besides proteins, such as nucleic acids

Ion-Exchange Chromatography

the beads in the column are coated with charged substances, so they attract or bind compounds that have an opposite charge; salt gradient is used to elute the charged molecules that have stuck to the column

Size-Exclusion Chromatography

the beads used in the column contain tiny pores that allow small compounds to enter the beads, thus slowing them down; Large compounds can’t fit into the pores, so they will move around them and travel through the column faster

Affinity Chromatography

coating beads with a receptor that binds the protein or a specific antibody to the protein; eluted by washing the column with a free receptor (or target or antibody)

ex. nickel - histidine tags; antibodies/antigens, enzyme substrate analogues

X-ray crystallography

most reliable and common method; An X-ray diffraction pattern is generated in this method, small dots in the diffraction pattern can then be interpreted to determine the protein’s structure

Edman degradation

uses cleavage enzymes to sequence proteins of up to 50 to 70 amino acids; selectively and sequentially removes the N-terminal amino acid of the protein, which can be analyzed via mass spectroscopy

nuclear magnetic resonance (NMR) spectroscopy

spectroscopic technique based on re-orientation of atomic nuclei with non-zero nuclear spins in an external magnetic field

UV spectroscopy

absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum; since proteins have aromatic side chains; used to determine concentration

bicinchoninic acid (BCA) assay

a biochemical assay for determining the total level of protein in a solution. total protein concentration is exhibited by a color change of the sample solution from blue to purple in proportion to protein concentration, which can then be measured using colorimetric techniques

Lowry reagent assay

a biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using colorimetric techniques.

Bradford protein assay

colorimetric protein assay; most common, reliable and simple