proteins: structure, function, and analytical techniques

Enzymes

Catalyze covalent bond breakage or formation.

Structural Proteins

Provides mechanical support to cells and tissue.

Transporting Proteins

Carry small molecules or ions.

Motor Proteins

Generate movement in cells and tissue.

Storage Proteins

Store amino acids or ions.

Signal Proteins

Carry extracellular signals from cell to cell.

Receptor Protein

Detect signals and transmit them to the cells response machinery.

Transcription Regulator

Bind to DNA to switch genes on or off.

Special-Purpose Proteins

Highly variable.

Peptide Bonds

Link amino acids by a condensation (dehydration) reaction.

Polypeptide Backbone

Formed by a chain of amino acids joined by peptide bonds.

Amino Acid Sequence

Specifies the shape of a protein.

Hydrophobic Effect

Drives folding by burying nonpolar side chains in the protein's interior.

Secondary Structures of Proteins

Include α Helix and β Sheet.

α Helix

Polypeptide folds into a coiled, repeating structure.

Hydrogen Bonds

Form between the N-H of one peptide bond and the C=O of a bond four residues away in α Helix.

Nonpolar Side Chains

Collapse inward to form a tightly packed hydrophobic core.

Polar/Charged Side Chains

Remain on the surface, where they hydrogen-bond or ionically interact with water.

Van der Waals Attractions

Close-packing contacts among nonpolar side chains.

Electrostatic (Ionic) Attractions

Form salt bridges between oppositely charged side chains.

Amino Acids

Proteins use 20 standard amino acids.

Charged Amino Acids

Divided into negative (acidic) and positive (basic) at ~pH 7.

β sheet

strands aligned, H-bonds between strands, side chains alternate, arrows in diagrams.

Serine proteases

enzymes with serine in active site, cleave peptide bonds.

Dimer

2 subunits, identical binding sites.

Tetramer

4 subunits, combination of binding sites forms ring.

Actin filaments

Composed of identical actin subunits in a helical array, can extend micrometers in length.

Tubulin

Forms hollow microtubules.

Viral capsids

Many copies of a small set of protein subunits form spherical shells enclosing viral DNA/RNA (e.g., SV40 virus).

Disulfide bonds

Covalent disulfide bonds form between cysteine -SH groups, significantly stabilizing the protein's folded structure.

Protein-Ligand Binding

Proteins bind tightly and selectively to specific molecules (ligands) through many weak, noncovalent interactions.

Binding Site Structure

Folding of the polypeptide chain forms a crevice or cavity on the protein surface.

Cyclic AMP binding

Involves hydrogen bonds and electrostatic interactions.

Enzyme-substrate complex

Formed when substrate binds to the enzyme's active site.

Enzyme-product complex

Formed when a covalent bond is made or broken.

Vmax

Maximum reaction rate when all enzyme active sites are fully occupied.

KM (Michaelis constant)

Substrate concentration at half-maximal velocity.

Michaelis-Menten Kinetics

Equation: v=Vmax[S]/(KM+[S]).

Reaction Rate

Enzyme reaction rate (v) increases with substrate concentration [S] until Vmax is reached.

Equation for Michaelis-Menten Kinetics

v=Vmax[S]/(KM+[S])

Hyperbolic Curve

Plotting v vs [S] gives a hyperbolic curve approaching Vmax.

Lineweaver-Burk Plot

Double-reciprocal plot of 1/v vs 1/[S] results in a straight line.

Lineweaver-Burk Equation

1/v=(KM/Vmax)(1/[S])+1/Vmax

Y-intercept in Lineweaver-Burk Plot

1/Vmax

X-intercept in Lineweaver-Burk Plot

-1/KM

Competitive Inhibition

Inhibitor binds the active site, blocking substrate; effect can be overcome by increasing [S].

Effect of Competitive Inhibition on Vmax and KM

Vmax is unchanged, but KM appears larger (weaker apparent binding).

Function of Enzymes

Enzymes bind to substrates and chemically alter them.

Lysozyme Mechanism

Enzyme action includes substrate binding, bond distortion, transition state formation, and bond cleavage.

Retinal

Light-sensitive molecule covalently attached to rhodopsin in the eye; essential for vision.

Heme

Contains a carbon ring with central iron; noncovalently bound to each polypeptide chain in hemoglobin; essential for oxygen transport.

Feedback Inhibition

A form of negative regulation in biosynthetic pathways.

Mechanism of Feedback Inhibition

When end product Z accumulates, it inhibits the first enzyme of its own synthesis.

Protein Phosphorylation

Covalent addition of a phosphate group to a protein to regulate its activity.

Mechanism of Protein Phosphorylation

Protein kinase transfers a phosphate from ATP to an amino acid side chain; protein phosphatase removes the phosphate.

Effect of Phosphorylation

Phosphorylation can increase or decrease protein activity, depending on the site and protein structure.

Multi-Site Protein Modification

Proteins can be covalently modified at multiple sites, regulating their activity, interactions, and degradation.

Example of Multi-Site Modification

p53, a key transcription regulator in response to cellular damage, has distinct domains and modifications.

GTP-Binding Proteins

Act as molecular switches; activation requires GTP binding.

Inactivation of GTP-Binding Proteins

Protein hydrolyzes GTP to GDP + Pi, switching to an inactive conformation.

Reactivation of GTP-Binding Proteins

GDP must dissociate, and GEFs accelerate GDP release for GTP binding.

Breaking Open Cells

Purpose is to release cell contents for protein purification.

Methods of Homogenization

Includes high-frequency sound, mild detergent, and high pressure to break cells.

Shearing force

Cells are sheared between a rotating plunger and vessel walls.

Homogenate

A mixture containing cytosolic molecules and intact organelles.

Centrifugation

Separates components by size and density using centrifugal force.

Pellet

Larger, denser components at the bottom of the centrifuge.

Supernatant

Smaller, lighter components remaining above the pellet.

Fixed-angle rotor

Holds tubes at a fixed angle during centrifugation.

Swinging-arm rotor

Tubes swing outward for even separation during centrifugation.

Centrifuge speeds

Can reach up to 100,000 rpm (~600,000× g).

Differential Centrifugation

Sequential centrifugation steps at increasing speeds.

Low-speed centrifugation

Pellets whole cells, nuclei, cytoskeletons (Pellet 1).

Medium-speed centrifugation

Pellets mitochondria, lysosomes, peroxisomes (Pellet 2).

High-speed centrifugation

Pellets endoplasmic reticulum fragments, vesicles (Pellet 3).

Very high-speed centrifugation

Pellets ribosomes, viruses, macromolecules (Pellet 4).

Velocity Sedimentation

Separation by sedimentation rate using a sucrose gradient.

Fast-sedimenting particles

Form lower bands during velocity sedimentation.

Slow-sedimenting particles

Form higher bands during velocity sedimentation.

Protein Separation Basics

Proteins differ in size, shape, charge, hydrophobicity, and binding affinity.

Column Chromatography

Proteins pass through a column filled with a solid matrix while a solvent flows through.

Ion-Exchange Chromatography

Matrix carries charged beads; proteins with the opposite charge stick to the beads.

Gel-Filtration Chromatography

Matrix contains porous beads; small proteins enter the pores and are delayed.

Affinity Chromatography

Matrix has specific ligands that bind to the protein of interest.

Gel Electrophoresis (SDS-PAGE)

Proteins move in an electric field through a gel at speeds depending on size and charge.

Sodium Dodecyl Sulfate (SDS)

Binds to proteins, gives uniform negative charge, and denatures proteins.

Isoelectric Focusing

Proteins migrate in a pH gradient until they reach their isoelectric point (pI).

Two-Dimensional Gel Electrophoresis

Combines isoelectric focusing and SDS-PAGE to separate proteins by pI and size.

Mass Spectrometry

Measures mass-to-charge ratios (m/z) of peptides to identify proteins.