Biology and Biochemistry: Non-enzymatic Protein Function and Protein Analysis

Structural Proteins

Compose the cytoskeleton, anchoring proteins, and much of the extracellular matrix

Most common structural proteins

Collagen, elastin, keratin, actin, and tubulin

Structural proteins are generally what texture?

Fibrous in nature

Motor Proteins

Have one or more heads capable of force generation through a conformational change

• They have catalytic activity, acting as ATPases to power movement

Most Common Applications of Motor Proteins

Muscle contraction, vesicle movement within cells, and cell motility

Most Common Motor Proteins

Myosin, Kinesin, and Dynein

Binding Proteins

Bind a specific substrate, either to sequester it in the body or holds its concentration at a steady state

Cell Adhesion Molecules (CAM)

Allows cells to bind to other cells or surfaces

Cadherins

Calcium-dependent glycoproteins that hold similar cells together

Integrins

Have two membrane-spanning chains and permit cells to adhere to proteins in the extracellular matrix. Some also have signaling capabilities.

Selectins

Allow cells to adhere to carbohydrates on the surface of other cells and are most commonly used in the immune system

Types of CAMs

Cadherins, Intergrins, Selectins

Antibodies (Immunoglobulins, Ig)

Used by the immune system to target a specific antigen

• Contain a constant region and a variable region; the variable region is responsible for antigen binding

• Two identical heavy chains and two identical light chains form a single antibody; they are held together by disulfide linkages and noncovalent interactions

Antigen

Protein or toxin on the surface of a pathogen

Ion Channels

Can be used for regulating ion flow into or out of a cell

How many types of ion channels are there?

3

What are the three types of ion channels?

Ungated channels, voltage-gated channels, and ligand-gated channels

Ungated channels

Always open

Voltage-gated Channels

Open within a range of membrane potentials

Ligand-Gated Channels

Open in the presence of a specific binding substance, usually a hormone or neurotransmitter

Enzyme-linked Receptors

Participate in cell signaling through extracellular ligand binding and initiation of second messenger cascades

G protein-coupled receptors

Have a membrane-bound protein associated with a trimeric G protein. They also initiate second messenger systems

Ligand binding engages…

G protein

• GDP is replaced with GTP; a alpha subunit dissociates from the beta and Rho subunits

• The activated alpha subunit alters the activity of adenylate cyclase or phospholipase C

• GTP is dephosphorylated to GDP; the alpha subunit rebinds to the beta and Rho subunits

After activation from the replacement of GDP with GTP, the alpha subunit alters th activity of…

Adenylate cyclase or phosphate lipase C

Adenylate cyclase

A membrane-bound enzyme that catalyzes the conversion of ATP to cAMP (cyclic AMP)

phosphate lipase C

An enzyme that cleaves phospholipids, specifically phosphatidylinositol 4,5-bisphosphate (PIP2), in the plasma membrane, producing two important signaling molecules: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). 

Electrophoresis

Uses a gel matrix to observe the migration of proteins in response to an electric field

Native PAGE

Type of electrophoresis that maintains a protein’s shape, but results are difficult to compare because the mass-to-charge ratio differs for each protein

SDS-PAGE

Type of electrophoresis denatures the proteins and masks the native charge so that comparison of size is more accurate, but the functional protein cannot be recaptured from the gel.

Isoelectric focusing

Type of electrophoresis that separates proteins by their isoelectric point (pI); the protein migrates toward an electrode until it reaches a region of the gel where pH=pI of the protein

Chromatography

Separates protein mixtures on the basis of their affinity for a stationary phase or a mobile phase

Column Chromatography

Uses beads of a polar compound, like silica or alumina (stationary phase), with a nonpolar solvent (mobile phase)

Ion-exchange Chromotography

Uses a charged column and a variably saline eluent

Size-exclusion chromotography

Relies on porous beads. Larger molecules elute first because they are not trapped in the small pores

Affinity Chromatography

Uses a bound receptor or ligand and and eluent with free ligand or receptor for the protein of interest

X-ray crystallography

Process that determines the structure of a protein after the protein is isolated, although NMR can also be used

Amino acid composition can be determined by…

Simple hydrolysis

Amino acid sequencing determination requires…

sequential degradation, such as Edman degradation

Edman Degradation

A chemical reaction that is executed in a series of steps and removes N-terminal amino acids in a sequential manner from a peptide or protein

Activity levels for enzymatic samples are determined by…

Following the process of a known reaction, often accompanied by a color change

Protein concentration is determined by…

• Colorimetrics, either by UV spec or through color change reaction

• Ex: BCA assay, Lowry reagent assay, Bradford protein assay

• Bradford protein assay is most common

BCA Assay

A colorimetric method used to quantify protein concentrations in a sample. It relies on the reaction of proteins with Bicinchoninic Acid to form a stable, purple-colored complex, which can be measured spectrophotometrically. 

• Advantages: Compatibility with detergents, Protein-to-protein uniformity, Sensitivity and linear range, and Stability of reagents

• Disadvantages: Interference from non-protein components, Incubation time, Cost and complexity, and Specificity issues

Lowry Reagent Assay

A colorimetric method used to quantify protein concentrations, particularly in biological samples. It's based on the biuret reaction, where copper ions react with peptide bonds in alkaline conditions, followed by a second reaction with the Folin-Ciocalteu reagent to produce a blue-green color. This color is then measured spectrophotometrically

Bradford Protein Assay

A simple and widely used method for quantifying protein concentration in samples. It involves a colorimetric reaction where the Coomassie Brilliant Blue G-250 dye binds to protein, causing a brown-green to blue color change with shift in absorbance that can be measured spectrophotometrically. 

• Advantages: Simplicity and Speed, Sensitivity, and Compatibility

• Disadvantages: Interference, Specificity, Linearity Limitations, pH Sensitivity, and Protein-to-Protein Variation