Kaplan Biochemistry Chapter 3 : Nonenzymatic Protein Function and Protein Analysis

Motif

Repetitive organization of secondary structural elements together

Structural Proteins

Proteins that take a role in intracellular support and extracellular matrices that support the body : Tendons, Ligaments, Cartilage and Basement Memebranes

Collagen

Elastin

Keratins

Actin

Tubulin

Collagen

Characteristic tri-helical fiber that makes up most of the extracellular matrix of connective tissue

Provides strength and flexibility of tissues

Elastin

Component of the extracellular matrix or connective tissue, that strecthces and recoils retaining the original shape of the tissue

Keratins

Intermediate filament proteins found in epithelial cells

Contribute to the mechanical integrity of the cell

Also function as regulatory proteins

Actin

Protein that makes up microfilaments and thin filaments in myofibrils

Most abundant protein in eukaryotic cells

Have positive and negative ends allowing motor proteins to travel unidirectionally along an actin filament

Tubulin

Protein that makes up microtubules

Important to providing structure, chromosome separation (mitosis & meiosis) and intracellular transport

Motor Proteins

Proteins hydrolyze ATP to interact with cytoskeletal elements and other cell components producing movement of the whole cell or parts of the cell.

ATPases

Hydrolyze ATP, power the conformational change necessary for motor functions

Myosin

Primary motor protein that interacts with actin

Thick Filament in myofibrils

Single Head and neck, movment at the neck responsible for power stroke of sarcomere contraction

Kinesins & Dynein

Motor protein associated with microtubules

Have two heads - one of which is always attached to the

Important for vesicular transport (often seen in neurotranmitter transport in axons)

Kinesins - Bring vesicles toward the positive end of the microtubule - synaptic terminus

Dynein - Bring vesicles toward the negative end of the microtubule - cell body

Binding Proteins

Proteins that bind to the molecule of interest allowing for the sequestration or transport of the bound substrate

Each protien has specific affinity for each molecule

Act to stabilize cells environment, Hold concentration of substrate to steady level (ex: hemoglobin, DNA binding proteins)

Cell Adhesion Molecules - CAMs

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

Cadherins

Integrins

Selectins

Cadherins

Groups of glycoproteins that mediate calcium-dependent cell adhesion

Often hold similar types of cells together (ex. Epithelial cells)

Differ for each type of cell bound

Integrins

Groups of protein that have 2 membrane spanning chains (Alpha and Beta)

Important in binding to and communicating with the extracellular matrix

Play important role in cellular signaling, and can promote cell division, apoptosis, or other processes

Selectins

Group of proteins that bind to carbohydrate molecules that project from other cell surfaces

Weakest of the CAMs

Expressed in white blood cells and endothelial cells that line the blood vessels

Important role in host defense, including inflammation, and white blood cell migration

Antibodies / Immunoglobulins - Ig

Y-shaped proteins produced by B-cells that function to neutralize targets in the body such as toxins and bacteria

Also recruit other cells to help eliminate the threats

Composed of 2 identical heavy chains and 2 identical light chains

Antigen

A substrate that, when introduced into the blood, triggers the production of an antibody

Antigen Binding Region

Region found at the y-tips of the antibody that binds to the substrate

Will bind to one and only one specific polypeptide sequence

The remaining part of the antibody, Constant Region, that recruits and bind cells

Antibody Defense Mechanisms

Neutralization

- the antibodies neutralize the pathogen/toxin through binding it making it unable to exert its effect on the body

Opsonization

- Marks the pathogen for destruction by other white blood cells immediately

Agglutinating

- Clumping together forming insoluble protein/antigen complex that will be digested by macrophages.

Biosignaling

Process in which cells receive and act on signals

Proteins participate in several different ways through : extracellular matrix ligands, trnasporters, receptor proteins, and second messengers

Ion Channels

Proteins that create specific pathways for charged molecules to pass through the membrane

Several types of channels, all allow for facilitated diffusion

Facilitated Diffusion

Type of passive transport that allows for diffusion of molecule down a concentration gradient

Ungated Channels

Channels that have no gate and therefore are unregulated

Potassium channels

Voltage-Gated Channels

Channels that are regulated by the membrane potential changes near the channel

Closed under resting conditions but open when membrane depolarization occurs

Ex : Action potential in nerves

Ligand-Gated Ion Channels

Channels that require the binding of a specific substance/ligand to open or close

Example : Neurotransmitters

Km and Vmax is applicable to ion channels as well

Km refers to solute concentration where channel is funcitoning at half of its activity

Enzyme Linked Receptors

Membrane receptors can also have catalytic activity in response to ligand binding

Composed of 3 Protein domains:

Membrane-Spanning Domain

Ligand-Binding Domain

Catalytic Domain

Membrane-Spanning Domain

Protein domain in enzyme-linked receptors, that anchors the receptors in the cell membrane

Ligand Binding Domain

Protein domain in enzyme-linked receptors, that is stimulated by the binding of the appropriate ligand

Induces conformational change that activates the catalytic domain

Catalytic Domain

Protein domain in enzyme-linked receptors, activated by a conformational change induced by the ligand-binding domain, causes the initiation of a second messenger cascade

Second Messenger Cascade

Cascade that is activated by the catalytic domain that typically causes a cellular response

Example: receptor tyrosine kinases will dimerize when the ligand binds and phosphorylate many molecules, including itself (autophosphorylation)

G-Protein Coupled Receptors -GPCR

Large family of integral proteins involved in signal transduction

Have 7 membrane spanning alpha helices

Each differs in their specificity of the ligand binding area

All utilize a heterotrimeric G protein structure

G-Protein pathways

3 main types of G-Proteins:

- Gs stimulates adenylate cyclase increasing cAMP

- Gi inhibits adenylate cyclase decreasing cAMP

- Gq activates phospholipase C, which elevates phospholipid form the mambrane to form PIP2 and PIP3 - increasing intracellular calcium

Gs - stimulate cAMP

Gi - inhibit cAMP

Gq - increases calcium

Heterotrimeric G Protein

Protein complexes composed of three subunits, usually alpha, beta, and gamma. Activated by G-protein coupled receptors. Gs, which is a stimulator of signal transduction, and Gi, which inhibits signal transduction. Gq, activates phospholipase C

Alpha Subunit

Subunit that binds GDP in complex with Beta and Gamma subunits

Once ligand binds Alpha subunit is bound to GTP resulting in active protein

Alpha subunit then dissociates from Beta/Gamma units to activate adenylate cyclase

Beta Subunit

Where alpha subunit of a G protein will normally be bound to when inactivated

Gamma Subunit

Adenylate Cyclase

Activated or inhibited by the alpha subunit

Activated by a G-protein. Converts ATP to cyclic AMP in response to an extracellular signal.

Protein Isolation

Allows for better understanding of a protein

Homogenization

Crushing, grinding, blending tissue into an evenly distributed mixture/solution

Centrifugation

Allows for the isolation of proteins from smaller molecules prior to further isolation

Electrophoresis

Method of separating proteins by subjecting proteins to an electric field moving proteins according to net charge and size

Negatively charged proteins will move towards the positive anode

Allows to determine the velocity of migration, separating proteins

Migration Velocity

The velocity of migration is dependent on electric field strength - E

, charge on the protein- z , and friction (affected by the size/shape of the molecule ) - f

Polyacrylamide Gel

Standard medium for protein electrophoresis,

Is slightly porous matrix that allows smaller molecules to pass faster and causing larger/ convolutedly shaped molecules to pass slower

Polyacrylamide Gel Electrophoresis - Native PAGE

Method of electrophoresis that analyzes proteins in their native state

Limited to separation based on MASS-to-CHARGE and MASS-to-Size ratios

Many of these proteins experience the same level of migration

Sodium Dodecyl Sulfate - SDS PAGE

Method of electrophoresis that is useful as it separates molecules based on MASS alone

Starts with page but adds SDS - which disrupts the noncovalent interactions of the protein - denaturing the protein

Electric field strength and Friction are the only variables in SDS PAGE

Isoelectric Point

The pH at which a protein will not have any net charge

For individual amino acids, is the zwitterion

Isoelectric Focusing

Separation of a protein that exploits the acidic and basic properties of amino acids by separation of a protein based on isoelectric point

Proteins are placed in a gel with pH gradient

Electric field is generated and the (acidic gel at the positive anode and basic gel at the negative cathode.)

Proteins with a positive charge will migrate towards the cathode

Proteins with a negative charge will migrate towards the anode

Chromatography

Separation technique that uses physical and chemical properties of proteins

Several techniques - often require homogenized mixture to be fractionated through porous matrix

Stationary Phase / Adsorbent

Solid medium in which the sample is placed onto

Elute

Extraction of protein run the mobile phase through the stationary phase

Depending on afffinty of sample, different substances will migrate at different rates

Retention Time

Amount of time a compound spends in the stationary phase

Partitioning

Varying retention times of each compound in the solution results in the separation

Results in the separation of components within the stationary phase

Column Chromatography

Column is filled with polarized silica or alumina beads for the adsorbent

Gravity then moves solvent and compounds down the column

Size and polarity play a role in the retention time of the eluent

Less polar compounds will pass through faster

Solvent polarity, pH, or Salinity can be changed to help elute protein

Differing fraction of collected eluent are then evaporated and solvent of interest is isolated

Ion-Exchange Chromatography

Beads are coated with charged substances so they bind or attract or bind compounds that have an opposite charge

Separation depends on the charge of the column

After solvent has passed a salt gradient will be used to elute bound molecules

Size-Exclusion Chromatography

Beads are used in tiny pores of varying sizes, pores are varied so that different molecular weights can be fractionated.

Small compounds will enter the beads thus slowing them down

Large compounds do not fit and will thus travel through the column faster

Affinity Chromatography

Columns can be customized to bind any protein of interest by creating column with high affinity for specific protein

Beads coated with receptor or antibody that binds specific protein

Wash column with free receptor or pH/Salt to separate protein from beads

Protein Structure

Important to know the structure of protein to determine interactions of that protein

The function of a protein depends on the protein's 3D shape

Largely studied through X-ray Crystallography 75% and Nuclear Magnetic Resonance - NMR Spectroscopy 25%

X-ray Crystallography

Most reliable method of protein structure analysis measures electron density on extremely high-resolution scale

75 % of total protein analysis

Amino Acid Composition

Requires the complete hydrolysis of peptide bonds in a protein to separate amino acids into individual units

If not in specific order, sequence cannot be determined

Must be done via Edman Degradation to determine sequence

Edman Degradation

Uses cleavage to sequence proteins of to 50-70 amino acids

Selectively and sequentially removes the N-terminal amino acids

to be analyzed via mass spectroscopy

Larger Proteins

For larger proteins digest with chymotrypsin , tripsin, cyanogen bromide to break into smaller sequences and follow with Edman degradation

Activity Analysis

Typically determined by monitoring a known reaction with given concentration of substrate

Activity correlated with concentration but affected by purification methods

Reaction with color change are applicable to microarrays that allow for chromatographic analysis of compound of interest

Concentration Determination

Determined exclusively through spectroscopy

UV sepctrocopy is not used as it is sensitive to sample contaminants

Largely through these methods : Bicinchoninic Acid Assay - BCA, Lowry Reagent Assay, Bradford Protein Assay

Bradford Protein Assay is the most common due to reliability and simplicity

Bradford Protein Assay

Mixes protein in solution with Coomassie Brilliant Blue Dye

In protonated form the dye is originally brownish-green

When dye give protons to ionizable groups in the protein it becomes blue

Increased protein concentrations correlate to large concentration of blue color

Is less accurate when more than 1 protein is present