Chapter 3: Nonenzymatic Protein Function and Protein Analysis

Ion channels are classified into three main groups that have different mechanisms of opening, but all permit facilitated diffusion of charged particles:

1. Ungated channels 2. Voltage-gated channels 3. Ligand-gated channels

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

Actin

We can also customize columns to bind any protein of interest by creating a column with high affinity for that protein. This can be accomplished by coating beads with a receptor that binds the protein or a specific antibody to the protein; in either case, the protein is retained in the column. Common stationary phase molecules nickel, which is used in separation of genetically engineered proteins with histidine tags; antibodies or antigens; and enzyme substrate analogues, which mimic the natural substrate for an enzyme of interest. Once the protein is retained in the column, it can be eluted by washing the column with free receptor (or target or antibody), which will compete with the bead bound receptor and ultimately free the protein from the column. Eluents can also be created with a specific pH or salinity level that disrupts the bonds between the ligand and protein of interest. The only draw back of the elution step is that the elutent can be bound to the recovery substance. If, for example, the eluent was an inhibitor of an enzyme, it could be difficult to remove.

Affinity chromatography

__________________, also called immunoglogulins (Ig) are 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.

Antibodies

Proteins that act in transporting or sequestering molecules by bonding to them. These include hemoglobin, calcium-binding proteins, DNA binding proteins (often transcription factors), and others.

Binding proteins

_______________ is a process in which cells receive and act on signals. Proteins participate in this differently (in different capacities), including acting as extracellular ligands, transporters for facilitated diffusion, receptor proteins, and second messengers. The proteins involved in this can have functions in substrate binding or enzymatic activity.

Biosignaling

The _____________ mixes a protein in solution with coomassie brilliant blue dye. The dye is protonated and green-brown in color prior to mixing with proteins. The dye gives up protons upon binding to amino acid groups, turning blue in the process. Ionic attraction between the dye and protein then stabilize this blue form of the dye. Thus, increased protein concentrations correspond to a larger concentration of blue dye. Samples of known protein concentrations are reacted with bradford reagent and the absorbance is measured to create a standard curve. The unknown sample is then exposed to the same conditions, and the concentration is determined based on the standard curve. This is a very accurate method when only 1 type of protien is present in solution, but because of variable binding of the coomassie dye with different AA, it is less accurate when more that one protien is present. The bradford assay is limited by presence of detergent in the sample or excessive buffer.

Bradford protein assay

___________ are a group of glycoproteins that mediate calcium-dependent cell adhesion. They often hold similar cell types together, such as epithelial cells. Different cells usually have type-specific ones, for example, epithelial cells use E-cadherin while nerve cells use N-cadherin.

Cadherins

___________________ are proteins found on the surface of most cells and aid in binding the cell to the extracellular matrix or other cells. While there are a number of different types, they are all integral membrane proteins. Adhesion molecules can be classified into 3 major families: cadherins, integrins, and selectins

Cell adhesion molecules (CAM)

____________________ can then isolate proteins from much smaller molecules before other isolation techniques must be employed. The most common isolation techniques are electrophloresis and chromotagraphy, either of which can be used for native or denatured proteins.

Centrifugation

__________________ is another tool using physical and chemical properties to separate and identify compounds form a complex mixture. Chromatography refers to a variety of techniques that require the homogenized protein mixture to be fractionated through a porous mixture. One of the reasons chromatography is a valuable tool is that the isolated proteins are immediately available fro identification and quantification. In all forms of chromatography discussed here, the concept is identical: the more similar compound is to its surroundings (by polarity, charge and so on), the more it will stick to and move slowly through its surroundings. Chromatography is preferred over electrophoresis when large amounts of protein are being separated. The process beings by placing the sample onto a solid medium called the stationary phase or adsorbent. The next step is to run the mobile phase through the stationary phase. This will allow the sample to run through the stationary phase, or elute. Depending on the relative affintiy of the sample for the stationary and mobile phase, different substances will migrate through with different speeds. Components with high affinity for the stationary phase will barely migrate at all, while components with high affinity for the mobile phase will migrate much more quickly.

Chromatography

_____________ has a characteristic trihelical fiber (three left-handed helices woven together to form a secondary right-handed helix) and makes up most of the extracellular matrix of connective tissue. It is found throughout the body and is important in providing strength and flexibility.

Collagen

____________ linkages and noncovalent interactions hold the heavy and light chains of antibodies together

Disulfide

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

Elastin

A method of separating proteins, and is one of the most important analytical techniques in molecular biology. This technique works by subjecting compounds to an electric field, which moves them according to their net charge and size. Negatively charged compounds will migrate toward positively charged anodes and positively charged compounds will migrate towards the negatively charged cathode.

Electrophloresis

__________________, a type of passive transport, is the diffusion of a molecule down a concentration gradient through a pore in the membrane created by this transmembrane protein. It is used for molecules that are impermeable to the membrane (large, polar, charged). It allows integral membrane proteins to serve as channels for these substrates to avoid the hydrophobic fatty acid tails of the phospholipid bilayer.

Facilitated diffusion

_____________________ are a large family of integral membrane proteins involved in signal transduction. They are characterized by their seven membrane alpha helices. The receptors differ in specificity of the ligand binding area found on the extracellular surface of the cell. In order for GPCRs to transmit signals to an effector in the cell, they utilize a heterotrimeric G protein. G proteins are named for their intracellular link to guanine nucleotides (GDP and GTP). The binding of a ligand increases the affinity of the receptor for the G-protien. The binding of the G-protien represents a switch to the active site and affects the intracellular signaling pathway. There are several different G protiens that can result in either stimulation or inhibition of the signaling pathway.

G protein-coupled receptors (GPCR)

3 main types of G proteins:

Gs-stimulates adenylate cyclase, which increases levels of cAMP in the cell. Gi-inhibits adenylate cyclase, which decreases levels of cAMP in the cell. Gq-activates phospholipase C, which cleaves a phospholipid from the membrane to form PIP2. PIP2 is then cleaved into DAG and IP3; IP3 can open calcium channels in the ER, increasing calcium in the cell.

_______________ are a group of proteins that all have 2 membrane-spanning chains called alpha and beta. These chains are very important in binding to and communicating with the extracellular matrix. These also play a very important role in cellular signaling and can greatly impact cellular function by promoting cell division, apoptosis, or other processes. For example, integrin aIIbB3 allows platelets to stick to fibrinogen, a clotting factor, which causes activation of platelets to stabilize the clot. Other integrins are used for white blood cell migration, stabilization of epithelium on its basement membrane, and other processes.

Integrins

_____________ are proteins that create specific pathways for charged molecules.

Ion channels

In this method the beads in the column are coated with charged substances, so they attract or bind compounds that have an opposite charge. For instance, a positively charged column will attract and hold negatively charged protein as it passes through the column, either increasing its retention time or retaining it completely. After all other compounds have moved through the column, a salt gradient is used to elute the charged molecules that have stuck to the column.

Ion exchange chromatography

_______________ exploits the acidic and basic properties of amino acids by separating them on basis of isolectric 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). An electric field is then generated across the gel. Proteins that are positively charged will begin migrating toward the cathode and proteins that are negatively charged will begin migrating toward the andoe. As the protein reaches the portion of gel where the pH is equal to the protein's pI, the protein takes on a neutral charge and will stop moving. Ex. We'll start with protein of pH 9. When the protein is in an environment with pH 9 it will carry no net charge. If we place this protein on the gel at pH 7, there will be more protons around the protein, creating a net positive charge on the molecule. This charge will then cause the protein to be attracted to the negatively charged cathode, which is located in the basic side of the gradient. As the protein moved closer to the cathode, the pH of the gel slowly increases. Eventually, as the protein nears a pH of 9, the protons creating the positive charge will dissociate, and the protein will become neutral again.

Isoelectric focusing

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

Keratin

____________ and ____________ are the motor proteins associated with microtubules. They have two heads, at least one of which remains attatched to tubulin at all times. Kinesins play key roles in aligning chromosomes during metaphase and depolymerizing microtubules during anaphase of mitosis. Dyneins are involved in the sliding movementof cilia and flagella. Both proteins are important for vesicle transport in the cell, but have opposite polarities: kinesins bring vesicles towards the positive end of the microtubule, and dyneins bring vesicles toward the negative end. In neurons, we see a classic example of these motor proteins' polarities. Kinesins bring vesicles of neurotransmitter to the positive end of the axonal microtubules (toward the synaptic terminal). In contrast, dyneins bring vesicles of waste or recycled neurotransmitter back toward the negative end of the microtubule (toward the soma) through retrograde transport.

Kinesins, dyneins

For _______________, the binding of a specific substance or ligand to the channel causes it to open or close. For example, neurotransmitters act as a ligand-gated channels at the postsynaptic membrane: the inhibitory neurotransmitter GABA (v-aminobutyric acid) binds to chloride channels and opens it.

Ligand gated channels

___________ proteins also display enzymatic activity, acting as ATPases that power the conformational change necessary for motor function. Motor proteins have transient interactions with either actin or microtubules

Motor

___________ is the primary motor protein that interacts with actin. In addition to its role as the thick filament in a myofibril, it can be involved in cellular transport. Each myosin subunit has a single head and neck; movement at the neck is responsible for the power stroke of sarcomere contraction.

Myosin

___________________ is the standard medium for protein electrophoresis. The gel is a slightly porous matrix mixture, which solidifies at room temperature. Proteins travel through this matrix in relation to their size and charge. The gel acts like a sieve, allowing smaller particles to pass through easily while retaining large particles. Therefore, a molecule will move faster through the medium if it is small, highly charged, or placed in a large electric field. Conversely, molecules will migrate slower, or not at all when they are bigger and more convoluted, electrically neutral, or placed in a small electric field. The size of a standard polyacrilimide gel allows multiple samples to run simultaneously.

Polyacrylamide gel

___________________ is a method for analyzing proteins in their native states. Unfortunately, it is limited by varying mass-to charge and mass-to-size ratios of cellular proteins because multiple different proteins may experience the same level of migration. In PAGE, the functional native protein can be recovered from the gel after electrophoresis, but only if the gel has not been stained because most stains denature proteins. PAGE is most useful to compare the molecular size or the charge of proteins known to be similar in size from other analytic methods like SDS-PAGE or size exclusion chromatography.

Polyacrylamide gel electrophoresis (PAGE)

____________________________-polyacrylamide gel electrophoresis is a useful tool because it separates proteins on the basis of relative molecular mass alone. The SDS-Page technique starts with the premise of PAGE but adds SDS as a detergent that disrupts all noncovalent interactions. It binds to proteins and created large chains with negative charges, thereby neutralizing the protein's original charge and denaturing the protein. As the proteins move through the gel, the only variables affecting their velocity are E, the electric field strength, and f, the frictional coefficient, which depends on mass. After seperation, the gel can be stained so the protein bands can be visualized and results recorded.

Sodium dodecyl sulfate (SDS)

_________ is the protein that makes up microtubules. Microtubules are important for providing structure, chromosome seperation in mitosis and meiosis, and intracellular transport with kinesin and dynein. Like actin, this protein has polarity: the negative end of a microtubule is usually located adjacent to the nucleus, whereas the positive end is usually in the periphery of the cell.

Tubulin

______________ have no gates and are therefore unregulated. For example, all cells possess ungated potassium channels. This means there will be a net efflux of potassium ions through these channels unless potassium is at equilibrium.

Ungated channels

Antibodies are ______ -shaped proteins that are made up of two identical heavy and 2 identical light chains.

Y

The three subunits that comprise the G protien are

a, B, and v

Protein _______________ is generally determined by monitoring a known reaction with a given concentration of substrate and comparing it to a standard. Activity is correlated with concentration but is also affected by purification methods used and the condition of the assay. Reactions with a color change have particular applicability because microarrays can rapidly identify the samples from a chromatographic analysis that contains the compound of interest.

activity

Each binding protein has an ____________ curve for it's molecule of interest; the oxyhemoglobin dissociation curve is one well-known example. This curve differs depending on the goal of the binding protein. When sequestering of a molecule is the goal, the binding protein usually has high affinity for its targets across a large range of concentrations so it can keep it bound at nearly 100 percent. A transport protein, which must be able to bind or unbind its target to maintain steady-state concentrations, is likely to have varying affinity depending on the environmental conditions.

affinity

The most prominent type of protein found in the immune system

antibody

Each antibody has an _____________ region at the tips of the Y. Within this region, there are specific polypeptide sequences that will bind one, and only one specific antigen sequence. The remaining part of the antibody molecule is known as the constant region, which is involved in recruitment and binding of other cells of the immune system, such as macrophages. Thus when the antibodies bind to their targets, called antigens, they can cause one of three outcomes. : 1. Neutralizing the antigen, making the pathogen or toxin unable to exert its effect on the body 2. Marking the pathogen for destruction by other white blood cells immediately, this marketing function is also called opsonization 3. Clumping together (agglutinating) the antigen and antibody into large insoluble protein complexes that can be phagocytized and digested by macrophages.

antigen-binding regions

Concentration is determined almost exclusively through spectroscopy. Because proteins contain aromatic side chains, they can be analyzed with UV spectroscopy without any treatment, however, this method is particularly sensitive to sample contaminants. Proteins also cause colorimetric changes with specific reactions, particularly the _______________________.

bicinchoninic acid (BCA) assay, Lowry reagent assay, and Bradford protein assay. The bradford method is most common because of its reliability and simplicity in the basic analyses.

In chromatography, for protein seperation, common properties include

charge, pore size, and specific affinities

Main bodily structural proteins:

collagen, elastin, keratins, actin, tubulin

In ______________________ chromatography, a column is filled with silica or alumina beads as an adsorbent, and gravity moves the solvent and compounds down the column. Both size and polarity have a role in determining how quickly proteins move through the polar silica or alumina beads: the less polar the compound, the faster it can elute through the column (short retention time). In column chromatography, the solvent polarity, pH, or salinity can easily be changed to help elute the protein of interest. Eventually the solvent will elute out of the end of the column, and different fractions that leave the column are collected over time. Each fraction contains bands that correspond to different compounds. After collection, the solvent can be evaporated and the compounds of interest kept. Column chrom is particularly useful in bichem because it can be used to separate and collect other macromolecules such as nucleic acids.

column

Membrane receptors may also display catalytic activity in response to ligand binding. These ____________ have three primary protein domains: a membrane spanning domain, a ligand-binding domain, and a catalytic domain. The membrane spanning domain anchors receptors in the cell membrane. The ligand stimulating domain is stimulated by appropriate ligand and induces a conformational change that activates the catalytic domain. This often results in the initiation of a second messenger cascade. Receptor Tyrosine Kinases (RTK) are classic examples. RTKs are composed of a monomer that dimerized upon ligand binding. The dimer is the active form that phosphorylates additional cellular enzymes, including the receptor itself (autophosphorylation). Other classes of enzyme-linked receptors include serine/threonine-specific protein kinases and receptor tyrosine phosphotases.

enzyme-linked receptors

Regularity gives structural proteins a __________ nature.

fibrous

Proteins and other biomolecules are isolated from body tissues or cell cultures by cell lysis and _______________-crushing, grinding, or blending the tissue of interest into an evenly mixed solution.

homogenization

In its inactive form, the a subunit binds GDP and is in complex with B and v subunits. When a ligand binds to the GPCR, the receptor becomes activated and, in turn, engages the corresponding G protein. Once GDP is replaced with GTP, the a subunit is able to dissociate from the B and v subunits. The activated a subunit alters the activity of adenylate cyclase. If the a subunit is as, then the enzyme is activated; if the a subunit is a1, then the enzyme is inhibited. Once GTP on the activated a subunit is dephosphorylated to GDP, the a subunit will rebind to the B and V subunits, rendering the G protein _____________.

inactive

Proteins can be separated on the basis of their _____________________. The pI is the pH at which the protein or amino acid is electrically neutral, with an equal number of positive and negative charges. For individual amino acids this electrically neutral form is called zwitterion, in which amino group is protonated, the carboxyl group is deprotanated and the side chain is electrically neutral. For polypeptides the isoelectric point is primarily determiend by relative numbers of acidic and basic compounds.

isoelectric point (pI)

For __________ proteins, digestion with chymotrypsin, trypsin, and cyanogen bromide, a synthetic reagent, may be used. This digestion selectively cleaves proteins at specific amino acid residues, creating smaller fragments, that can be analyzed by electrohphloresis or the Edman degradation. Because disulfide links and salt bridges are broken to reduce the protein to its primary structure, their proteins can not be determined by these methods.

larger

Varying retention times of each compound in solution results in separation of the components within the stationary phase, or ____________________. Each component can then be isolated individually for the study.

partitioning

the amount of time a compound spends in the stationary phase

retention time

Structural proteins generally have highly repetitive _____________ structure and a super-secondary structure- a repetitive organization of secondary structural elements together sometimes refered to as motif.

secondary

_____________ are unique because they bind to carbohydrate molecules that project from other cell surfaces. These bonds are the weakest formed by CAMs discussed here. Selectins are expressed on white blood cells and the endothelial cells that line blood vessels. Like integrins, they play an important role in host defense, including inflammation and white blood cell migration.

selectins

The amino acids that compose a protein can be determined by complete protein hydrolysis and subsequent chromatographic analysis. However, the random nature of hydrolysis prevents amino acid ______________. To determine the primary structure of a protein, sequential digestion of the protein with specific cleavages enzymes is used. Small proteins are best analyzed with Edman degradation, which uses cleavage to sequence proteins of 50-70 AA. The edman degradation selectively and sequentially removes the N-terminal amino acid of the protein, which can be analyzed via mass spectroscopy.

sequencing

In this method the beads used in the column contain tiny pores of varying sizes. These tiny pores 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. It is important to remember that in this type of chromatography, the small compounds are slowed down and retained longer- which may be counterintuitive. The size of the pores may be varied so that molecules of different molecular weights can be fractionated. A common approach in protein purification is to use an ion-exchange column followed by a size-exclusion column.

size-exclusion chromatography

Some ___________ proteins also have motor functions in the presence of motor proteins. The motile cilia and flagella of bacteria and sperm are prime examples, as is contraction of the sarcomere in muscle.

structural

Protien ____________ can be determined through X-Ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Before crystallographic analysis, the protein must be isolated and crystallized. X-ray crystalogrpahy is the most reliable and common method; 75 percent of the protein structures known today were analyzed through this method (25% by NMR). Crystalography measures electron density on an extremely high-resolution scale and can be used for nucleic acids. An X-ray diffraction pattern is generated in this method. The small dots in the diffraction pattern can be interpreted to determine the proteins structure.

structure

Purpose of cells and proteins of the immune system:

to rid body of foreign invaders

The Km and Vmax parameters that apply to enzymes are also applicable to _______________ such as ion channels. The kinetics of transport can be derived from the Michaelis-Menten and Lineweaver-Burk equations, where Km refers to the solute concentration at which the transporter is functioning at half of its maximum activity.

transporters

In _____________________, the gate is regulated by the membrane potential change near the channel. For example, many excitable cells such as neurons possess VG sodium channels. The channels are closed under resting conditions, but membrane depolarization causes a protein conformation change that allows them to quickly open and then quickly close as the voltage increases. Voltage-gated nonspecific sodium-potassium channels are found in cells of the sinoatrial node of the heart. Here, they serve as a pacemaker current; as voltage drops, these channels open to bring the cell back to threshold and fire another action potential.

voltage-gated channels