Nonenzymatic Protein Function and Protein Analysis Flashcards
Typical Protein Functions
- supporting intracellular func and aidng w/ organization
- acting as enzymes
3.
Intracellular Protein Functions
- Adhesion Molecules
- Immunoglobins
- Structural molecules
- Motor molecules
- Binding Proteins
Structural Proteins
Proteins in the intracellular or extracellular matrices regions such as actin, tubulin, elastin, collagen and keratin that contribute to constitution of ligaments, tendons, cartilage and basement membranes.
***They have repetitive secondary structures referred to as Motif that give them a fibrous appearance.
All Primary Structural Protein Types
- Collagen
- Keratin
- Elastin
- Actin
- Tubulin
***They give rise to motifs or repetitive secondary structural proteins
Collagen
A primary structural protein that consists of 3 l-handed helices giving rise to a secondary right-handed helix. It constitutes the major extracellular matrices of connective tissues and is considered a fibrous tissue that gives the body strength and flexibility.
Elastin
A primary structural protein that like collagen, constitutes the extracellular matrix of connective tissue. It allows the skin to stretch and recoil.
Keratins
Intermediate filament protein mainly found in the epithelial tissues that gives rise to the hair, nail and skin.
***contributes to mechanical integrity of cell in addition to serving as a regulator. (for what??)
Actin
Primary structural protein that gives rise to the microfilaments and thin filaments of the myofibrils. It is polar and its polarity allows for unidirectional movement of motor proteins along its borders.
Is the most abundant protein in the body
Tubulins
Primary structural proteins that give rise to microtubules. They are polar in nature like actins and specifically have their negative end adjacent to the their nuclei while having their positive ends somewhere in the periphery of the cell.
Microtubules
Cytoskeletal proteins that
- help with chromosal separation in meiosis and mitosis
- help with intracellular transport with dyesin and kinesin
- provide structure and support for the cell’s internal structure
Motor Proteins
One of the nonenzymatic protein types that can contribute to motor function by
- providing enzymatic activity; ex. ATpase
- providing structural support
- providing motor activity: ex. flagella, celia
Main motor proteins:
- Myosin
- Kinesin
- Dyenin
ATpase
Enzyme that triggers necessary conformational change for motor function
Myosin
one of the major types of motor proteins that
- interacts with actin in myofibril contraction
- helps w/ cellular transport
Is composed of
- head
- neck
Kinesin
Motor protein that is primarily known for its interaction with microtubules. It:
- aligns chromosomes in the cell during metaphase
- depolymerizes microtubules during anaphase
& also - participates in vesicle transport intracellularly
It has two heads structurally, one of which is always tied to microtubule and it brings vesicles toward the positive end of the microtubule.
Dyenin
Another motor protein similar to kinesin with two heads, one of which is always tied to microtubules.
It:
- helps with flagella and celia’s sliding movement
- transport of vesicles intracellularly toward the negative end of microtubules
Binding Proteins
Proteins that bind other material for purpose of transport.
Ex:
1. hemoglobins
2. Ca-carrying proteins
3. DNA [transcription-factor] binding proteins
Can help sequester molecules they bind and often have a dissociation curve indicating their affinity for the molecules they bind depending on environmental conditionsex: oxyhemoglobin dissociation curve
Cell Adhesion Molecules
Proteins on the surface of cells that aid in binding of the cells to extracellular matrix or to other cells.
Have 3 primary categories:
- cadherins
- Integrins
- Selectins
Cadherins
Glycoproteins that mediate Ca dependent cell adhesion. Have specific types that contribute to adhesion of similar cell types.
Integrins
one type of adhesion molecules with two spanning chains of alpha and beta that participate in sell signalling, in binding to and communicating with extracellular matrix, and in promoting cell division and apoptosis in addition to other processes.
participate in cell defense and wbc migration
Selectins
Third type of CAD that bind to carbohydrates on surface of other cells. Present on WBC and on epithelial lining of blood vessels and participate in host defence, inflammation and wbc migration.
Immunoglobins
aka antibodies, are one type of non-enzymatic protein that help with elimination of pathogenic invaders and toxins by
- neutralizing the antigen, making it unable to exert its effect
- opsonizing the antigen; marking it and accumulating other immune cells to neutralize it
- aggregating the antigen with immune cells into an unsoluble mass; allowing it to be phagocytized by macrophages.
Structure:
- y shaped immune cell with a constant region that signals to and accumulates other immune cells and an antigen-binding region that binds to a specific antigen
- is stabilized by disulfide and non-covalent bonds
Biosignaling
The biological process of receiving and acting on signals.
Proteins participate in bio-signaling by
- serving as receptors
- serving as extracellular ligands
- acting as second messengers (enzymes)
- acting as transporters in facilitated diffusion (binding)
Ion Channels
- Proteins that create pathways for passage of certain charged particles;
- Even though they all share the similarity of participating in facilitated diffusion, they vary depending on their opening mechanism
The 3 types consist of:
- Ungated
- Voltage-gated
- Ligand-gated
Can display km & vmax similar to enzymes and can be graphed using Lineweaver-Burk plots
Ungated Channels
Self-explanatory; protein channels that are ungated; ex. k channels [e.c. there is always an efflux of potassium ions outside of cells unless [k] is at equilibrium inside and outside the cell
Voltage-Gated Channels
Channels which open/close based on a cell membrane’s potential/voltage which inc with depolarization of the cell membrane
Ex. voltage gated channels are found in neurons, muscles, & SA node of the heart
Ligand-Gated Channels
Protein ion channels which open/close as a result of binding/releasing of specific molecules to/from the channels
ex. channels that open as a result of binding neurotransmitters.
Type of Biosignaling Proteins
- Ion Channels [Types: 1. ligand-gated, 2. voltage
gated, 3. ungated] - Enzyme-Linked Receptors
- G-protein-Coupled Receptors
Enzyme-Linked Receptors
Receptors with 3 domains of
1. membrane spanning–[allows for binding of a
ligand]
2. ligand binding–[activates the catalytic domain]
3. catalytic domain–[activates a second messenger
cascade]
- **ex.
1. Receptor Tyrosin Kinases
2. Receptor Tyrosin phosphatases
3. Serine-threonie specific protein kinases
GPCR
aka G protein that cascades an inhibitory or a stimulating signal inside a cell once a ligand binds to the membrane receptor.
there is reception specificity for the ligand*
3 Types:
- Gs
- Gi
- Gq
Structure:
GPCRs have 3 subunits (alpha, beta, & Y)
Function:
(Alpha subunit is bound to GDP in its inactive form, and remains associated with b * Y subunits in a complex);
1. Once a ligand binds to GPCR, the alpha subunit activates the g protein, binds GTP, and dissociates from b & Y subunits.
2. When GTP dephosphorylates, the alpha subunit of GPCR rebinds to the b & Y subunits and renders the G-protein inactive.
If a is ai, the enzyme is inhibitory; if a is as, the enzyme is stimulating
Gs
One type of GPCR that stimulates adenylate cyclase, increasing cAMP levels in the cell
Gi
One type of GPCR that inhibits adenylate cyclase, decreasing cAMP levels in the cell
Gq
One type of GPCR that increases Ca level in the cell by activating phospholipase C which cleaves a phospholipid from the cell membrane, transforming it into PIP2. PIP2 is then cleaved into DAG & IP3. IP3 then forces the calcium channels to open in the endoplasmic reticulum, releasing calcium in the cell.
Protein Isolation
A biological technique used for studying the physical and chemical properties of proteins and for identifying the protein of interest from a given cell/tissue
Process & Types:
1. Homogenization–crushing and grinding tissue of interest
2. Centrifugation–separating protein from other small particles
3 either electrolysis or chromatography
Common Protein Isolation Strategies
- Electrolysis
- Chromatography
Homogenization & centrifugation are required steps before electrolysis and chromatography*
can be used for both denatured & native proteins
Electrolysis
- A common analytic technique employed for protein separation
- Uses a porous polyacrylamide gel and an electrical field with a negatively charged cathod and a positively charged anode to separate proteins based on their
I. charge
2. shape & size & friction
***small, charged particles migrate faster in a strong electrical field as opposed to large, neutral particles in a weak electrical field.
Types of Electrolysis
- Native PAGE
- SDS-PAGE
- Isoelectric Focusing
Native-Page
Polyacrylamide-Gel-Electrophoresis: analytic tool that helps in separation of proteins based on charge & size
Advantages
—Protein can be recovered from gel after electrolysis
unless it has been stained
—separates proteins even when they have been demonstrated to have similar sizes by other analytic techniques.
Disadvantages:
—limits mass to charge and mass to size ratios that can be used for protein particles
SDS-PAGE
Page with SDS that separates proteins based on mass alone.
SDS is a detergent that neutralizes the proteins and denatures them
Iso-electric Focusing
Separation of proteins based on their PI (isoelectric points)
exploits acidic & basic properties of proteins
Process:
Proteins placed in a gel with pH gradient move toward the cathod/basic end or toward the anode/acidic end until they reach their pI, the pH at which they are electrically neutral.
Chromatography
Analytic technique for separating proteins that involves homogenization and fractioning of the proteins through a porous mixture.
**Logic behind this technique is that particles similar to each other in nature, move more slowly and stick together. Therefore, identity of protein particles can be determined based on their polarity, charge, pH, etc. after separation.
- **Preferred technique to electrophoresis b/c
1. it allows for immediate identification of proteins after separation
2. allows for separation and identification of large amounts of protein
Process:
- placing a sample in a stationary/adsorbent phase
- running the mobile phase through the stationary phase
- varying sample retention times in the stationary/elute phase allow for partitioning of the sample components [aka separation of protein types]
Common stationary phase properties:
- pore size
- charge
Types of Chromatography
- Column
- Ion-Exchange
- Size-Exclusion
- Affinity
Column Chromatography
- Sample is run through silica or alumina adsorbent in a column;
- components are separated based on
- polarity
- pH
- salinity
*used for separation and collection of proteins and nucleic acids.
Ion-Exchange Chromatography
Charged adsorbent is used to separate protein components based on their charge.
opposite charges attract
Size-Exclusion Chromatography
Distinctly-sized porous adsorbent collects smaller sized protein components while allowing larger particles to move through
- Counterintuitive**
- Could be followed by ion-exhchange chromatography*
Affinity Chromatography
Adsorbent coated with a compound that has high affinity for a given protein component can be used to separate particles of interest from the rest of the mobile phase.
Constituting Factors of Protein Analysis
After Isolation
- Protein Structure
- Amino Acid Composition
- Activity Analysis
- Concentration Determination
Protein Structure Analysis
- One of the ways for analyzing an isolated protein;
2. Uses x-ray crystallography (75%) or NMR spectroscopy (25%) as techniques for determining a protein’s structure
NMR Spectroscopy
Nuclear Magnetic Resonance Spectroscopy: One of the techniques used for analyzing the structure of an isolated, crystalized protein
Protein’s Amino Acid Composition Analysis
- One of the ways for analyzing an isolated protein;
- Uses Edman degradation or other digestive enzymes to cleave proteins before identifying them using chromatography
Does not yield correct sequence of amino acids constituting the intact protein
Edman Degradation
Small protein (50-70 amino acid) cleavage technique used in amino acid composition analysis that cleaves amino acids off the primary structure of proteins at the N-terminus
Digestive Enzymes Used for Separation of Amino Acids in Amino Acid Composition Analysis
- Trypsin
- Chymotrypsin
- Cyanogen Bromide
Protein Activity Analysis
Protein activity is determined by monitoring a known rxn with a given concentration and by comparing it to a standard.
Impacted by concentration level**
Protein Concentration Determination
Concentration is determined exclusively with spectroscopy.
Bradford Protein Assay
Bradford Protein Assay
An essay that helps with determination of the concentration of one type of protein in a mixture by mixing the mixture with a green/brown solution that protonates the ionizable protein components in that mixture in addition to forming bonds with them.
In this process, the green/brown mixture turns blue.
To determine concentration, absorbent becomes quantified and gets graphed as a bradford curve.
