Non-Enzymatic Protein Function and Protein Analysis Flashcards
Structural Proteins
- Function: Stabilize and protect the integrity of the cell; compose the cytoskeleton, anchoring proteins and extracellular matrix
- Examples:primary structural proteins in the body are collagen, elastin, keratin, actin, and tubulin
- Properties: Fibrous proteins; Repetitive secondary structure and super-secondary structure
Collagen (structure, location, and function)
Structure: trihelical fiber (three alpha helices woven together to form a secondary helix)
Location: makes most of the extracellular matrix of connective tissue
Function: strength & flexibility`
Elastin ( location, and function)
Location: makes most of the extracellular matrix of connective tissue.
Function: stretches an recoils like a spring, restores original shape of the tissue
Keratin (structure, location, and function)
Intermediate filament proteins found in epithelial cells
Function: mechanical integrity of the cell and regulatory protein
Location: primary protein that makes up hair and nails
Actin (structure, location, and function)
Most abundant protein in eukaryotic cells
Structure: it has a positive and negative side so can travel unidirectionally
Location: It makes up thin filaments and microfilaments in myofibrils
Function: polarity allows motor proteins to travel
unidirectionally.
Tubulin
Make up microtubules
-provide structure, chromosome seperation and intracellular transport with kinesin and dynein
Motor Proteins
- Definition: Proteins capable of force generation through conformational change
- Function: Serve in muscle contraction, cellular trafficking and cell motility
- Examples:Myosin, kinesin and dynein
- Properties: contain one or more heads for force generation’ have catalytic ability to break down ATP for force generation (ATPase)
Motor porteins also display ____ activity
enzymatic activity
When motor proteins display enzymatic activity, they act as ____
They act as ATPases, that power the conformational change for motor function
Motor proteins have transient interactions with either ____ (a) or ____ (b)
(a) actin
(b) microtubules
Primary Motor Proteins
- Myosin
- Kinesins
- Dyneins
Myosin
- Primary motor protein that interacts with actin
- It is the thick filament in myofibril
- It also plays a role in cellular transport
- Each myosin subunit has a single head and neck: movement at the neck powers sarcomere contraction
Kinesins and Dyneins
- Motor proteins associated with microtubules
- Have two heads, one remains attached to tubulin at all times
- Polarity: kinesins (move towards positive end of microtubule) and dyneins (move towards negative end of microtubule)
Key roles of Kinesins and Dyneins
Key roles:
- Alignment of chromosomes during metaphase
- Depolymerization of microtubules during anaphase
- Sliding movement of cilia and flagella
- Vesicle transport in the cell (opposite polarities
- Kinesins bring vesicles of neurotransmitters to the synaptic terminal (positive end of axonal microtubules)
- Dyneins bring vesicles of waste or recycled neurotransmitter back toward the soma (negative end of microtubule)
Binding proteins ___ (a) or ___ (b) molecules by binding to them
(a) transport
(b) sequester
Examples of Binding Proteins
- Hemoglobin
- Calcium-binding proteins
- DNA-binding proteins (often transcription factors)
Cell Adhesion Molecules (CAMs)
Proteins found in the surface of most cells
CAMs function
Bind cells to matrix or other cells
All CAMs are ___ proteins
Integral membrane proteins
Three major families of CAMs
- Cadherins
- Integrins
- Selectins
Cadherins
- Glycoproteins
- Mediate calcium-dependent cell adhesion
- Hold SIMILAR cell types together (e.g. epithelial cells)
- Different cells have type-specific cadherins
Integrins
- Group of proteins.
- All have two membrane spanning chains (alpha and beta)
- Chains are important for binding and communicating to the extracellular matrix.
- Cellular signaling (promote cell division,apoptosis and other processes)
- Host defense
Selectins
- Bind to carbohydrate molecules that project from cell surfaces (weakest bonds formed by CAMs)
- Expressed on white blood cells and endothelial cells (line blood vessels)
- Host defense
Immunoglobulins
- Y-shaped proteins produced by B-cells
Key roles:
- Neutralize targets (toxins and bacteria)
- Recruit other cells (such as macrophages) to eliminate the threat.
Structure of immunoglobulins
- Y-shaped proteins
- Two identical heavy chains and two identical light chains
- Disulfide linkages and noncovalent interactions hold them together)
- Antibody binding region at the tips of the Y (sequence specific) the binds antigen
- Constant region, rest of the antibody.
Possible outcomes of antibodies binding to antigen
- Neutralization of antigen
- Marking the pathogen or Opsonization for destruction by white blood cell
- Clumping together or agglutinating into large insoluble protein complexes that can be phagocytized and digested by macrophages.
Protein act in biosignaling by acting as:
- extracellular ligands
- transporters (for facilitated diffusion)
- receptor proteins
- second messengers
Three main types of ion channels
- Ungated
- Voltage-gated
- Ligand-gated
Ion channels permit ____ diffusion of charged particles
Facilitated diffusion-A type of passive transport in which a molecule is transported down a concentration gradient through a pore in the membrane created by a transmembrane protein.
Ungated Ion Channels
- They are unregulated, always open
- Example: ungated K+ channels. There will be a net efflux of K ions through these channels unless K+ is at equilibrium
Voltage-Gated channels
- Regulated by membrane potential change near the channel
Examples:
- Voltage-gated Na+ channels in neurons. Closed at resting conditions. Depolarization opens them, which close again very quickly as voltage increases.
- K+/Na+ voltage-gated channels in cells of the sinoatrial node of the heart. Serve as the pacemaker current
- lower voltage= channels open
Ligand-Gated Channels
- Binding of a specific substance or ligand opens/closes the channel.
- Km and Vmax parameters also apply to transporters such as ion channels.
- They do not have Km values because there is no catalysis
Example:
Neurotransmitter GABA binds to chloride channel at the postsynaptic membrane and opens it.
Enzyme-linked receptors
- Membrane receptors that display catalytic activity in response to ligand binding
- participates in cell signaling through extra-cellular ligand binding and initiating a 2nd messenger
Structure of enzyme-linked receptors
Three primary protein domains:
1) membrane-spanning domain (anchors receptor to in the cell membrane
2) ligand-binding domain
3) catalytic domain
Initiation of a second messenger cascade
ligand-binding domain is stimulated by the appropriate ligand and induces a conformational change that activates the catalytic domain.
Examples of enzyme-linked receptors are:
- Receptor tyrosine kinases
- Serine/threonine specific protein kinases
- Receptor tyrosine phosphatases
G protein-coupled receptors
- Large family of integral membrane proteins
- Involved in signal transduction
- Have 7 membrane-spanning alpha helices
- Differ in specificity of the ligand-binding area located on the extracellular surface of the cell.
- They utilize heterotrimeric G protein
- Binding of a ligand increases affinity of the receptor for the G protein.
- Binding of the G protein represents a switch to the active state and affects the intracellular signaling pathway
G protein
- They have an intracellular link to guanine nucleotides (GPD and GTP)
- It has 3 subunits: alpha, beta, and gamma
In the G proteins inactive form:
The alpha subunit binds GDP and is in a complex with beta and gamma subunits.
Trimeric G protein cycle
(1) inactive α subunit of G protein binds GDP and forms complex with β and γ subunits.
(2) inactive α subunit protein binds GTP then it dissociates from β and γ subunits
(3) activated α subunit alters the activity of adenyl cyclase*
(4) Once GTP in activated α subunit is dephosphorylated to GDP, it will rebind to the β and γ subunits, rendering the G protein inactive
- as will activate the enzyme, a1 will inhibit the adenyl cyclase
Gs _____ (a) adenylate cyclase, which _____ (b) cAMP levels in the cell
(a) stimulates
(b) increases
G1 _____ (a) adenylate cyclase, which _____ (b) cAMP levels in the cell
a) inhibits
(b) decreases
Gq _____ (a) phospholipase C, which _____ (b) calcium channels in the ER, _____ (c) calcium levels in the cell
(a) activates
(b) opens
(c) increasing
homogenization
crushing, grinding or blending the tissue of interest into an evenly mixed solution
centrifugation
isolation of proteins from much smaller molecules before other isolation techniques are employed
Polyacrylamide gel
the standard medium for protein Electrophoresis; pourous matrix that solidifies at room temperature and acts like a sieve allowing smaller particles to move faster through medium
PAGE (Polyacrylamide gel electrophoresis)
analysis of proteins in native states - limited by varying charge:mass and size:mass ratios but allows native protein recovery
-maintains protein shape (non-denaturing)
SDS-PAGE (sodium dodecyl Sulfate)
separates proteins on mass alone by denaturing + forming negative charge on all; followed by staining to make protein bands visual
Isoelectric focusing
separates on the basis of PI point by migrating proteins through gel with pH gradient; protein stops moving when reaches point where pH = pI and the protein is neutral
Mnemonic for isoelectric focusing: A+
Anode has acid (H+ rich) gel and a (+) charge.
Chromatography
fractionation of a homogenized mixture through a porous matrix - the more similar the compound is to the matrix, the slower it will move through
Column chromatography
used for other macromolecules besides just proteins - column of alumina/silica beads as adsorbent and gravity moves compounds and solvent down at vary speeds based on polarity and size
ion exchange chromatography
beads in column coated w/ charged substances - attract or bind substances w/ different charge. then a salt gradient is used to elute remaining charged particles that stuck
size exclusion chromatography
beads in column contain pores of different sizes. small compounds go through pores slowly, large do not fit and elute quickly. used after ion exchange.
affinity chromatography
coat beads w/ specific receptor of protein of interest to bind it. can be difficult to remove particle of interest from receptor then though.
What are the most prevalent extra cellular proteins?
Keratin, elastin and collagen
What receptor types use second messengers
Enzyme linked receptor and G protein coupled receptor
