Non-Enzymatic Protein Function And Protein Analysis

Question 1

Generally describe the cytoskeleton

Answer 1

Three dimensional web of proteins anchored to the cell membrane by embedded protein complexes

Question 2

What are the primary structure of proteins in the body?

Answer 2

Collagen, elastin, keratin, actin, tubulin

Question 3

Describe the structure of structural proteins

Answer 3

Generally highly repetitive secondary structure and a super secondary structure- a repetitive organization of secondary structural elements together sometimes referred to as a motif

This regularity gives many structural proteins of fibrous nature

Question 4

Collagen

Answer 4

Structural protein
Trihelical fiber, three left-handed helices woven together to form a secondary right hand helix
Makes up most of the extra cellular matrix of connective tissue
Found throughout the body and is important in providing strength and flexibility

Question 5

Elastin

Answer 5

Structural protein
Important component of extra cellular matrix of connective tissue
Stretch and recoil

Question 6

Keratin

Answer 6

Structural protein
Intermediate filament proteins found in epithelial cells
Contribute to the mechanical integrity of the cell and also function as regulatory proteins

Question 7

Actin

Answer 7

Structural protein
Composes microfilaments, and the thin filaments in myofibrils
Most abundant protein in eukaryotic cells
Have a positive side and a negative side allowing motor proteins to travel, unidirectionally along an actin filament like a one-way street

Question 8

Tubulin

Answer 8

Structural protein
Mixup microtubules
Micro tubules are not only important for mitosis and meiosis but also for intracellular transport with kinesin and dyein
Polarity
Negative end adjacent to the nucleus and the positive end peripheral

Question 9

Motor protein activity

Answer 9

Act as ATPases to fund their activity and power conformational chance necessary for motor function
Have transient interactions with either actin or microtubules

Question 10

Myosin

Answer 10

The primary motor protein that interacts with actin
Thick filament in myofibril
Can be involved in cellular transport
Each subunit has a head and neck, the neck being responsible for power stroke of sarcomere contraction

Question 11

Kinesins and dyneins

Answer 11

Motor protein associated with microtubules
They has two heads, at least one remaining attached to tubulin at all times
Vesicle transport
Opposite polarities

Question 12

Kinesins

Answer 12

Play key roles in aligning chromosomes during metaphase and depolymerizing microtubules during anaphase of mitosis
bring vesicles toward the positive end of the microtubule
Bring vesicles of neural transmitter to the positive end of the axon of microtubules (toward the synaptic terminal)

Question 13

Dyneins

Answer 13

Involved in the sliding movement of cilia and flagella
Bring vesicles of waste or recycled neurotransmitter back to the negative end of the microtubule (toward the soma) through retrograde transport

Question 14

Binding proteins

Answer 14

Transport or sequester molecules by binding to them
Hemoglobin, calcium binding proteins, DNA binding proteins, transcription factors, and others
Affinity curve

Question 15

Affinity curve of binding proteins

Answer 15

When sequestration of a molecule is the goal, the binding protein usually has high affinity for its target a Crossing large range of concentration so you can keep it bound at nearly 100%
A transport protein is likely to have varying affinity, depending on the environmental conditions

Question 16

Cell adhesion molecules

Answer 16

Found on the surface of most cells and aid in binding the cell to the extracellular matrix or other cells
There are a number of different types of cams
All integral membrane proteins
Adhesion molecules can be classified into three major families: integrins, cadherins, selectins

Question 17

Cadherins

Answer 17

Cell adhesion molecules
Groups of glycoproteins that mediate calcium dependent cell adhesion
Specific cadherin types for specific tissue types (E-cadherin for epithelial and N-cadherin for nerve cell)

Question 18

Integrin

Answer 18

Cell adhesion molecules
Group of proteins that all have two membrane spanning chains called alpha and beta
These chains are very important in the binding and communities with the extra cellular matrix
Cell signaling, apoptosis, cell division
Blood clotting, white blood cell migration, basement membrane stabilization

Question 19

Selectin

Answer 19

Cell adhesion molecule
Bind to carb molecules that project from other cell surfaces
These bonds are the weakest formed by the CAMs
Expressed on white blood cells and endothelial cells that line blood vessels
Host defense

Question 20

Immunoglobulins

Answer 20

Rid body of foreign invaders
Also called antibodies
Produced by B cells
Neutralize targets
Recruit others
Y-shaped
Made up of two heavy identical chains and two identical light chains
Disulfide linkages and noncovalent interactions hold the heavy and light chains together
Each antibody has an antigen-binding region at the tips of the Y
Antigenic sequence specific
Remaining portion is the constant region which recruits binding of other immune cells

Question 21

What happens when antibodies bind to antigens?

Answer 21

1) neutralize
2) opsonization
3) agglutinating to be phagocytized

Question 22

ion channels

Answer 22

facilitated diffusion, passive transport

used for impermeable molecules (large, polar, uncharged)

3 types: ungated, voltage-gated, ligand-gated

Question 23

ungated channels

Answer 23

no gates, unregulated
K+ leak channels all cells possess

Question 24

voltage-gated channels

Answer 24

• regulated by membrane potential change
• possessed by excitable cells
• closed under resting conditions, but membrane depolarization causes protein conformation change that allows them to quickly open and then quickly close as voltage inc
• sodium-potassium channels found in SA node, pacemaker current

Question 25

ligand-gated channels

Answer 25

- specific ligand binding causes channel to open or close - NT act at LGC

Question 26

enzyme-linked receptors

Answer 26

- 3 primary protein domains: 1. membrane-spanning domain (anchor) 2. ligand-binding domain (induces conformational change to activate catalytic domain) 3. catalytic domain (second messenger cascade) RTK dimerization and autophosphorylation RTP serine/threonine-specific protein kinases

Question 27

GPCR

Answer 27

- 7 membrane a-helices - Gs: stim adenylate cyclase, inc cAMP - Gi: inhibit adenylate cyclase, dec cAMP - Gq: activate phospholipase C, cleaves a phospholipid from membrane to form PIP2 ~> cleaved into DAG and IP3 ~> IP3 opens calcium channels in the ER inc calcium levels in cell - a inactive and bound to b and g but when ligand binds, GDP replaced with GTP and a dissociates, does its thing as mentioned before, and then becomes inactive and bound again after GTP dephosphorylated

Question 28

what do we do before isolation techniques employed?

Answer 28

homogenization and centrifugation

Question 29

electrophoresis

Answer 29

- isolation technique - electric field moves compounds according to net charge and size - neg will migrate to pos anode and pos with migrate to neg anode v = Ez/f v: migration velocity E: electric field strength z: net charge f: frictional coefficient - medium is polyacrylamide gel which is slightly porous, solid at room temp, movement dependent on size and charge, large particles easily retained - small highly charge particles move faster or placed in large electric field -multiple samples can run simultaneously

Question 30

native PAGE

Answer 30

- analyze protein in native state - functional native protein recovered from gel after electrophoresis only if it hasn’t been stained - limited by mass-to-charge or -size rations of cellular proteins

Question 31

SDS PAGE

Answer 31

- separates proteins on basis of relative molecular mass alone - SDS detergent disrupts all noncovalent interactions by binding to proteins and creating large chains w net negative charges neutralizing and denaturing the protein - E and f affect velocity and after separation gel can be stained so that protein bands can be visualized and the results recorded

Question 32

isoelectric point

Answer 32

the pH at which the protein or amino acid is electrically neutral zwitterion exceptions: arg and lys bc of basic side chains so their zwitterion occurs when the amino group is deprotonated for polypeptides the isoelectric point is primarily determined by the relative numbers of acidic and basic amino acids

Question 33

isoelectric focusing

Answer 33

the mixture of proteins is placed in a gel w a pH gradient an electric field is then generated across the gel pos proteins will migrate towards cathode and neg toward anode as protein reaches portion where pH is equal to its pI, protein takes on a neutral charge and stop moving

Question 34

chromatography

Answer 34

sample placed on stationary phase mobile phase runs through and elutes sample diff substances move at diff speed affinity of sample observed amount of time spent in stationary phase by sample is called retention time causes compounds to partition

Question 35

column chromatography

Answer 35

- column filled w silica or alumina beads - gravity moves everything through beads - size and polarity determine speed of compound moving through polar beads - less polar, shorter retention time - solvent polarity, pH, or salinity can easily be changed to help elute the protein of interest - solvent drips out fraction by fraction - solvent can be evaporated and compounds of interest kept - can also collect nucleic acids

Question 36

ion-exchange chromatography

Answer 36

the column are coated w charged substances so they attract or bind compounds that have an opposite charge a pos column will attract and hold a neg protein as it passes through the column either inc its retention time or retaining it completely after all other compounds have moved through column a salt gradient is used to elite the charged molecules that have stuck to the column

Question 37

size-exclusion chromatography

Answer 37

- porous beads - small compounds enter the beads thus slowing them down - large compounds move through the beads quickly

Question 38

affinity chromatography

Answer 38

- customize columns to bind any protein of interest by creating a column w high affinity for that protein - accomplished by coating beads w a receptor that binds the protein or a specific antibody to the protein; protein retained in column - nickel and histidine tags - once protein retained it can be elites w free receptor, target, or antibody - can be different to remove substance from eluent

Question 39

x-ray crystallography

Answer 39

- measure electron density on an extremely high resolution scale and can be used for nucleic acids - x-ray diffraction pattern is generated in this method

Question 40

Edman degradation

Answer 40

- amino acids composition - sequential digestion of protein w specific cleavage enzymes is used - sequences up to 50-70 amino acids - selectively and sequentially removes N-terminal amino acids of protein, which can be analyzed via MS - synthetic reagent cleaves at specific amino acid residues - positions cannot be measure using this method bc of disulfide and salt bridges being broken

Question 41

activity analysis

Answer 41

- rxns w color change have particular applicability bc microarrays can rapidly identify the samples from a chromatographic analysis that contains the compound of interest

Question 42

concentration determination

Answer 42

- spectroscopy - proteins have aromatic side chains and thus can be analyzed w UV spectroscopy w/o treatment (sensitive to contaminants) - also cause colorimetric changes w specific rxns, particularly BCA, Lowry, and Bradford assays

Question 43

Bradford Protein Assay

Answer 43

- mixes a protein in solution w Coomassie Brilliant Blue dye - dye is protonated and green-brown in color prior to mixing w proteins - dye gives up protons upon binding to aa groups turning blue in the process - ionic attractions between dye and protein stabilize this form of dye; thus inc protein concentrations are reacted w the Bradford reagent and then absorbance is measured to create a standard curve - the unknown sample is then exposed to the same conditions and concentration is determined based on standard curve - very accurate when only one type of protein is present in solution bc of variable binding of Coomassie dye - this assay is limited by the presence of detergent in the sample or by XS buffer