Chapter 3: Nonenzyme Protein Function and Analysis Flashcards Preview

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Flashcards in Chapter 3: Nonenzyme Protein Function and Analysis Deck (42):


  • Separating technique that uses an electrical field to move charged particles toward a side opposite to their charge. 
    • Particles travel through a Polyacrylamide gel
  • Positive charged particles move to the negative cathode
  • Negative charged particles move to the positive anode
  • Faster moving molecules are the small and have strong charges
  • Stronger electrical fields allow molecules to move faster
  • Separates particles based on their charge, shape, and molecular weight.


Define G-protein coupled receptors



What are the 3 types of G proteins, and what are their functions?

Do hormones bind to these?

  • Transmembrane protein receptors that work with a trimeric G protein.
  • Has seven membrane-spanning α-helices
  • The G protein has 3 subunits: α, β, ɣ (gamma)
  • Steps:
    1. G-protein is inactivated
      • G-protein is not yet connected to the receptor inside the cell
      • Its α-subunit has GDP bound to it
    2. Ligand binds to the receptor and activates the G-protein, causing it to bind to the receptor.
    3. GDP ⇒ GTP on the α-subunit.
    4. The G-protein separates from the receptor and its      α-subunit breaks away from the β and ɣ subunits
    5. α-subunit with GTP-bound goes to activate a target protein, which causes a second messenger cascade
    6. The GTP on the α-subunit is dephosphorylated to GDP 
    7. α-subunit reconnects with the β and ɣ subunits of the G-protein and is inactive
  • Then the α-subunit detaches and alters the activity of adenylase cyclase or phospholipase C
  • 3 types of G proteins
    • Gs = stimulates adenylate cyclase, increasing levels of cAMP 
    • Gi = inhibits adenylate cyclase, decreasing levels of cAMP
    • Gq = activates phospholipase C, which leads to increased calcium levels in the cell
  • Hormones do bind to these because hormones initiate secondary messenger cascades


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Column chromatography

What factors does it use to separate?

Stationary phase and Mobile phase?


Polarity (high/low)  vs Speed?

  • Technique to separate mixtures based on size and polarity of the compounds
  • Stationary phase of the column is silica or alumina beads
  • Mobile phase is the mixture itself, which flows through the stationary phase due to gravity
  • Speed down the column depends on size and polarity of the compounds
  • High polar compounds: move slow
  • Low polar compounds: move fast 


Enzyme-linked receptors

  • Transmembrane receptor proteins that initiate a secondary messenger cascade once a ligand binds to it.
  • Hormones are the ligands that bind to these receptors and cause secondary messenger cascades


What type of receptors do hormones attach to and why?

  • Hormones attach to:
    • Enzyme-linked receptors
    • G protein coupled receptors
  • Hormones need to use secondary messengers to alter the activity of cells, therefore they use receptors that iniatiate secondary messengers


Size-exclusion chromatography 

how does it work?

what factor does it depend on for separation?

  • Technique to separate compounds of a mixture depending on their size.
  • Small pores are placed in the stationary phase, which trap small molecules
  • Big molecules do not get trapped in the pores and move faster 
    • Big molecules elute out of the column first
  • Usually used in combination with ion-exchange chromatography to separate compounds based on their charges and size



structural proteins that is found in epithelial cells, which lines the internal and external parts of the body.

Provides epithelial cells with structural support.

Main protein that makes up hair and nails.

It is a Intermediate Filament protein.


Cell Adhesion Molecule proteins.

What are the 3 types:

proteins that are on the surface of the cell and adhere (attach) to the extracellular matrix or to other cells

________________________________________3 types:

  1. Cadherins - depend on calcium (Ca2+) and bind cells together in tissues
  2. Integrins - bridges for cell-cell and cell-extracelullar matrix interactions
  3. Selectins - proteins on the surface of cells that bind only to carbs (sugars) that stick out of other cells



made by what cells?

also known as?


specific or nonspecific?

  • Immune system proteins made by B-cells that neutralize antigens and recruit other cells to help eliminate them.
  • Also known as Immunoglobulins.
  • Y-shaped with 2 heavy chains and 2 light chains which are connected by disulfide bonds
  • There are antigen-binding regions at the tips of their Y-shaped structure
  • Each antibody only binds to very specific antigens


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Basic compounds are what charge? (+ or -)

Basic compounds are (+) charge because they readily gain a proton, gaining a (+) charge



  • structural protein that works with myosin to form myofibrils (muscle fibers) and allows cells to travel on it.
  • Actin makes up the thin filaments of myofibril (muscle fibers)
  • Myosin makes up the thick filaments
  • it has a positive side and a negative side, which cause polarity
    • This polarity allows motor proteins to travel along actin filaments in one direction



The most abundant structural protein in the body.

It makes up most of the extracellular matrix and provides strength/flexibility.


Voltage-gated ion channels



  • Transmembrane protein channels that only open when a certain charge builds up near the channel. 
  • They are closed during resting conditions
  • Open when depolarization occurs 
  • Example: depolarization of neurons and opening of voltage-gated ion channels in axons, which causes action potentials to fire.




Dehydration Synthesis

Hydrolysis: Putting H2O into a compound so that is breaks into two compounds


Dehydration Synthesis: Taking a H+ off one compound and an OH- off the other. Then the two compounds combine to form one. 


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Ion channels


  • Channel proteins that allow charged particles to pass through the membrane.
  • Ions cannot pass through the membrane without these channels.
  • Allows the facilitated diffusion of charged particles


Ligand Gated Channels



  • Transmembrane protein channels that open/close once a specific ligand binds to a receptor on it
  • Example: neurotransmitters bind to ligand-gated channels at the post-synaptic neuron, causing it to open or close


Define structural proteins

proteins that provide internal structure of the cell and sometimes are involved in cell movement



motor protein that works with actin to make myofibrils (muscle fibers).

Allows muscles to contact.

Makes up the thick filaments of myofibrils.

Actin makes up the thin filaments of myofibrils.



motor proteins that work with microtubules to move the flagella and cillia of cells.


Also involed in intracellular transport, moving vesicles toward the nucleus



structural protein in the extracellular matrix of connective tissue that allows it to stretch and return to its original shape


What is the best pH to use when using electrophoresis?

  • Electrophoresis is a technique that separates proteins based on their charges.
  • A pH that separates the compounds into negative, neutral, and positive groups is best.
    • Causes some to be deprotonated, and other to be protonated
    • Allows the compounds to go to opposite sides and to be separated


facillitated diffusion

  • type of passive transport in which molecules diffuse down their concentration gradients
  • Cross the membrane via openings made by channel proteins


What must proteins have in order for them to be analyzed via UV Spectroscopy.

What is UV spectroscopy used to determined?

  • To use UV Spectroscopy, proteins must have aromatic side chains.
  • UV spectroscopy determines the concentration of the protein.


Affinity chromatography 

What factor does it use to separate molecules?

How is it done?


Example of this done in my biochem lab?

  • Technique for separating compounds of a mixture depending on affinity 
  • Beads in the column are coated with receptors that have an affinity for a specific protein
    • Ex: Nickle-coated beads that I used in my biochem lab to bind histidine of galactosidase
  • The protein of interest is slowed down due to the column bead's high affinity for it
  • Disadvantage:
    • The protein may bind to the column and never elute out of it



Motor protein that acts with microtubules to allign chromosomes at the metaphase plate and to remove microtubules during anaphase of mitosis.


Also involved in intracellular transport, moving vesicles to the border of the cell




structural protein that forms the mitotic spindle, provides support, and intracellular transport


Makes up microtubules.



Motor proteins?



  • proteins that act as ATPases to get energy and move along a surface. 
  • They are enzymes, because they catalyze the hydrolysis of ATP for energy. 
  • Functions: muscle contraction. vesicle movement within cells, and cell motility 


Ion-exchange chromatography

how does it work?

what factor does it use to separate?

  • Technique to separate compounds of a mixture depending on the different charges of the compounds.
  • Beads in the column are coated with charged substances
    • They attract opposite charged compounds.
  • Compounds w/ opposite charge of beads: move slower because they are attracted to the opposite charge
  • Compounds w/ same charge move faster 




Type of cell adhesion molecule (CAM) protein that binds cells together in tissues

They are dependent on calcium


Remember: "Ca"lcium__"d"ependent




  • Uses centripital force to separate components of a mixture.
  • Uses to separate proteins from other molecules


Ungated ion channels.

How does this affect the nervous system?

  • Transmembrane protein channels that are always open.
  • Allow ions to diffuse down their concentration gradients.
  • This, as well as the sodium potassium pump, create membrane resting potential of the nervous system



What are the two molecules most bound to proteins?

  • Calcium and Magnesium are required for many physiological functions
  • They are most often bound to proteins


Isoelectric Focusing

What is it used for?

How is it done?

Which side is acidic/basic?

Draw what happens when two proteins (protein 1 has pI =9; protein 2 has pI = 4) are put on a gel at a pH of 7.

What happens if isoelectric focusing is done to these proteins?

  • Technique that uses gel with a pH gradient to separate proteins by their isoelectric points. 
  • Isoelectric point = the pH when a protein or amino acid is neutrally charged

  • Negative charged molecules migrate toward the Anode

  • Positive charged molecules migrate toward the Cathode

  • Anode is acidic (positive charged) because it attracts anions

  • Cathode is basic (negative charged) because it attracts cations

  • Proteins/amino acids stop moving once they reach their isoelectric point and become neutrally charged


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Stationary phase

Mobile phase


factors used for separation?

  • Technique to separate compounds depending on their affinity for the stationary or mobile phase.
  • Stationary phase - the solid or liquid where a sampled is placed on
  • Mobile phase - the fluid that carries the mixture as it flows through the stationary phase
  • High affinity to stationary phase: moves slow
  • High affinity to mobile phase: moves fast 
  • Factors used to separate compounds:
    • Charge
    • Size
    • Affinity to mobile phase and stationary phase


Acidic compounds are what charge? (+ or -)

Acidic compounds are (-) charged because they readily give up their protons, becoming (-) charged


The main functions of proteins? (9)

  1. enzymes
  2. structural proteins
  3. motor proteins
  4. binding proteins
  5. cell adhesion molecule proteins
  6. immunoglobulin (antibody of immune system)
  7. Receptors
  8. Channels 
  9. Transporters



type of cell adhesion molecule (CAM) protein

bridges for cell-cell and cell- extracelullar matrix interactions



type of cell adhesion molecule (CAM) protein

Selectins - proteins on the surface of cells that bind only to carbs (sugars) that stick out of other cells


Facilitated diffusion


what traits of molecules cannot pass through the membrane itself?

what must they do?

the passive transport of molecules down their concentration gradient through a protein pore in the membrane


Large, polar, or charged molecules cannot pass through the membrane.

They must pass through a membrane protein pore via facilitated diffusion



Enzyme-linked receptors



membrane receptors that have catalytic activity when a ligand binds


example: Receptor Tyrosin Kinase, which dimerizes when a ligand binds and autophosphorylates itself.



Sodium Dodecyl Sulfate

What does it do? How does it work?

What is it used for?

Use of SDS in this experimental technique allows for the separation of proteins by factor?

SDS is a detergent that disrupts all noncovalent interactions, thus denatures proteins.


It is used to separate proteins with electrophoresis.

It is used to separate proteins only by their molecular weights


what two techniques can be used to determine protein structure?

x-ray crystallography

nuclear resonance (NMR) spectroscopy