Nonenzymatic Protein Function and Protein Analysis Flashcards Preview

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Flashcards in Nonenzymatic Protein Function and Protein Analysis Deck (54):
1

Typical Protein Functions

1. supporting intracellular func and aidng w/ organization
2. acting as enzymes
3.

2

Intracellular Protein Functions

1. Adhesion Molecules
2. Immunoglobins
3. Structural molecules
4. Motor molecules
5. Binding Proteins

3

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.

4

All Primary Structural Protein Types

1. Collagen
2. Keratin
3. Elastin
4. Actin
5. Tubulin

***They give rise to motifs or repetitive secondary structural proteins

5

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.

6

Elastin

A primary structural protein that like collagen, constitutes the extracellular matrix of connective tissue. It allows the skin to stretch and recoil.

7

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??)

8

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****

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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.

10

Microtubules

Cytoskeletal proteins that
1. help with chromosal separation in meiosis and mitosis
2.help with intracellular transport with dyesin and kinesin
3. provide structure and support for the cell's internal structure

11

Motor Proteins

One of the nonenzymatic protein types that can contribute to motor function by
1. providing enzymatic activity; ex. ATpase
2. providing structural support
3. providing motor activity: ex. flagella, celia

Main motor proteins:
1. Myosin
2. Kinesin
3. Dyenin

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ATpase

Enzyme that triggers necessary conformational change for motor function

13

Myosin

one of the major types of motor proteins that
1. interacts with actin in myofibril contraction
2. helps w/ cellular transport

Is composed of
1. head
2. neck

14

Kinesin

Motor protein that is primarily known for its interaction with microtubules. It:

1. aligns chromosomes in the cell during metaphase
2. depolymerizes microtubules during anaphase
& also
3. 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.

15

Dyenin

Another motor protein similar to kinesin with two heads, one of which is always tied to microtubules.

It:
1. helps with flagella and celia's sliding movement
2. transport of vesicles intracellularly toward the negative end of microtubules

16

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 conditions****ex: oxyhemoglobin dissociation curve

17

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:
1. cadherins
2. Integrins
3. Selectins

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Cadherins

Glycoproteins that mediate Ca dependent cell adhesion. Have specific types that contribute to adhesion of similar cell types.

19

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***

20

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.

21

Immunoglobins

aka antibodies, are one type of non-enzymatic protein that help with elimination of pathogenic invaders and toxins by
1. neutralizing the antigen, making it unable to exert its effect
2. opsonizing the antigen; marking it and accumulating other immune cells to neutralize it
3. aggregating the antigen with immune cells into an unsoluble mass; allowing it to be phagocytized by macrophages.

Structure:
1. 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
2. is stabilized by disulfide and non-covalent bonds

22

Biosignaling

The biological process of receiving and acting on signals.

Proteins participate in bio-signaling by
1. serving as receptors
2. serving as extracellular ligands
3. acting as second messengers (enzymes)
4. acting as transporters in facilitated diffusion (binding)

23

Ion Channels

1.Proteins that create pathways for passage of certain charged particles;
2. Even though they all share the similarity of participating in facilitated diffusion, they vary depending on their opening mechanism

The 3 types consist of:

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

***Can display km & vmax similar to enzymes and can be graphed using Lineweaver-Burk plots***

24

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

25

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

26

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.

27

Type of Biosignaling Proteins

1. Ion Channels [Types: 1. ligand-gated, 2. voltage
gated, 3. ungated]
2. Enzyme-Linked Receptors
3. G-protein-Coupled Receptors

28

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

29

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:
1. Gs
2. Gi
3. 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

30

Gs

One type of GPCR that stimulates adenylate cyclase, increasing cAMP levels in the cell

31

Gi

One type of GPCR that inhibits adenylate cyclase, decreasing cAMP levels in the cell

32

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.

33

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

34

Common Protein Isolation Strategies

1. Electrolysis
2. Chromatography

***Homogenization & centrifugation are required steps before electrolysis and chromatography****

***can be used for both denatured & native proteins***

35

Electrolysis

1. A common analytic technique employed for protein separation
2. 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.

36

Types of Electrolysis

1. Native PAGE
2. SDS-PAGE
3. Isoelectric Focusing

37

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

38

SDS-PAGE

Page with SDS that separates proteins based on mass alone.

***SDS is a detergent that neutralizes the proteins and denatures them***

39

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.

40

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:
1. placing a sample in a stationary/adsorbent phase
2. running the mobile phase through the stationary phase
3. 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:
1. pore size
2. charge

41

Types of Chromatography

1. Column
2. Ion-Exchange
3. Size-Exclusion
4. Affinity

42

Column Chromatography

1. Sample is run through silica or alumina adsorbent in a column;
2. components are separated based on
1. polarity
2. pH
3. salinity

*used for separation and collection of proteins and nucleic acids.

43

Ion-Exchange Chromatography

Charged adsorbent is used to separate protein components based on their charge.

***opposite charges attract***

44

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***

45

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.

46

Constituting Factors of Protein Analysis
After Isolation

1. Protein Structure
2. Amino Acid Composition
3. Activity Analysis
4. Concentration Determination

47

Protein Structure Analysis

1. 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

48

NMR Spectroscopy

Nuclear Magnetic Resonance Spectroscopy: One of the techniques used for analyzing the structure of an isolated, crystalized protein

49

Protein's Amino Acid Composition Analysis

1. One of the ways for analyzing an isolated protein;
2. 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***

50

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

51

Digestive Enzymes Used for Separation of Amino Acids in Amino Acid Composition Analysis

1. Trypsin
2. Chymotrypsin
3. Cyanogen Bromide

52

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***

53

Protein Concentration Determination

Concentration is determined exclusively with spectroscopy.

***Bradford Protein Assay***

54

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.