Biochemistry Fall 08- Ch. 1&2: Protein Structure And Characterization

Front 1

structure of an amino acid? identify groups and protein polarity and type of bonds involved.

Back 1

-carboxylic group and amino group.
-polar with 2 distinct ends: alpha carbonyl end on the right side and alpha amino end terminal on left side.

-peptide bonds

Front 2

1) the atoms involved in peptide bonds are _____.
2) there is ____ about the peptide bond.

3) the oxygen atom of the carbonyl group and the hydrogen atom of the NH- group are generally ____ to one another.

Back 2

1) planar
2) restricted motion
3) trans

Front 3

describe the four levels of protein structure

Back 3

1) primary- the amino acid sequence and is most impt as it dictates higher level of protein organization

2) secondary-the regular repeated folding of primary structures.

3) tertiary-globular shape of protein involving one polypeptide

4) quarternary-ONLY oligonaric (large proteins/multi sub unit proteins, ex: hemoglobin). not all proteins are quarternary.

Front 4

what types of secondary structures are there and what type of bonding stabalizes secondary structures?

Back 4

1) alpha helix
2) beta sheet conformation
3) beta turns or beta bends

HYDROGEN BONDING

Front 5

there are 3 additional secondary structures of proteins:
1) parallel and anti parallel beta helix, what is the difference?
2) beta turns or beta bends involving random coils and turns/loops/bends [no question]
3) super secondary structure, what are these?

Back 5

1) parallel-all end terminals on same side (more stable)
antiparallel- alternating arrangment of peptide chain

3) collection of secondary structures forming motifs. common motiffs include beta-alpha-beta and alpha-alpha

Front 6

describe all bonding involved in tertiary structure

Back 6

1) ionic linkages
2) H-bonds
3) hydroPHOBIC interactions- b/w non-polar AA in water solution
4) Van der Waals (weak)
5) disulfide bonds (strong, covalent)-found in extracellular proteins and are formed by the oxidation of two *CYSTEINE* residues.

Front 7

describe the two classes of proteins including fcn, solubility, and give an example

Back 7

1)GLOBULAR
-polypeptide chains arranged in a globule
-fcn: as ENZYMES
-water SOLUBLE
-HYDROPHOBIC core of AA
-ex: catalase, hemoglobin

2) FIBROUS
-extended simple structures
-fcn: structural, supportive
-INSOLUBLE in water
-ex: collagen, verratin

Front 8

protein seperation using electrophoresis when not denatured:
1) how? what determines the speed of the seperation? what two things oppose seperation? do smaller or larger molecules move further?

2) what is the most popular seperation medium used?

Back 8

a mixture of proteins are dissolved in a buffer which creates an overall net charge
-seperated in an applied electric field
-speed of seperation based on *strength of applied field AND net charge on protein*
seperation is opposed by friction and size. proteins migrate to opposite charged plates

-for the medium, gels and paper are used. most widely used is POLYACRYLAMIDE GEL which is a polymer of acrylamide.
-molecular sieve-smaller molecules move further than larger ones
-can be prepared with a range of pore sizes

Front 9

when electrophoresis is performed under DENATURING CONDITIONS such as in the presence of __1__. in this case protein seperation is based solely on __2__.
3) describe the technique. and the distance moved by the protein is inversely proportional to what?

Back 9

1) sodium dodexyl sulfate (SDS)
2) protein SIZE only
3) protein seperation is achieved by running the sample through a vertical polyacrylamide gel. all the SDS-protein complxes migrate toward the anode to a degree that is inversely proportional to the LOG OF THEIR MOLECULAR WEIGHT. large proteins slow down and smaller proteins move rapidly through it. (on a graph with molecular mass on y-axis and relative mobility on x-axis it forms a slanted line that is decreasing).

Front 10

ISOELECTRIC FOCUSING is an electrophoretic technique where the electrophoretic seperation is determined by what? describe.

Back 10

determined by the *relative amounts of acidic and basic moieties.
-this is a high resolution technique because the seperation of proteins that differ in their pH by only 0.0025 units
-seperation of charged proteins on a gel with a pH gradient
-proteins migrate until they reach a point in the gel where the pH is equal to their ISOELECTRIC POINT (pl). at this point, the protein remains stationary and the net charge on the protein will be *ZERO*

Front 11

list the 3 techniques in seperating proteins that are on the basis of SIZE only

Back 11

1) dialysis-small molecules travel outside and big molecules stay in the semi-permeable membrane.

2) Gel filtration chromatography

3) Molecular exclusion chromatography

Front 12

molecular exclusion chromatography

Back 12

-means of seperating proteins based on SIZE (and shape)
-pass sample through a column of hydrolyzed polymer such as SEPHADEX
-gel filtration occurs--large molecules move quickly right through while smaller molecules travel through the medium and take longer to pass down the column.

Front 13

what techniques are used in seperating proteins based on net charge and binding affinity?

Back 13

-ion exchange chromotography
-affinity chromotography

Front 14

affinity chromotography

Back 14

-seperates protein based on net charge and affinity
-CONCANAVALIN-A has an affinity for glucose and will bind to glucose beads if passed from soln

-based on a highly specific biologic interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.

Front 15

ion-exchange chromotography

Back 15

seperation of proteins on basis of charge
-cations bind negatively charged beads
-influenced by pH and electrostatic interactions
-anions are best seperated on postively charged columns such as *diethylaminoethyl-cellulose* (note how amino is positive)
-cations best seperated on negatively charged columns such as *carboxymethyl-cellulose* (notice how carboxy is negative)...opposites attract!

Front 16

NAME THE TECHNIQUE USED TO SEPERATE/ANALYZE PROTEINS
1) ____ is a chromatographic method of separating biochemical mixtures, based on a highly specific biologic interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.

2)____ is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.

3) ____ is a technique for separating different molecules by their electric charge differences. It is a type of zone electrophoresis, usually performed in a gel, that takes advantage of the fact that a molecule's charge changes with the pH of its surroundings.

4) ____ is the motion of dispersed particles relative to a fluid under the influence of an electric field that is space uniform.

5) ____ is a chromatographic method in which particles are separated based on their size, or in more technical terms, their hydrodynamic volume. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. When an aqueous solution is used to transport the sample through the column, the technique is known as gel filtration chromatography.

Back 16

1) Affinity chromatography
2) Ion-exchange chromatography
3) Isoelectric focusing
4) Electrophoresis
5) molecular exclusion chromatography (aka size exclusion chromatography)

Front 17

Edman degradation

how do you improve the reliability of this procedure?

Back 17

Edman degradation is a method of sequencing amino acids in a peptide. In this method, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues.
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-ONLY LABELS THE AMINO TERMINAL residue. this residue is cleaved from the peptide leaving the remainder of the peptide intact, then the free amino terminal is covalently labeled with PHENYLISOTHIOCYANATE AT A pH of 9.0.
*ONLY WORKS FOR 20-25 AMINO ACIDS, longer than that can't be accurately sequenced.

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IMPROVING RELIABILITY:
-specific cleavage by chemicals or enzymes
-chemicals (CYANOGEN)
-enzymic: *tryptic and chymotrypsin*

Front 18

what are 3 potential uses of protein sequence data?

Back 18

1) family resemblence
2) homologous protein
3) protein targetting

Front 19

why is the genetic code superior to Edman degradation

Back 19

the genetic code makes it possible to predict the sequence of AA in a given protein.
-faster and more accurate than the Edman rxn when the gene is available
-PROBLEM: the predicted AA sequence may be altered in the final protein