Quiz 5

Question 1

the primary structure of a protein

Answer 1

provides information about its function

Question 2

in cells, this potential variety

Answer 2

is limited by the efficiency of protein synthesis and by the ability of the polypeptide to fold into a functional structure

Question 3

the peptide backbone

Answer 3

-N-Ca-Co

Question 4

N

Answer 4

the amide nitrogen of the amino acid

Question 5

Ca

Answer 5

is the alpha carbon of the amino acid

Question 6

Co

Answer 6

is the carbonyl carbon of the amino acid

Question 7

peptide formation is the creation of an

Answer 7

amide bond between the carboxyl group of the first amino acid and the amino group of the next amino acid

Question 8

condensation reaction

Answer 8

results in a low of water

Question 9

the peptide bond

Answer 9

• adopts a trans conformation
• due to the partial double bond character of the peptide bond, the six atoms of the peptide-bond group define a plane the AMIDE PLANE

Question 10

environmental conditions that affect a proteins stability during purification

Answer 10

• pH
• temperature

Question 11

an assay based on a proteins chemical or binding properties

Answer 11

may be used to quantify a protein during purification

Question 12

fractionation procedures

Answer 12

take advantage of a proteins unique structure and chemistry in order to separate it from other molecules

Question 13

” salting out”

Answer 13

• increasing the salt concentration causes selective salting out [precipitation ] of proteins with different solubilities

Question 14

what influences a proteins chromatographic behavior

Answer 14

• charge
• polarity size
• ligand-binding

Question 15

ultracentrifugation

Answer 15

what the overall size and shape of macromolecules and larger assemblies can be assessed through.

Question 16

salting out [1]

Answer 16

separates based on protein solubility in high ammonium acetate concentration

Question 17

chromatography [1]

Answer 17

separates based on a proteins ionic charge, polarity, size or ligand binding ability.

Question 18

gel electrophoresis [1]

Answer 18

and its variations can separate proteins according to charge, size, and isoelectric point

Question 19

ultracentrifugation [1]

Answer 19

separates based on the overall size and shape of macromolecules and larger assemblies

Question 20

below the isoelectric point or PI

Answer 20

[the pH at which there is a net charge of 0] the protein will carry a net positive charge

Question 21

above the pl

Answer 21

a net negative charge

Question 22

positively charged proteins

Answer 22

bind cation exchange columns

Question 23

negatively charged proteins

Answer 23

bind anion exchange columns

Question 24

proteins are eluted [released] by

Answer 24

high salt concentration

Question 25

dialysis

Answer 25

semipermeable membrane bag is immersed in beaker of solution, diffusible solutes in the dialysis bag equilibrate across the membrane, while your protein stays inside

Question 26

small proteins are impeded

Answer 26

by access to the inside of the gel beads, delaying travel through the column

Question 27

large proteins

Answer 27

have no access to the matrix of the gel beads and flow easily around gel beads

Question 28

proteins are eluted in the order

Answer 28

largest to smallest

Question 29

small-molecule targets (ligands, black)

Answer 29

are immobilized through covalent attached to a solid matrix (tan) column

Question 30

absorbance

Answer 30

dated the presence of proteins, but it does not distinguish between different proteins

Question 31

proteins in a small sample can be separated by

Answer 31

electrophoresis, based on their size, using a semi-solid phase gel.

Question 32

after separation, proteins are detected by staining with

Answer 32

coomassie brilliant blue for visualization of "bands" representing the proteins of different sizes

Question 33

SDS page

Answer 33

sodium dodecyl sulfate poly acrylamide gel electrophoresis. small samples are taken from fraction tubes, SDS is added, samples are boiled to denature and coat all of the amino acids with negative charges and separated by electrophoresis on a gel

Question 34

260 nm is problematic, why?

Answer 34

at 280nm there is a strong absorbance by Tyr and Top but not Phe

Question 35

A reducing agent, such as DTT or BMe

Answer 35

is needed to disrupt structure but does not break disulfide bonds

Question 36

a proteins mobility in the gel

Answer 36

is inversely proportional to the molecular weight

Question 37

separation is based on

Answer 37

size, without irreversibly denaturing the protein

Question 38

to be sequenced

Answer 38

a protein must be separated into individual polypeptides that can be cleaved into sets of overlapping fragments

Question 39

the amino acid sequence can be determined by

Answer 39

Edman degradation, a procedure for removing N-terminal residues at one time

Question 40

mass spectromy

Answer 40

can identify amino acid sequences from the mass-to-charge ration of gas-phase protein fragments

Question 41

proteins can be sequenced in two ways

Answer 41

1. direct amino acid sequencing 2. sequencing the corresponding dna of the gene today, gene databases provide these deduced protein sequences

Question 42

acid hydrolysis

Answer 42

liberates the amino acids of a protein - some amino acids are partially or completely destroyed by acid hydrolysis especially, trp

Question 43

chromatography is used

Answer 43

to separate amino acids

Question 44

the amino acid compositions

Answer 44

of different proteins are different

Question 45

the sequence of amino acids in a protein is

Answer 45

distinctive but composition is not

Question 46

subunit interactions depend on weak forces, which can

Answer 46

- be denatured

Question 47

denaturation can be achieved with

Answer 47

- extremes of pH - high urea concentration - high guanidine HCL concentration - high salt concentration

Question 48

disulfide reducing agents

Answer 48

-2-mercaptoethanol, B-me - dithiothreitol, DTT

Question 49

to prevent disulfide bridges from reforming, follow with

Answer 49

an irreversible alkylating agent, like iodoacetate, IOAc

Question 50

n terminal analysis, uses

Answer 50

edmans reagent - derivatives are PTH

Question 51

c-terminal analysis is enzymatic with carboxypeptidases

Answer 51

- carboxypeptidase A cleaves any residue except PRO, ARG, AND LYS - carboxypeptidase B cleaved only works on arg and lys

Question 52

enzymatic fragmentation

Answer 52

- trypsin - chymotrypsin - staphylococcal protease

Question 53

chemical fragmentation

Answer 53

cyanogen bromide

Question 54

trypsin

Answer 54

cleavage on the C side of Lys, Arg [basic]

Question 55

chymotrypsin

Answer 55

C side of Phe , Tyr, Trp, less so Leu [large nonpolar]

Question 56

staphylococcal protease

Answer 56

- c side of glu, asp[acidic] in phosphate buffer pH7 - specific for glu, in acetate [pH 5] and bicarbonate. buffer [pH 10]

Question 57

cyanogen bromide

Answer 57

- CNBr acts only on methionine residues - CNBr useful because proteins usually have only a few Met residues - a peptide with a C-terminal homoserine lactone

Question 58

compare and align overlapping peptide sequences

Answer 58

to learn the sequence of the original polypeptide chain

Question 59

mass spec [MS]

Answer 59

mass spectrometry separates particles on the basis of mass-to-charge ratio