Biochem midterm 2 protein structure and isolation methods

Question 1

heterotrimer protein’s are composed of what type of sub unit

Answer 1

Protein complexes can consist of individual protein subunits encoded by separate genes

Question 2

homodimer proteins are composed of what kind of subunit

Answer 2

Identical protein subunits encoded by the same gene (a complex of identical protein subunits

Question 3

bond structure of amino acids central carbon

Answer 3

Tetrahedral

Question 4

L vs D conformation of amino acids

Answer 4

in the L conformation the amino group is facing the Left while the carboxyl group is facing the right

in the D conformation the Amino group will be facing the right while the carboxyl group is facing the left

(both conformations have the side chain pointing up)

Question 5

Charged amino acids

Answer 5

Neg: aspartate, glutamate
pos: lysine, arginine, histidine

Question 6

hydrophilic amino acids

Answer 6

Serine, threonine, cysteine, asparagine, glutamine

Question 7

hydrophobic amino acids

Answer 7

Glycine, alanine, proline, valine, leucine, isoleucine, methionine

Question 8

Aromatic amino acids

Answer 8

Phenylalanine, tyrosine, tryptophan

Question 9

how are amino acids joined together

Answer 9

Peptide bonds formed between the carboxyl group of the first amnio acid and the amino group of the second

Question 10

peptide bond

Answer 10

due to resonance the peptide bond has some double bond character thus lacks rotation round the C-N bond

Question 11

what is a Ramachandran Plot

Answer 11

a Ramachandran Plot shows the allowable torsional angles of amino acids φ (phi) and ψ (psi) in a protein sequence due to steric hinderance. the dark blue areas show the locations where you are most likely to find a compatible angle

Question 12

small structural fluctuations

Answer 12

part of protein structure, areas where the protein is able to move more freely this aids in ligand binding and other mechanisms

Question 13

primary protein structure

Answer 13

made from the sequence of amino acids which composes a polypeptide chain
this structure will determine how the polypeptides will fold

Question 14

Secondary structure

Answer 14

local confirmation of part of the polypeptide
repetitive patterns of local regions of the polypeptide backbone
B strand
A helix
B turn

Question 15

Tertiary protein structure

Answer 15

overall shape of a single protein chain
includes spatial location of all atoms in the polypeptide
depends heavily on the R group interactions

Question 16

Quaternary protein structure

Answer 16

only applies to multimeric proteins which are proteins composed of more then one polypeptide chain

Question 17

amphipathic alpha helix characteristic

Answer 17

will have both a hydrophobic and a hydrophilic face this allows for more effective interactions with membranes

Question 18

beta strands

Answer 18

arrow runs from Amino group to C terminus end
often rotated rather then strictly planer
form of secondary structure

Question 19

B sheets

Answer 19

formed via the combination of 2 or more beta strands is a quaternary structure

Question 20

Type 1 and 2 B turns

Answer 20

In the type 1 beta turn the carbonyl oxygen is oriented inward
in the type 2 beta turn the carbonyl oxygen is oriented outward
the function of these structures is to connect A helices and B strands

Question 21

Loops

Answer 21

share same function as B turns however they are much larger the B turns, these types of structures are usually found on the surface of proteins

Question 22

do secondary structures increase or decrease steric hindrance

Answer 22

secondary structures in proteins often are oriented so that they minimize hindrance thus will often be found in low energy region on the Ramachandran plot.

Question 23

how do Quaternary Structures provide functionality to proteins

Answer 23

1. many subunits provide structural properties that are impossible with just single subunits
2. many subunits allow for regulation though conformational changes that alter subunit interfaces
3. having many subunits increases efficiency of processes by linking functional components into close proximity (can preform many more tasks with more subunits)

Question 24

keratin

Answer 24

fibrous coiled protein which is made from 2 helical polypeptides linked via disulfide bonds (covalent)
the reason why hair is curly

Question 25

Where does silk get its strength?

Answer 25

silk is composed of 3 subunits which is almost inertly made from B sheets providing it with its strength

Question 26

Collagen

Answer 26

major connective tissue and found in the cornea of the eye
structure consists of 3 interwound left handed helices

Question 27

causes of scurvy

Answer 27

ascorbate (vit C) is used in a reaction which converts proline to hydroxyproline, in the absence of vit C Hydroxyproline cannot be made, this is a very important molecule as it provides stability to the collagen helix, so in the absence of Hydroxyproline the symptoms of scurvy are observed

Question 28

the 6 key themes to protein folding

Answer 28

1. 3D structure observed in proteins are determined via there amino acid sequence
2. function depends on form
3. most proteins exist in one stable structure which is energetically favorable
4. most important factor is protein stabilizing is noncovalent interactions
5. there are patterns which aid in understanding protein structure
6. proteins are not static structures rather they are quite dynamic

Question 29

proper Denaturation and renaturation of proteins

Answer 29

in a process which used Urea and BME to chemically break down the protein to renature back to full function first urea must be removed followed by BME

Question 30

Assisted folding

Answer 30

molecular chaperones are proteins which interact with improperly or partially folding proteins and facilitate correct folding
Hsp70/Hsp40 family (DnaK/DnaJ in bacteria)
Chaperonins (GroEL/GroES in bacteria)

Question 31

The GroEL–GroES Protein-Folding Cycle

Answer 31

GroES and ATP bind to GroEL trapping the unfolded protein within the folding chamber

conformational changes in the folding structure allow for the release of GroES and a folded protein

a new unfolded protein will now enter the empty chamber and the process repeats

Question 32

3 main methods used to disrupt protein structure

Answer 32

sonication -sound
shearing -use of a pestle to break structure
detergents - chemically break down structure

Question 33

how does centrifugation work?

Answer 33

spins at high RPM’s Appling high G’s to the sample, this separates the componets based on density

Question 34

preparative centrifugation

Answer 34

after disruption of cell structure the contains can be centrifuged at varying RMP’s resulting in the isolation of 4 fractions which contain progressively less dense cellular components as G’s increase

Question 35

Gel filtration chromatography

Answer 35

Separates proteins based on size
uses gel beads with pores, these if the protein can fit in the pore it will elute much slower as it takes a longer path then the larger proteins which are able to avoid the pores and take a more direct rout through the gel

Question 36

ion-exchange chromatography

Answer 36

separates proteins based on size
uses charged beads, oppositely charged proteins with respect to the beads will stick and same charge proteins will elute quickly

Question 37

affinity chromatography

Answer 37

separates proteins based off ligand interactions

Question 38

SDS-PAGE

Answer 38

detergent is used to denature the protein then migrates though the PAGE via gel electrophoresis the separation seen is based on MW

Question 39

2-D PAGE

Answer 39

separates proteins based on both pl point and MW

Question 40

Isoelectric Focusing Electrophoresis

Answer 40

separates proteins based on pl point

Question 41

The Edman Degradation method

Answer 41

the protein is chemically tagged at the terminal amino group with PITC in the presence of a base, acid is then added to the solution cleaving the carboxyl group of the tagged amino acid, this can then be isolated and identified this process can be repeated for the for the remaining amino acids in the protein

Question 42

Differential protease cleavage

Answer 42

since Edman degration is only accurate to to a certain extent it is more effective to use enzymes to cleave the polypeptide sequence these smaller segments then using a overlapping method fill in the blanks and create the full polypeptide

Question 43

Tandem Mass spectrometry

Answer 43

composed of 4 chambers first being used for electrosapry ionozation, second petide separation, thrid petide fragmentation and 4th fragment seperation

Question 44

X-ray Crystallography

Answer 44

protein is first crystalized and a x ray beam is shot threw it and the diffracted rays are captured on a sheet the points of diffraction can then give information on the electron dense locations within the protein

Question 45

NMR

Answer 45

provides information of the locations of atoms within a protein based on nuclear spin

Question 46

Cryo-electron microscopy

Answer 46

uses electron beams to cast a shadow of the protein this is done to the protein in many orientation the images which are captured can then be put together via a computer program.

Question 47

polyclonal vs monoclonal antibodies

Answer 47

poly- recognize many epitopes on a antigen whereas mono only recognize one