Biochem midterm 2 protein structure and isolation methods Flashcards

(47 cards)

1
Q

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

A

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

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2
Q

homodimer proteins are composed of what kind of subunit

A

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

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3
Q

bond structure of amino acids central carbon

A

Tetrahedral

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4
Q

L vs D conformation of amino acids

A

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)

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5
Q

Charged amino acids

A

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

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6
Q

hydrophilic amino acids

A

Serine, threonine, cysteine, asparagine, glutamine

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7
Q

hydrophobic amino acids

A

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

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8
Q

Aromatic amino acids

A

Phenylalanine, tyrosine, tryptophan

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9
Q

how are amino acids joined together

A

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

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10
Q

peptide bond

A

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

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11
Q

what is a Ramachandran Plot

A

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

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12
Q

small structural fluctuations

A

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

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13
Q

primary protein structure

A

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

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14
Q

Secondary structure

A

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

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15
Q

Tertiary protein structure

A

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

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16
Q

Quaternary protein structure

A

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

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17
Q

amphipathic alpha helix characteristic

A

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

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18
Q

beta strands

A

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

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19
Q

B sheets

A

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

20
Q

Type 1 and 2 B turns

A

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

21
Q

Loops

A

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

22
Q

do secondary structures increase or decrease steric hindrance

A

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.

23
Q

how do Quaternary Structures provide functionality to proteins

A
  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)
24
Q

keratin

A

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

25
Where does silk get its strength?
silk is composed of 3 subunits which is almost inertly made from B sheets providing it with its strength
26
Collagen
major connective tissue and found in the cornea of the eye structure consists of 3 interwound left handed helices
27
causes of scurvy
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
28
the 6 key themes to protein folding
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
29
proper Denaturation and renaturation of proteins
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
30
Assisted folding
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)
31
The GroEL–GroES Protein-Folding Cycle
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
32
3 main methods used to disrupt protein structure
sonication -sound shearing -use of a pestle to break structure detergents - chemically break down structure
33
how does centrifugation work?
spins at high RPM's Appling high G's to the sample, this separates the componets based on density
34
preparative centrifugation
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
35
Gel filtration chromatography
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
36
ion-exchange chromatography
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
37
affinity chromatography
separates proteins based off ligand interactions
38
SDS-PAGE
detergent is used to denature the protein then migrates though the PAGE via gel electrophoresis the separation seen is based on MW
39
2-D PAGE
separates proteins based on both pl point and MW
40
Isoelectric Focusing Electrophoresis
separates proteins based on pl point
41
The Edman Degradation method
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
42
Differential protease cleavage
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
43
Tandem Mass spectrometry
composed of 4 chambers first being used for electrosapry ionozation, second petide separation, thrid petide fragmentation and 4th fragment seperation
44
X-ray Crystallography
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
45
NMR
provides information of the locations of atoms within a protein based on nuclear spin
46
Cryo-electron microscopy
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.
47
polyclonal vs monoclonal antibodies
poly- recognize many epitopes on a antigen whereas mono only recognize one