Test 1 Ch. 3

Front 1

What conformational change occurs in pathogenic proteins?

Back 1

Alpha helical regions reform into beta-sheets that readily aggregate into oligomers and amyloid fibrils

Front 2

How does the ER respond to the presence of unfolded proteins?

Back 2

The size of the ER is increased to increase capacity of cell to process secretory proteins and aid proper folding

Front 3

How does Spec techniques intersect with the use of an Assay?

Back 3

Spectroscopy can be used to follow the formation of product by observing changes in abosorbance

Front 4

What purification method seperates by charge, size, shape, and oligemeric state?

Back 4

Native PAGE

Front 5

What are some homogenization methods to lyse cells?

Back 5

Blender, French Press, and Sonication

Front 6

What is the most commonly used salt for salting out and why?

Back 6

Ammonium sulfate because it is unusualy soluble is cold buffers, and it yields a precipitated protein slurry that is stable

Front 7

What is the effect of a low salt concentration (.1-.5M) in protein solutions?

Back 7

Increased ionic strength which shields unfavorable electrostatic interactions thereby increasing solubility

Front 8

What happens to water/protein interactions at >1M salt concentrations

Back 8

Water that normally would have solvated the protein now solvates the salt ion

The protein loses its hydration shell and precipitates out

Front 9

How is the surface of beads charged in cationic exchange chromatography?

Back 9

Negatively charged beads

Front 10

In anion exchange chromatography, protons with a weak negative charge will elute _____ than proteins with strong negative charge. Why?

Back 10

Earlier, because the weak negative charges will bind to the positive beads less strongly than those with a stronger negative charge

Front 11

Describe Nickel-Affinity Chromotography

Back 11

Proteins of interest have a Histidine tag attached that can be trapped by nickel compounds on beads

Front 12

Gel Filtration separates based on ____. How does it work?

Back 12

Size, it works because small molecules can enter the porous beads but large ones cannot, thus the smaller ones elute later since they're trapped

Front 13

What filtration method can indicate the presence of a quarternary structure?

Back 13

Gel filtration

Front 14

How does gel filtration volume indicate quarternary structure?

Back 14

If the protein elutes earlier than expected at some multiple of the expected monomer, we can assume it is part of a multimeric complex

Front 15

What purification method effectuates the highest purity? Which effectuates the lowest purity?

Back 15

Affinity chromatography, Homogenization

Front 16

What purification method gives the highest protein yield? Which gives the lowest?

Back 16

Homogenization, Affinity Chromatography

Front 17

How does the specific activity of an enzyme change as it is subsequently purified?

Back 17

Specific activity increases with purity levels

Front 18

What are the steps in protein sequencing?

Back 18

Free amino acid analysis --> C-terminus and N-terminus end determinations --> Sequence determination --> Proteolytic digestion to sequence

Front 19

What compound, time, and temperature do we use for amino acid analysis?

Back 19

We hydrolyze the protein at 6M HCl, for 25 hours, at 100* Celcius

Front 20

How do we separate amino acid for analysis?

Back 20

Ion-exchange chromatography

Front 21

What reagent do we use to detect amino acids? What conditions are needed for this reaction?

Back 21

Ninhydrin for 2-5 minutes at 100* Celcius gives nanomole level detection

Front 22

What reagents do we use for N-terminal determination?

Back 22

1) DNFB = Sanger's Reagent = dinitrofluorobenzene

2) DANSYL Chloride

Front 23

What are the steps to an Edman's degradation?

Back 23

1) Treat peptide with PITC at pH = 9 to form a (PTC)-peptide

2) Treat PTC-peptide with TFA (trifluoroacetic acid) to cleave N-terminal peptide

3) Rearrange thazoline derivative to a PTH-amino acid with HCl then chromatograph

Front 24

What happens to the N-terminus upon raction with PITC (phenylisothiocyanate)?

Back 24

N-terminus forms a (PTC)-peptide

Front 25

What happens to the PTC-peptide upon reaction with TFA?

Back 25

N-terminal PTC-peptide is cleaved and forms a thizaoline derivative

Front 26

What does CNBr do?

Back 26

Preferentially cleaves at the C-terminal side of methionine

Front 27

What does Trypsin do?

Back 27

Preferentially cleaves at C-terminal side of Lysine or Arginine

Front 28

What does Chymotrypsin do?

Back 28

Cleaves at C-terminus side of aromatic structures (Y, T, F) and bulky structures (L, M)

Front 29

What does Carboxypeptidase A do?

Back 29

Cleaves N-terminus side of C-terminal amino acid

Front 30

What amino acid with Carboxypeptidase not cleave?

Back 30

R, L, and P

Front 31

What are the steps of MALDI-TOF Mass Spec?

Back 31

1) Peptides are mixed with matrix and applies to well on target plate

2) Peptide ions are generated by a LASER

3) TOF allows us to extrapolate relative masses

Front 32

NOE effect identifies paired protons that are <___angstoms apart

Back 32

5

Protons closer than 5 angstroms apart will perturb the spins of other protons

Front 33

What is the premier technique for studying protein dynamics?

Back 33

NMR!

Front 34

What information does NMR give?

Back 34

Inter-atomic distances, not absolute position of atoms

Front 35

What are the size limitations of NMR?

Back 35

Limited to small proteins of 150-200 residues

Up to 20kDa

Front 36

How do we proceed from protein to atomic model with X-ray crystallography?

Back 36

Protein crystal --> Diffraction pattern --> Electron density map --> Atomic model

Front 37

What is Bragg's law for diffraction?

Back 37

n(lambda) = 2d sin (theta)

n= refractive index

lambda = wavelength of X-ray

d = spacing between atoms

thera = angle of incidence

Front 38

What assumption of from Bragg's law allows us to calculate structure with X-ray crystallography?

Back 38

The diffraction pattern is determined by the spacing between atoms, and thus we can calculate structure from diffraction pattern

Front 39

What raw data does X-ray crystallography provide?

Back 39

Position of reflection points on the reciprocal lattice

Intensity of reflection

Front 40

What is the relationship between the reciprocal lattice and real lattice?

Back 40

Reciprocal lattice is the coordinate system of the diffraction pattern

Real lattice is the coordinate system within a unit cell

Front 41

How do we generate X-rays?

Back 41

Bombard a metallic anode with electrons

Front 42

What is the spacing of spots in an x-ray crystallography reciprocal lattice due to?

Back 42

Due to size and symmetry of your crystal lattice

Front 43

What are the steps in X-ray Crystallography?

Back 43

1) Create Crystal

2) Space group determination to get crystal symmetry

3) Get diffraction data

4) Extrapolate electron density from diffraction with a Fourier Transformation

5) Phase determination by Isomorphous Replacement due to phase problem

6) Refinement of atomic model

Front 44

What is a Fourier Transform

Back 44

An equation that transforms a function with some variables into a function with reciprocal variables

It's how we go from reciprocal lattice to real lattice

Front 45

What is the phase problem?

Back 45

When extrapolating electron density with fourier transformations, we lose phases of electrons, so electron density cannot be directly calculated and instead we need to estimate them

Front 46

How do we overcome the phase problem?

Back 46

We do phase determination by isomorphous replacement (MIR)

We get diffraction data for heavy atom derivatives which diffract x-rays stronger than light atoms and compare them to the native data.

Using a patterson function, we can then calculate the phases of the rest of the molecules

Front 47

What is considered High Resolution crystallography? Why can we visualize at high res?

Back 47

1 - 2.5 angstroms

We can see side chains and interchain interactions

Front 48

What is considered Low Resolution crystallography? Why can we visualize at low res?

Back 48

3.5 - 4 angstom

We can see secondary structures

Front 49

How do we get highly ordered crystals for good x-ray crystallography results?

Back 49

Vapour diffusion

Front 50

What characteristics of a crystal lead to good x-ray crystallography data?

Back 50

High purity and conformation homogeneity

Front 51

What are the limitations of X-ray crystallography?

Back 51

1) Not all proteins crystallize

2) Not all crystals diffract high resolution structures

3) Protein packing with a crystal can distort 3* structure

4) Proteins can only be studied as crystals rather than in vivo

Front 52

What x-ray crystallography data is collected for structure determination?

Back 52

Coordinates and intensities of reflection {h,k,l}

Front 53

What are the methods of chromatography?

Back 53

Gel-filtration, Ion-exchange, affinity chromatography, High-pressure liquid, Hydrophobic interaction chromatography

Front 54

What are the methods of electrophoresis?

Back 54

Gel electrophoresis, Isoelectric focusing, and 2-D electrophoresis

Front 55

Define Hydrophobic interaction chromatography

Back 55

Separates on hydrophobicity, has good resolution, and high capacity