Proteins: General and spectroscopic properties

Question 1

All amino acids are chiral, with one exception. Which one?

Answer 1

Glycine (R-group = Hydrogen).

If a molecule is chiral, it can NOT be superimposed on its mirror image. Chiral centers are carbon atoms with four different ligands. The ligands positioning in space is what prohibits chiral reflections from being superimposed on one another.

Question 2

Do naturally formed amino acids host a D- or L-chirality?

Answer 2

L-chirality (left-handed). This means that the “left mirror image” is the natural form of the amino acids.

Question 3

Which are the positive polar amino acids?

Answer 3

Arginine
Lysine
Histidine (~10% of histidine is positive @ pH7)

Question 4

Which are the negative polar amino acids?

Answer 4

Glutamate
Aspartate

Question 5

What’s special about histidine with regards to polar charge?

Answer 5

Histidine can be positive or negative depending on the pH context.

Question 6

What’s special about cysteine compared to other amino acids?

Answer 6

Two cysteins can form a disuflite bridge. This can occur within a protein or between two separate proteins.

Question 7

What’s special with proline compared to other amino acids?

Answer 7

The R-group is a ring structure, which inhibits its ability to form hydrogen bonds, this makes proline a helix breaker.

Question 8

What’s special about glycine?

Answer 8

1. It’s not chiral (like all other amino acids)
2. The R-group is hydrogen, this allows glycine to act like a hinge in helixes.

Question 9

Explain what is displayed in the primary, secondary, tertiary and quarternary structures of a protein.

Answer 9

1. Primary structure: Linear (displayed as a sequence)
2. Secondary: local regularity (alfa helix, beta strands). Displayed linearly displayed as a sequence
   2*. Super-secondary: local regularities (displayed in 2D)
3. Tertiary: The overall folding of a protein
   4.Quarter: The interactions between multiple protein domains to make up the final protein complex.

Question 10

Is the following statement true?: In alfa-helixes, the hydrogen groups are pointed outwards, the R-groups are pointed inwards.

Answer 10

No. The R-groups are pointed outwards. This makes sense as the R-groups are what facilitate the chemical reactions in which the protein can take place.

Question 11

How many amino acid residues are there per turn in an alfa-helix?

Answer 11

3.6 resideus / turn

Question 12

True or false: Are there parallel AND anti-parallel beta-sheets?

Answer 12

Yes.

Question 13

What chemical interaction is instrinsic to disulfite bridges, zinc finger protein interactions and cytochrome interactions?

Answer 13

They are all covalent interactions.

Question 14

Which two chemical interactions take part in the formation of salt bridges?

Answer 14

H-bonds, electrostatic bonds, ionic bonds

Question 15

Why does base stacking improve the stability of a protein structure?

Answer 15

Base stacking generally occurs between residues which have planar ring structures, like tryptophan. The ring structures are generally hydrophobic, thus, they will be “attracted” to one another.

Question 16

What are van der Waal interactions?

Answer 16

Short lived interactions between temporary dipole formations.

Question 17

What are Edman degradation and Tandem mass spectrometry used for?

Answer 17

Edman degradation and tandem mass sspectrometry are used for assaying protein composition.

Question 18

What are HPLC based analysis (glycosylation analysis) and mass spectronomy used for?

Answer 18

HPLC and mass spectronomy methods are used for assaying post-translational modifications. MS is used to find the mass / charge ratio, from which you can extrapolate the mass.

Question 19

What are atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS) used for?

Answer 19

AAS and ICP-MS are used for metal ion analyses.

Question 20

What does spectrofluorimetry measure?

Answer 20

The emitted light from the sample. Individual wavelengths are tested in order to characterize where absorption and emission occur.

Fluorescence is measured by a spectrometer. There is no detector behind the sample, thus, no spectrophotometer that observes transmitted light.

Question 21

What does spectrophotometry measure?

Answer 21

The light absorbed by the sample.

Question 22

What is the relationship between absorption and transmission?

Answer 22

Absorption: The amount of light which transfers energy to sample.

Transmission: The amount of light which passes through the sample.

Transmission = 1 - (absorption + scatter)

Question 23

What’s the relationship between luminescence, fluorescence and phosphorescence?

Answer 23

Fluorescence + phosphorescence = Luminescence.

Phosphorescence remains when the light source is removed. Fluorescence only refers to the immediate light reaction after having excited a chromophore.

Question 24

What’s the difference between a spectrophotometer and a spectrometer?

Answer 24

A spectrometer has its detector 90 degrees off-angle from the illumination source. Transmitted light is not observed. Detects sample luminescence.

A spectrophotometer has its measurement device straight behin the sample, observing transmittance.

Question 25

what’s “quantum yield” a measurement of?

Answer 25

it measures the efficiency of fluorescence.

Photons emitted from sample / samples absorbed by the sample.

Question 26

What three mechanims can cause fluorophore quenching?

Answer 26

Dynamic queenching (via collisions): The fluorophore is deactivated through interactions with other molecules.

Static quenching: The fluorophore is statically bound in a complex.

Resonance energy transfer: The fluorophore absorbs light, gets excited, emits a wavelength, which in turn is absorbed by another fluorophore, that fluoresces.

Question 27

Name an application of fluorophore quenchning.

Answer 27

Assaying whether a protein is folded or not. The less folded the protein, the more fluorescence.

Question 28

What’s is FRET? what’s the mechanism of FRET? Give an example of two fluorophores which have this relationship.

Answer 28

FRET = Fluorescence resonance energy transfer.

Two fluorophores are near each other, one of them is excited, which in turn excites the other one. CFP and YFP have this relationship.

FRET can be used to see if two proteins are in interactive range by ex fusing CFP and YFP to different proteins and assaying whether FRET can occur.