LT3 NMR Side-Chain Assignment

Question 1

What don’t we know after doing HNCACB and CBCAA(CO)NH?

Answer 1

We know the correct order of the residues

But don’t know which amino acid the peak is yet

Question 2

What does TOCSY stand for

Answer 2

Total Correlation Spectroscopy
For ALIPHATIC side chain assignment

Question 3

What is TOCSY used for?

Answer 3

Correlates amide groups (N-15 labelled) to all aliphatic H (protons) in the SAME amino acid residue

Question 4

What will each amide strip of a TOCSY show?

Answer 4

Each amide strop will show H chemical shift of Ha, Hb, Hy from the same residue in the 3D

Question 5

In a TOCSY, can we always see all aliphatic protons?

Answer 5

No

If transfer is not enough, sometimes only Ha and Hb can be observed

Question 6

What are the axes of the TOCSY?

Answer 6

H1 vs NH

Question 7

How do we get secondary structure from chemical shift index?

Answer 7

Secondary shift = different between H/C chemical shift for each residue and that reported for the same residue type in a “random coil” conformation

Experimental - random coil = secondary

Question 8

What does the Chemical Shift Index (CSI) tell us?

Answer 8

Correlates between chemical shift tendencies and secondary structure

Question 9

What are the values of a Ha or Cb secondary shift?

Answer 9

-ve for alpha helix
+ve for beta-sheet

Question 10

What are the values of a Ca secondary shift?

Answer 10

+ve for alpha helix
-ve for beta-sheet

Question 11

What does NOE stand for and what is it?

Answer 11

Nuclear Overhauser Effect = exist only between two H separated by less than 5 angstrom

Question 12

What does an observable NOE tell us?

Answer 12

Means the two H are in close vicinity to each other

Question 13

Describe the difference in NOE for the secondary structures

Answer 13

Alpha-helix = characterized by the close approach between residue i and i+3

Extended beta-sheet = excludes medium range distances
Close proximity of 2 strands brings about inter-strand distances

Question 14

What is the difference between N-15 TOCSY and NOESY?

Answer 14

TOCSy will only protons on that SAME residue

NOESY will only show protons within 5 angstrom = including side chains from other residues

Question 15

What does intensity on a NOESY tell us?

Answer 15

How close the H are to each other

Question 16

What is NOE cross-peak intensity proportional to?

Answer 16

1/r^6

Question 17

Why do HSQC-TOCSY and HSQC-NOESY show different things?

Answer 17

TOCSy = through bond expt, gives chemical shift of all H within same residues as the HN

NOESY = through space expt, gives chemical shift of all H within a distance of 5A from particular HN (amide proton)

Question 18

What do the different dxy(i, i+z), dNN(i,i+1), daN, dbN show?***

Question 19

Define intra-residue NOE

Answer 19

Between H within the same residue

Question 20

Name the 3 inter-residue NOE

Answer 20

Sequential = adjacent residues
Medium range = i to i+4
Long range = more than 4 residues apart

Question 21

Definer inter-molecular NOE

Answer 21

Between two molecules = dimer or beta-strands

Question 22

What NOEs define what structures of proteins?

Answer 22

Secondary = short and medium range
Tertiary = long range
Quaternary = inter-molecular

Question 23

Are daN and dNa different in NOE?

Answer 23

YES

Depict different places between residues

Question 24

What NOEs can we find in alpha-helix?

Answer 24

Sequential and medium range

dNN(i, i+1), daN(i, i+3), daN (i, i+4) and dbN(i, i+1)

Question 25

What NOEs can we find in beta-sheet?

Answer 25

Sequential and inter-strand daN (i, i+1)

Question 26

What type of NOE defines tertiary structures?

Answer 26

Long-range NOEs

Question 27

How can we tell if beta-sheets are parallel or anti-parallel?

Answer 27

Parallel = N and N terminals will be close and C-C terminals will be close Antiparallel = N-terminus close to C-terminus

Question 28

Are anti- or parallel beta-sheets closer and why?

Answer 28

Antiparallel sheets have closer beta-strands because the H bonds are more linear

Question 29

What is the coupling constant between?

Answer 29

J = measured between pairs of protons separated by 3 covalent bonds Contains information about intervening torsion angle

Question 30

Which dihedral angle is used for J and why?

Answer 30

Phi angle Because related to both secondary and tertiary structure

Question 31

What is the coupling constant for alpha-helices and beta-sheets?

Answer 31

a-helices = 4Hz b-sheets = 9Hz

Question 32

What are the phi angles for right-handed a-helix and anti-parallel b-sheet?

Answer 32

a-helix = -57 b-sheet = -139 Larger angle = larger distance in Hz of the peak

Question 33

What are labile protons, and what happens to them in the presence of heavy water (D2O)?

Answer 33

Labile protons = easily exchangeable (--NH or -OH) In presence of heavy water, H-1 get exchanged for H-2 H-2 cannot be detected by HSQC because gives out different resonance freq

Question 34

How are hydrogen bonds useful in determining standard secondary structure and tertiary structures in NMR?***

Question 35

What 3 restraints are usually used for NMR structure calculation?

Answer 35

NOE distance restraints=strong, medium, weak Dihedral angle restraints from coupling constant Hydrogen bond restraints

Question 36

What does the computer do to generate an ensemble of structures that obey the constraints?

Answer 36

Use simulated annealing and energy minimization

Question 37

What happens if the number of constraints is larger?

Answer 37

The ensemble structure will be very similar to each other Having a SMALL root mean square deviation (rmsd) between their backbone and side chain

Question 38

Summarize the steps of protein NMR

Answer 38

Get a solution of proteins with C-13 and N-15 Triple resonance 3D experiments Backbone and side chain sequential assignment by through-bond experiments Distance and dihedral angle restraints through-space and coupling constant experiments

Question 40

What are the cons to NMR?

Answer 40

Protein size is limited High solubility is needed Conformational exchange problems Distance restraints assignment and structure refinement

Question 41

What are the prerequisites needed for NMR?

Answer 41

Over-expression of folded protein in minimal medium ~95% purity Highly soluble Stable at greater than or equal to 25 degrees for a week