Nuclear Magnetic Resonance of Proteins
[1] Tracing NOE Connectivities
Weber et al. (Weber PL, Wemmer DE, and Reid BR (1985) Proton NMR studies of λ cro repressor. 2. Sequential resonance assignments of the proton NMR spectrum Biochemistry 24 4553-62) reported sequential resonance assignments of the λ cro repressor protein, a small (66 amino acid) sequence-specific DNA binding protein. The sequence of this protein from amino acid seven through ten (inclusive) is LKDY. The NOESY spectrum of this protein in the amide proton (NH) region (chemical shifts of 6–10 ppm) is shown below . Beginning with L7 at 7.65 ppm, estimate the chemical shifts of the amide protons through amino acid Y10 (hint: K8 has been done for you, follow arrows 1 & 2, and continue the pattern).
Show Answer
The trace of NOE connectivities are shown in the figure below. Starting at K8 (8.9 ppm) and looking to the left shows a large area devoid of cross peaks. Thus the chemical shift of D9 must be lower than that of K8. Leaving the diagonal veritcally (arrow 3), there is a cross peak at 7.42 ppm (note that the arrows are offset by a bit so that you can still clearly see the cross peaks). Returning to the diagonal along arrow 4, notice that you pass through a cross peak (with Y10) before you reach the diagonal. To find Y10, it is back to the cross peak you've seen before and return to the diagonal at 7.75 ppm. Thus the chemical shifts are:
| Nucleus | Chemical Shift (ppm) |
| L7 | 7.65 |
| K8 | 8.90 |
| D9 | 7.42 |
| Y10 | 7.75 |
[2] Linking Spin Systems
Ribonuclease Sa (RNase Sa) is the smallest member of the microbial ribonuclease family. The sequence of RNase Sa is given below.
1 DVSGTVCLSA LPPEATDTLN LIASDGPFPY SQDGVVFQNR ESVLPTQSYG YYHEYTVITP 60
61 GARTRGTRRI ITGEATQEDY YTGDHYATFS LIDQTC 96
While using NOESY data to connect your COSY data, the following sequence of spin systems was elucidated.
[AMX][AMX][A3MX][AX][AMX][AMX][AMX]
Using the spin system idenifications below, identify the amino acids linked by NOE data.
| Spin System | Amino Acid |
| AX | G |
| A3X | A |
| A3B3X | V |
| A3MPT | I |
| A3B3MPTX | L |
| A3MX | T |
| AMX | C D F H Q S W Y |
| AM(PT)X | E M Q |
| A2(T2)MPX | P R |
| A2(F2T2)MPX | K |
Show Answer
Seeing so many AMX spin systems in the grouping, it might be quicker to look for the more unique portion of this grouping. The distinct region is [A3MX][AX], thus, we know that our sequence must have a threonine followed by a glycine. Scanning the sequence of RNase Sa, this dipeptide occurs in two positions 72–73 and 82–83. Positions 70 and 71 are both isoleucines (A3MPT spins systems) which do not match the two AMX spin systems in the connectivities in question. However, there are two AMX spin systems before (Y80 and Y81) and three following (D84, H85 and Y86) the second TG (82–83) combination. Thus these spin systems correspond to Y80 through Y86.
