NMR studies on the citrate free form of CitAP

Only 62% of the possible peaks were observed in the ¹⁵N-¹H HSQC spectrum of the citrate free form of CitAP. The average rotational correlation time (τ_c) measured for the protein was 7.54 ns, indicating a monomeric CitAP in solution. No improvement and no additional peaks were observed with TROSY-HSQC spectrum. These results suggest that line broadening due to conformational exchange might be the cause of the disappearence of around 40% of the peaks.

Usually, three dimensional experiments for protein backbone assignments are acquired with amide proton detection in the direct dimension. These amide protons are the most sensitive to line broadening due to chemical exchange. In order to limit line broadening due to conformational exchange, several non-standard NMR experiments were acquired. To reduce amide proton chemical exchange broadening, CPMG-HSQC experiments were measured.

CPMG sequence was applied during the INEPT (refer to Chapter 1 for description of INEPT) transfer period in the normal HSQC. Relaxation due to chemical exchange is reduced in the CPMG sequence. No additional peaks were obtained in the CPMG-HSQC spectra.

Rather than using amide detection in the direct dimension, H_α detected experiments were also tried. Back and forth HACACO experiment with H_α detection in the direct dimension did not give any additional peaks (refer to Chapter 2, section 2.2 for more details). Only 80 peaks were observed in the spectrum (Figure 4.26 panel A ). This implies that line broadening due to chemical exchange is not restricted to amide group only.

To determine the presence of conformational exchange in the side chain resonances in the protein,¹³C-¹H HSQC experiment was measured. Methyl groups have the most favorable T₂ relaxation time due to rapid rotation of its carbon-carbon single bonds. The ¹³C-¹H HSQC spectrum of the methyl region of the citrate free CitAP shows only 62 peaks (Figure 4.26 panel B ). 85 possible peaks were expected from the protein sequence. The missing number of methyl resonances fit well with the number of methyl peaks that were expected from the unassigned amino acids in the protein sequence. This proves that line broadening due to

Figure 4.26: (A)¹³Cα-¹Hα projection of three dimensional HACACO experiment. Only 80 peaks are observed in this spectrum. (B) ¹³C-¹H HSQC spectrum of the methyl region. Only 62 peaks out of 85 peaks are seen in the spectrum.

conformational interchange affect the side chain resonances as well. These results indicate that there must be different conformations of CitAP in solution. This is in accordance with the identification of two different structures for the monomers of CitAP in the asymmetric unit of the citrate free CitAP crystals.

Nevertheless, the backbone assignment of 72 of the 80 peaks observed in the HSQC spectrum were possible. The unassigned residues of CitAP is plotted onto the monomer B of the structure of citrate free form of CitAP (Figure 4.39 ). It is interesting to observe that most of the unassigned residues in the sequence are those residues which show large backbone r.m.s.d deviations between the two forms of the monomers of the citrate free structure of CitAP. These residues are mostly in the twoβ sheets and the parts of the major loop which also defines the binding pocket for citrate in the citrate bound structure. The three residues that are assigned in the major loop region have considerably high flexibility associated with

it (refer to discussion below), this could have reduced the relaxation process which leads to the disappearance of the other peaks in the major loop.

Figure 4.27: Plot of measured rotational correlation time against individual residue number of citrate free-CitAP. The plot gives a measure of flexibility of residues. Residues at the N and C terminal and the two residues that was assigned in the major loop region show low τc values indicating major flexibility in these region.

Usual T₁, T₂ methods for estimation of τ_c haveR_ex (relaxation rate due to conformational exchange) term associated with it. For proteins for which R_ex is the dominant relaxation rate, these methods give a very large value for τ_c. Hence cross correlated relaxation rate was used to determine the τ_c values. The τ_c values obtained using this method does not have contribution from chemical exchange. As indicated earlier, the average τ_c of CitAP determined using this method (described in 2.2.8) is 7.54 ns. Spectral density function of a rigid body is used in the calculation for τ_cs. Hence a lower bound for the values of τ_cs are obtained using this method. τ_c for individual amino acids are determined. As described in chapter 2, section 2.2.8, the τ_c values are a function of the amplitude of local motion associated with that amino acid residue (S²). Residues with lowerτ_c values than the average value have significant flexibility term associated with it. Most of the residues showing lowτ_c values are in the N and C terminals. The τ_c for the amino acids assigned in the major loop

region also have lower than average value, indicating high flexibility and deviation from rigid body approximation (Figure 4.27). Heteronuclear NOE experiment was also measured for the citrate free CitAP. The amplitude of motions in the pico- to nano-second time-scale is investigated with the steady state ¹⁵N-¹H NOE. ¹⁵N-¹H Het-NOE values therefore give a measure of residue flexibility. The three residues for which assignment was possible in the major loop (residues 81,83 and 84) , have lower Het-NOE value (lower than 0.6) indicating high flexibility in this region (Figure 4.28 ) . This correlates well with the lowerτ_c values of these residues (residues 81 and 83).

Figure 4.28: ¹⁵N-¹H NOE values measured for citrate free-CitAP are plotted against residue number. Residues near the N and C terminal and the residue in the main loop region have lower Het-NOE values indicating high flexibility.