Order Parameters (S2)
The model-free motional parameters for E-Lg C, derived from 15N relaxation values measured at 305 K, 313 K and 320 K are listed in Appendices D.1-D.3, respectively. The model-free derived order parameters for these experiments are plotted separately in Figure 3.14 (A), (B) and (C), and a comparison of these parameters is shown in Figure
CHAPTER 3. RESULTS AND DISCUSSION
showing similar gradual variations of flexibility across secondary structural elements and increased flexibility for backbone amides positioned in the N-terminus and for backbone amides positioned in loop regions. Ala34, right in the middle of A/B loop, still displays a significantly low order parameter at 313 K and 320 K.
When the S2 trends of the different temperature data sets are compared, two subtle changes in flexibility are detected. The first being that when the temperature is raised from 313 K to 320 K the 15N-1H vector of Ser30, located in the region of the 310-helix
within the A/B loop, becomes more constrained as indicated by a higher S2 value (Figure 3.15). This implies that Ser30 becomes more associated with another element, when the temperature is raised to 320 K, restricting its motion. Differences in the values of S2, among the different temperature data sets, are also observed across the major D-helix (D-2), suggesting that this helix (Asp129 - Lys141) becomes more rigid between 305 K and 320 K. These results also imply that the major D-helix becomes more associated with another nearby region/s of E-Lg, as the temperature is raised. However, 15N relaxation data is not available for the centre region of the helix for a full
S2 assessment, as seen by the absence of values for this region in Figure 3.14 (A), (B) and (C), to confirm that this was happening for the entire region.
These results show the flexibility of internal motions across the backbone of E-Lg C are all remarkably similar at 305 K, 313 K and 320 K, except for two subtle changes in the A/B loop and the D-2 helix, providing evidence that these regions are becoming more associated with other regions in E-Lg C. This lack of large changes in flexibility observed is perhaps not unexpected, when changes in amide protection were only observed for E-Lg A above 55 ˚C (Edwards et al., 2002).
Conformational Exchange (Rex)
Comparing values of Rex between temperature sets is not considered in this comparison,
as Rex is only determined at one static field strength, which can compromise the
precision of the resulting values. By measuring Rex, at least at two different field
3.5 Comparing Dynamics at 305 K, 313 K and 320 K 320 K. The total number of residues revealing Rex terms increases from 18 at 305 K to 27 at 320 K (Table 3.4). Not surprisingly, at all three temperatures tested, the conformational exchange constant, Rex, is significant for some of the backbone amides
that are located in loops and link regions (Figure 3.16). A higher occurrence of these motions are found in loops that are positioned at the open end of the barrel (A/B, C/D and E/F loops), and a lesser occurrence in loops located at the more closed end of the barrel (B/C, D/E, and F/G loops).
The Rex constant for Leu22 shows that its critical position at the hinge point of the E-A
strand, is still maintained when the temperature is raised to 313 K and then at 320 K. His59, located at substitution site between variant B and variant C, displays a relatively high Rex value at 313 K, but unfortunately 15N relaxation data measured at 320 K could
not be fitted to define its motions at this temperature. The highest density of Rex values observed at 305 K, are concentrated on the E-D strand at 305 K (Figure 3.16 (A)). The model-free parameters at increasing temperatures describe this E-strand as still experiencing these slower motions, which is not unexpected.
In comparison, 15N relaxation data from the E-E strand residues had optimal fits using simpler models at lower temperatures (models one and two for 305 K and 313 K respectively); whereas at 320 K, relaxation data from backbone amides in this region were fit with the more complex model four. These results suggest that the only two H-bonds formed between strands E-D (Glu74) and E-E (Lys83) (Qin et al., 1998b, Edwards et al., 2002) are disrupted at temperatures between 313 K and 320 K. The hypothetical ‘freeing’ of the E-E strand would contribute to its conformational mobility at 320 K. The relative length of the long E-D strand, coupled to its hydrogen bonding potentials not being completely fulfilled, are plausible explanations for why this E-strand is undergoing conformational exchange at the lower temperatures, before the possible disruption of the two H-bonds between 313 K and 320 K.
As noted previously, the interactions formed between the E-G and E-H strands are highly ordered, with a dense network of inter-strand H-bonds and a disulfide bridge connecting these strands at positions Cys106 and Cys119. At 305 K, the measured 15N
CHAPTER 3. RESULTS AND DISCUSSION
become more conformationally mobile due to an increase in temperature, which possibly contributes to a stretching motion of these E-strands in the regions closest to the open end of the barrel or caused by the disruption of H-bonds formed by residues located in these areas.
Correlation Times (We)
As stated earlier, the effective correlation times (We) are imprecisely determined, but are
useful for spotting mobile regions of the backbone (Palmer, 1993). As seen in Figure 3.17, when the temperature is raised to 313 K (Plot B) from 305 K (Plot A), residues with significant We contributions (> 500 ps) become more prevalent in the A/B loop, the
B/C loop, D/E loop G/H loop, the link regions around the major D-helix, E-F strand and the N- and C-terminal end of the major D-helix (D-2). These results indicate that many of the loop regions become more mobile when the temperature is raised to 313 K.
These trends are still seen at 320 K (Figure 3.17 (C)), but in some instances,
15
N relaxation data could not be fitted with a model or they were discarded from dynamical analysis as their assigned peaks were merging with others in the
15
N,1H-HSQC spectrum, so therefore, are absent in the 320 K plot.
Previously, when comparing the S2 values with increasing temperatures, it appears that the D-2 helix (major D-helix) becomes more rigid when the temperature is raised, therefore, the presence of nanosecond time-scale motions with relatively small amplitudes are assumed. In this case, model-free analysis can significantly under-estimate the effective correlation time (Chen et al., 2004), particularly for residues Leu133 and Ala139, whose S2 values are highest at 320 K, but display longer effective correlation times when temperature is increased to 313 K, but become shorter again at 320 K.
Two Time-Scale Spectral Density Function (Model Five)
A comparison at different temperatures of the extended model-free two time spectral density function is not considered in these studies, as 15N relaxation measurements need to be acquired at a number of magnetic field strengths to assess the quality of the
3.5 Comparing Dynamics at 305 K, 313 K and 320 K
Changes in Chemical Shifts
NMR spectroscopy chemical shifts are very sensitive to the local environment of the
residue’s atoms. Increases in temperature do not generally change the dispersion of peaks in the 15N,1H-HSQC spectrum, showing that the average conformation of the
protein’s structure is similar at all three temperatures (Figure 3.6). However, it is possible that minor conformational changes occur at some sites when the temperature is raised to 320 K, as detected by chemical shift perturbation of peaks in the 15N dimension between 305 K and 320 K (Figure 3.18). Residues positioned in the N-terminus (Val3, Thr4, Gln5, Lys8, and Gly9) show significantly higher than average changes in their backbone amide 15N chemical shifts (> 0.35ppm). However, this is not observed for Met7, which forms an H bond with Val94 (Qin et al., 1998b, Uhrínová et al., 2000). Significantly higher than average changes in 15N chemical shifts are also observed for Tyr18 and Leu22 (E-A), Glu42 (E-B), Asn63 (C/D), Ala67 and Ile71 (E-D). Notably, Tyr18, Glu42, Ala67 and Ile71 are all positioned in E-strands that form one E-sheet, which makes up half of the E-barrel and Leu22 is positioned at the midpoint of both E-sheets. This E-sheet is less ordered than the second E-sheet, which may account for its higher frequency of residues with 15N chemical shift changes greater than 0.35 ppm, causing subtle changes in conformation as the temperature is increased. The second E-sheet is formed by E-strands that possesses a more extensive inter-strand H-bond network and is further stabilised by a disulfide bridge. Changes in
15
N chemical shifts for Glu131 positioned in the D-2 helix and Glu158 positioned in the ill-defined D-3 helix, also suggest subtle changes in conformation when temperature is raised to 320 K.
3.5 Comparing Dynamics at 305 K, 313 K and 320 K
Figure 3.15 An Overlay of S2 Traces for EE-Lg C at 305 K, 313 K and 320 K.
Values are derived from the Lipari and Szabo model-free formalism (1982a, 1982b) from 15N relaxation parameters measured at 305 K (blue), 313 K (black) and 320 K (red). In this plot the positions of the nine E-strands (labelled A-I) and D-helices (labelled 1-3) are highlighted with teal and salmon, respectively.
Ser30 major
3.5 Comparing Dynamics at 305 K, 313 K and 320 K
CHAPTER 3. RESULTS AND DISCUSSION
Figure 3.18 Changes in 15N Chemical Shifts for EE-Lg C between 305 K and 320 K.
Values are calculated from the differences in chemical shifts of residues in the 15N-dimension between 305 K and 320 K. In this plot the positions of the nine E-strands (labelled A-I) and
3.7 Assigning the Backbone of EE-Lg Variants A & B