Structural Dynamic Study by H/DX Measurement

Figure 30. Workflow of an automated H/DX MS experiment. In the first step a protein of interest gets exposed to deuterated solvent for a period of time, followed by quenching in a low pH buffer where exchange rates are dramatically decreased, followed by fragmentation of the pro-tein and mass analysis.^[266]

In the presented thesis a successful H/DX study for the OYE family member DrER from Deinococcus radiodurans R1 is reported. The attempt to examine such a structural dynamic study with TsER proved to be not feasible with the availa-ble setup. Due to the thermostability and robustness of this enzyme, it was not possible to rapidly denature the protein by quenching and digest it to obtain small enough peptide fragments for the analysis. All possible parameter chang-es, which were undertaken, such as higher temperatures of the digest, did not lead to an analysable dataset. Consequently, the H/DX study was performed with the related, but not thermostable ene reductase DrER, to gain more struc-tural information about this family member, since to date no crystal structure is available.

The enzyme was analysed in three different states, one apo-state, that means just the holo-protein without any additive, the second state with calcium chlo-ride added and the third state with the addition of calcium chlochlo-ride and NADP⁺. The addition of a divalent metal ion was used, because it is known that

OYEs from group 2 benefit from the presence of calcium or magnesium during catalysis.^[99] But neither the location of the divalent metal binding site nor the role this ion plays is known.

Labelling of the protein samples occurred at four different time intervals, which were chosen to be t0 = 0 min, t1 = 0.5 min, t2 = 1 min and t3 = 5 min. During this time the amide hydrogens of the protein backbone were able to exchange with the deuterated solvent. Depending on its solvent accessibility and hydrogen bonding, the deuterium uptake of a local peptide fragment differs from the na-tive control fragment.To interrupt the exchange reaction, the medium becomes quenched with a cold acidic buffer and is injected into the H/DX manager at 0 °C. The quenching does not just stop the exchange, but also leads to a dena-turation of protein, which facilitates the following proteolysis by pepsin. To en-sure that the pepsin is not injected into the LC-MS system, it is immobilized to a stationary phase and packed into a column.^[267] These quantities of peptide fragments, which are a result of the unspecific digestion of pepsin, are of ran-dom size and therefore have to be identified by the PLGS Software of WATERS. Digestion of the unlabelled protein constructs gives the initial peptide map, which in combination of the digestion of labelled proteins results in a creation of a so-called heat map, representing the local uptake of deuterium (Figure 31).

For the analysis of DrER a total coverage of 96.8% by 259 peptides was calculat-ed and lcalculat-ed to a rcalculat-edundancy of 6.75. In the context of the heat map, remarkably high uptake of deuterium can be observed at the C- and N-terminus of the en-zyme, indicated by the red fields, but changes in uptake over time are marginal just as the differences to between each state. In addition, this is highlighted by the heat map, where all of the measured states are compared to each other, showing no significant differential uptake.

Through b-factors, which can be obtained from the software DYNAMX 3.0, the two dimensional heat map can be plotted on a user-chosen crystal structure to

get a three dimensional picture with dynamic features. No crystal structure of DrER wt is available so far, but with the online bioinformatic toolSWISS-MODEL

a homology model was created (Figure 32). The results show that the b-factors of H/DX calculation fit quite well to the homology structure. For example loops, which are dangling mainly at the outer face of the enzyme, have a higher up-take over time than conserved regions, like most α-helices and β-sheets. Quite low fractional uptake is present between N- and C-terminus with exception to a few fragments.

An interesting strong fractional uptake over time takes place at the position 29-32 (amino acids: ELPN), whereas broad and more moderate to low uptake over time can be observed for positions 87-94, 128-138, 157-161, 247-257, 273-301, 304-308, 314-326 (marked with black arrows in Figure 32).

DAUGHERTY et al. showed via a synthetic circular permutation library of the OYE1 from Saccharomyces pastorianus, that structural rearrangements of some flexible loops and domains play an important role in the catalytic function of OYE enzymes.^[168] During the strategy of circular permutation (CP), the original N- and C-termini of the protein are covalently linked by a peptide linker and new termini are introduced elsewhere in the protein structure through breakage of a peptide bond.^[268] The selected flexible regions for CP are comparable with the results from H/DX plotted b-factors to the homology model of DrER. They chose three different sites as locations for new termini. One is the exterior heli-cal subdomain (OYE1 residues 125-160), which is also highly flexible in DrER at positions 285-301. The second sector includes loop and helix in regions from 250-265 at OYE1, which is comparable to residues 317-325 in DrER. The third part represents a short loop, residues 375-380 in OYE1 that is also present with a big exchange in DrER (residues 87-94).^[168]

igure 31. Heat map generated with the Software DYNAMX3.0 for the sequence of DrER wild type. The coloring indicates the relative fractional uptake, which is aled to a data range of -077 to 63.54% within the given amino acid sequence for its three states Apo, Ca2+ and Ca2+/NADP+ in respect to the different time intervals f 0 min, 0.5 min, 1 min and 5 min. Deuterium exchange ranges from poor (blue) to strong (red) uptake

Figure 32. Homology model of DrER wt, based on pdb 1Z44^[94], representing fractional uptake of the peptide amino-backbone after 5 min within the Ca²⁺/NADP⁺ state, ranging from no/low (dark to light blue) to moderate/high (white to red, marked with black arrows and peptide numbers). Black color fragments indicate mapping gaps, for which no peptides were available.

Cofactor FMN is shown in grey sticks.

In addition, H/DX analysis was performed to find a potential divalent metal binding site in DrER. To identify the calcium binding site, it is expected that at this specific position in the peptide, less uptake will take place when Ca²⁺ is bound in the Ca²⁺ and Ca²⁺/NADP⁺ states compared to the apo state.

However the heat map did not show any difference in uptake in the different states. Even though it has not been possible to identify the divalent metal bind-ing site, the H/DX results give good insights into the structure and dynamics of DrER in solution.