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Lobster Metallothionein 113Cd3-b Domain Solvent Atom Exposure Patterns Studied by NMR Spectroscopy:
Impact on Metal-Thiolate Cluster Reactivity

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Hydrogen Bonding

While measuring temperature dependence of the amide protons and H/D exchange rates provides an indication of possible hydrogen bonds, scalar spin-spin couplings 2hJHNCd, typically in the order of 0.5-2.0 Hz [5] and not observed in standard experiments, can provide direct evidence of a NH...S hydrogen bond. A two dimensional 1H{113Cd}-HMQC experiment optimized for small couplings and with decoupling of 3JHNHa during the indirect detection period (Figure 4B) was used to obtain qualitative through hydrogen bond connectivity information between the HN and Cd atoms (Figure 5). Additionally, in the case of Cd3b N the magnitude of 2hJHNCd was obtained from one dimensional 1H{113Cd}HSED experiment [5] (Figure 4A).
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Figure 4: Pulse sequence for 1D 1H{113Cd} HSED experiment (A) and 2D long range 1HN{113Cd} HMQC (B) employing a jump and return pulse pair for selective decoupling of 3JHNHa during t1 and water suppression.
f1= x,y,-x,-y
f2= -x,-y,x,y
f3= 4(-x), 4x
f3'= 4 (-x)
y = 2(x, -x), 2(-x, x)
D = 1 / [2(dHN-dH2O)] (208 ms)
t = 1 / (2hJHN-Cd) (62 ms)
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Figure 5: HN regions of the 2D long range 1HN{113Cd} HMQC experiment. Cross peaks arise from couplings through NH...S hydrogen bonds (2hJHNCd) or from intra residual long range couplings (5JHNCd). Assignments of HN protons are shown in blue, and uniquely identified acceptor residues are shown in red. If measured coupling constants are also indicated.
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Inspection of a three dimensional structure then allows one to identify the acceptor sulfur among the four bound to the particular Cd atom, allowing the assignment of three NH...S hydrogen bonds in Cd3-b N and two in Cd3bC (Figure 5 and 6). This is the first example of a 2D HN-113Cd correlation through a hydrogen bond and the first time hydrogen bond are observed in a metallothionein in solution. bar.gif
Figure 6: Ribbon display of the three dimensional structures of lobster MT-bN and- bC. The Cd-S cluster and Cys- side chain are shown as ball and sticks. Experimentally established HN...S hydrogen bonds are shown as dashed lines with donor and acceptor residues indicated.
This figure was prepared using the program MOLMOL2k.1 by Koradi et al. [4]
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