D-Pantothenic acid sodium deuterium levels at 10 s also indicate that the region covered by residues 339�C353 has lost,3 hydrogen bonds, suggesting a loss of structure at the top of b-sheet A that is an important site in the early stages of RCL insertion. Additionally, there is disruption of hydrogen bonds between the central portion of b3A and the adjacent b2A and b5A. Loss of hydrogen bonds in these regions, together with smaller but still significant losses in helices A, B, and C, clearly demonstrates that the E342K Simetryn mutation disrupts native structure in areas both distant from and close to the mutation site. In addition to the loss of hydrogen bonds, deuterium uptake at 10 seconds also indicates the formation of additional hydrogen bonds in regions spanned by residues 127�C142, 191�C212 and 252�C272, in Z a1AT compared to M. These regions correspond to hE-b1A, b3A-b4C and hG-hH respectively. Taken together these results on deuterium uptake at 10 seconds clearly indicate that Z a1AT exists in an altered native conformation compared to M a1AT and that there is significant disruption of hydrogen bonding in much of b-sheet A which is in agreement with our previously published data using site single point mutations and molecular dynamic simulations. Significant differences in the extent of deuterium exchange at longer labeling times were found within 7 peptides, indicating dynamic and structural differences between the two proteins. One of the peptides includes the mutation site, Glu 342; this peptide was observed to be more mobile in Z a1AT. Also in this region was peptide 191�C212 which displayed decreased deuterium uptake indicating that this region contains additional hydrogen bonds and is more rigid in Z a1AT. This increased rigidity may be due to stabilizing interactions between Lys342 and Glu199. Trp194 is located in this region, and the increased rigidity may appear to be at odds with previous results showing differences in Trp fluorescence between M and Z a1AT. We note, however, that while the region covered by the peptide containing Trp194 shows decreased exchange at short times, the top of b5A, which is immediately adjacent to Trp194, shows increased exchange, indicating a more dynamic local environment. We therefore conclude that there is no inconsistency between the fluorescence and H/D exchange data. These changes in deuterium uptake suggests that the interactions within the vicinity of the mutation are altered by the removal of the salt bridge between Glu342 and K290, which allows this region to sample a conformation in which the top of s5A is open. This open conformation is maintained by new interactions formed 
between Lys342 and Val200, Thr203 present within peptide 191�C212. There are several regions, distant from the mutation site, whose structure and stability depend upon the residues they pack against such as helix A, B and H which are affected by the Z mutation. We observe a significant increase in the flexibility of peptic fragments corresponding to the helix B in Z a1AT. Peptide 38�C51 show a comparable behavior in both M and Z a1AT whereas an increase in exchange is seen for residues 38�C62 suggesting that the increase in exchange can be attributed to the B. The flexibility in this region suggests that the amide hydrogen bonds in these peptides are less stable and the packing around the helix is loosened in Z relative to M a1AT.