Exchangeable protons, assignments

Hoffman and collaborators (Doan et al. 1994) also performed ENDOR experiments on the oxidized [3Fe-4S] cluster in D. gigas hydrogenase. The authors detected resonances from strongly coupled protons which were assigned to the [3-CH2 of the cysteines and exchangeable protons that are probably involved in three different hydrogen bonds to the sulfurs of the cluster. Based on these data a model for the binding of the cluster to the protein was developed. 

This technique has been taken to new heights through the use of 2-D nuclear magnetic resonance " to assign many, if not all, exchangeable protons in terms of the residues to which they are attached. As a result, one can assess individual rate constants defining the kinetics of exchange. 

Proximal Histidyl NzH Resonances. In the H NMR spectrum of deoxy-Hb A in H2O two low-field resonances occur at + 58.5 and +71.6 ppm downfield from H20 (La Mar et al., 1977 Takahashi et al., 1980). These two resonances disappear in the presence of D20 and have been assigned to the hyperfine-shifted NsH-exchangeable protons of the proximal histidyl residues (F8) of the a and p chains of deoxy-Hb A (Takahashi et al., 1980 La Mar et al., 1980). In Fig. 17, it is clear that the resonance at + 71 ppm is missing in Hb M Milwaukee, suggesting that this resonance comes from the p chain and the resonance at +58.5 ppm from the a chain. Hb M Boston has only the resonance at +71 ppm, which confirms the assignment of the +71-ppm resonance to the N8H proton of the proximal histidine of the P chain and the + 58.5-ppm resonance to the corresponding residue in the a chain (Takahashi et al., 1980). The fact that the a- and P-heme resonances are clearly separated indicates that the conformations of the proximal histidyl residues are different in the a and P chains of deoxy-Hb A. 

As also shown in Fig 57A, for deoxy[a(des-Arg-Tyr)(3]A[a 3]cXL at pH 6.0, about half the intensity of the hyperfine-shifted proximal histidyl N8H exchangeable proton resonances has already shifted downfield by about 15 ppm, but the remaining resonance remains at a position close to that of the a-subunit proximal histidyl N8H proton resonance in intact Hb A. It is reasonable to assign the resonance that remains unchanged to the normal intact a subunit of [a(3]c in deoxy[a(des-Arg-Tyr)(5]A[a(5]cXL. In correspondence with the shifted N8H proton resonance of a(des-Arg-Tyr), both resonances at +9.2 and +6.3 ppm from H20 have lost about half of their respective intensities, and they disappear when the pH is raised to 8.6. The increase in pH also causes the a-subunit histidyl N8H exchangeable proton resonance, normally occurring around +57... 

The combined information from the l3C/DEPT, COSY, TOCSY, and HMQC enables us to assign all protons in the 3H spectrum except those that are exchanging rapidly (i.e., the carboxyl and free amino protons) and all of the non-quaternary carbon resonances in the 13C spectrum. There is no need to assign the rapidly exchanging protons, and the non-quaternary carbons will be assigned during the sequencing discussion. 

An exchangeable proton of ribonuclease A titrates with a pK of 5.8 and has been assigned [39] to the NH of the active-site histidine-119. A low field resonance can be observed for chymotrypsin in H2O [40] and its pH dependence (15 to 18 ppm, pKa 7.2) and response to chemical modification suggests that this is the hydrogen-bonded proton between His-57 and Asp-102 at the active site. 

The nuclear magnetic resonance (NMR) spectrum (Fig. 2) was obtained by preparing a saturated solution of meperidine hydrochloride, U.S.P. (Wyeth Lot No. F-665901) in deutero chloroform containing tetramethylsilane as internal reference. The only exchangeable proton is the hydrogen associated with HC1. The NMR proton spectral assignments are given in Table II. 

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