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III. ASSIGNMENT OF THE RESONANCES OF THE INHIBITED CATALYTIC DOMAIN OF STROMELYSIN-1

III. ASSIGNMENT OF THE RESONANCES OF THE INHIBITED CATALYTIC DOMAIN OF STROMELYSIN-1

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Figure 3 3-D and 4-D triple resonance experiments correlate interresidue or

intraresidue nuclei. (A) The efficiency of the experiments depend on the large

one-bond couplings; (B) atoms correlated in the 4-D HCANNH, an example of

an intraresidue experiment; (C) the 4-D HCA(CO)NNH, an interresidue experiment; (D) the 3-D HNCO, an interresidue experiment; and (E) the 3-D HCACO,

an intraresidue experiment.



of label is nonrandom such that Leu and Val residues are labeled as

13

Cy2H3-12CgH, 13Cy1H3-13CgH, and 13Cg2H3-12ChH, 13Cg1H3-13ChH,

respectively. Consequently, the 13Cy2H3 of Leu and 13Cg2H3 of Val

groups appear as singlets in the 1H,13C HSQC spectra and are thus readily stereoassigned. Measurement of 3JHNa in 3-D HNHA spectra [35] aids

determination of f torsion angles and stereoassignment of h-methylene

groups requires 3-D HNHB [36] and HACAHB [37] experiments. To

determine the fold of the protein, a large number of interresidue NOEs

must be assigned in 3-D 15N-NOESY [38], 3-D and 4-D 13C-NOESY experiments [39,40]. The assignment of the backbone resonances of the

13 15

C, N-enriched catalytic domain of stromelysin-1 were mostly accomplished with 4-D HCANNH and HCA(CO)NNH experiments (Fig.4) [5].

Side-chain atoms were assigned with 3-D HCCH-COSY and HCCHTOCSY experiments with the carrier located near 35 ppm for aliphatic

side chains and at 124 ppm for aromatic side chains. Stereospecific

assignment of the methyl groups were obtained with a 10% 13C-labeled



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Figure 4 Sequential assignment of the backbone atoms for the segment Pro-109

to Val-113 of inhibited sfSTR by 4-D HCANNH and 4-D HCA(CO)NNH. Four

planes are shown from each spectrum. The assigned backbone atoms are

indicated in (A). In (B) the upper four planes in solid lines are from the 4-D

HCANNH and the lower four planes in dashed lines are from the 4-D

HCA(CO)NNH. The chemical shifts for the four correlated nuclei in each case

are shown. The correlations continue for the segment Pro-109 to Pro-129. As Pro

lacks a protonated N, this residue serves as a ‘‘stop’’ signal. The correlation of 19

residues with Pro at the N- and C-terminal ends is unique for this segment in the

sequence of sfSTR, therefore these backbone atoms are specifically assigned

without having to further assign side chain atoms. (From Ref. 5.)



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



sample and a 2-D 1H, 13C HSQC spectrum, and couplings from 3-D

HNHA and HNHB experiments aided defining f and m1 angles. To

determine which specific ring nitrogens of the six histidine residues ligate

the catalytic and structural zincs a 2-D 1H, 15N HMQC spectrum was

acquired, where the delay for generating antiphase 1H, 15N magnetization

is set to 22 ms [41]. This experiment will favor the weak two- and threebond couplings between the histidine ring nitrogens and the Cy2H and

Cq1H protons (Fig. 5) and clearly showed the Nq2 of His-151, -166, 201,

-205 and -211, and the Ny1 of His-96 are the zinc ligands. Finally, the

critical NOEs that describe the tertiary structure of the protein were

assigned in 3-D 15N-NOESY, 3D and 4-D 13C-NOESY experiments.



IV.



ASSIGNMENT OF THE RESONANCES OF THE

INHIBITOR AND NOEs BETWEEN THE PROTEIN

AND THE INHIBITOR



To understand the interactions between a protein and a small ligand, we

take advantage of the fact that the protein is enriched with 13C and 15N and

the ligand is not. Pulse sequences can be designed to edit the spectrum of the

protein-ligand complex into spectra (2-D 1 H, 1 H-COSY, TOCSY,

NOESY) of the ligand or intermolecular NOEs between the labeled protein

and unlabeled ligand in either 2-D or preferably 3-D NOESY spectra.

These experiments are composed of X-half-filters [2,42,43] and either select

or filter the 13C,15N-attached protons (Fig. 6). Consequently, a 2-D 13C

doubly filtered NOESY spectrum will show intramolecular NOEs for the

ligand, whereas a 3-D 13C-filtered, 13C-selected NOESY will show intermolecular NOEs between the protein and the ligand. The latter experiment

is preferably acquired as a 3-D to minimize the ambiguities in assigning the

protons of the protein that are involved in the interaction with the ligand.

For the complex of the inhibited catalytic domain of stromelysin-1,

2-D doubly filtered 1H,1H COSY and TOCSY experiments performed

poorly. As these experiments depend on 1H, 1H couplings, the linewidths of

the stromelysin-inhibitor complex must be too large for efficient magnetization transfer. On the other hand, 2-D doubly filtered NOESY experiments acquired in 2H2O and H2O showed correlations for all protons of

the inhibitor (Fig. 7), and, as the inhibitor (Fig. 1) was quite simple, the

resonances were readily assigned. 3-D 13C-filtered, 13C-separated NOESY

experiments were also successfully acquired and assignment of these

NOEs were unambiguously obtained (Fig. 8).



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Figure 5 Part of a 2-D 1H, 15N HMQC spectrum of inhibited stromelysin-1

where the ring nitrogens of His resonate. The delay where antiphase magnetization evolves is set to 22 ms thus favoring the weak two- and three-bond

couplings of Ny1 and Nq2 to the protons of Cy2H and Cq1H [41]. The

deprotonated (h-type) nitrogen typically resonates near 249 ppm. These

resonances for inhibited sfSTR are near 200 to 210 ppm, shifted upfield by

ligation to the zinc ions. For the stable Ny1-H tautomer two strong couplings are

observed from the deprotonated Nq2 nucleus to the Cy2H and Cq1H protons. For

the stable Nq2-H tautomer only one strong coupling is observed from the

deprotonated Ny1 nucleus to the Cq1H proton. For the imidazolium tautomer the

resonances of the ring nitrogens are both near 176 ppm and equivalent couplings

from these nitrogens to both Cy2H and Cq1H protons are observed. For inhibited

sfSTR, His-151, -166, -201, -205 and -211 are in the Ny1-H tautomer, His-179 is

in the Nq2-H tautomer and His-96 and -224 are in the imidazolium tautomer.

Specific labeling of the Ny1 nucleus supports these assignments [5]. (From Ref. 5.)



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



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