JOURNAL OF MAGNEIIC RESONANCE 71,571-575 (1987)

SimpMation of Two-Dimensional NOE Spectra of Proteins by I3C Labeling

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Reozived November 6, 1486

Two-dimensional NOE spectroscopy provides a convenient method for measuring interproton distances in solution, permitting detailed conformational studies of nucleic acids and Small proteins in soIution. Unfortunately, many proteins of interest are not yet accessible by this type of experiment because of severe overlap in the 2D spectrum. Limit& resolution in the aliphatic region, for example, can obscure "long-range" interactions which can provide information regarding tertiary structure. To overcome these limitations, heternnuclear labels may be introduced (1-3). We demonstrate here the feasibility of a '3C-e&t& NOESY experiment in which only those htemctions involving at least one 13C-attachd proton are obsewd.

M e y and Redfield ( 4 ) have previously proposed a method for determining which NUE cross peaks involve a 13C-attached proton by using decoupling during the evolution period but not during the detection period Interactions involving 13Gat- tached protons appear as asymmetric moss paks in the resulting 2D spectrum. How- ever, the final spectrum is of the same complexity as an ordinary MOESY spectrum. Jn the application to pm?eins it is dkrable to keep the final 2D NOE spectrum as simple as possible. The pulse scheme we propose is outIined in Fig. 1. The scheme differs from the regular NOFSY experiment by the insertion of an extra delay, A, of duration 1 /Jm at the end of the prepasation period. A 'H 180" pulse is applied at the centex of this A in* in odd-numbered scam a 180' I3C pulse is also applied at the center of this interval, and data are fllMrrtcted from those acquired in even-num- bered scans. At time tl = 0, all 'H magnetization in even-numbered scans will be aligned dong the +y axis; in add-numbered scans magnetization of '3C-attached pro- tons dl be aligned along the -y axis and all other 'H magnetization will be dong the fy axis.

Subtraction of odd-numbered scam from even-numbered scans will thus mdt in the canoellation of 'H signals that do not involve an interaction with a '3C-athched proton. The basic eight-step phase cycle is presented in Table 1. In addition, phase cychg of the find 90" 'H pulse is used to reduce the magnitude of coherent cross peaks (5) and CYCLOPS phase cycling (6) is used to suppress quadrature artifacts. A "C 180" pulse at the center af the evolution period is used to remove the effect of heternnuclear coupling in the F, dimension of the final s-m, simplifying the

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572

I

I I3c I

C o M m c A n O N s

DECOUPLE 4

FIG. 1. Pulse scheme ofthe 'fceditd 2D NOE experiment. The phase cycling is given in TabIe 1. The dday A is adjusted to l/Jm or slightly shorter to minimize Iossas due to transverse relaxation. The phase 4 is cycled along the %r axis, d n g in 0 and 180' ''C pulses at the mnter of the A wncd in alter- nate sc8I1s.

spectrum and maximizing the signal--noise ratio of cross peaks. To allow measure- ment of c m peaks close to the diagonal it is also desirable to employ I3C decoupling during the detection period. Without t h i s Heternnuclear decoupling, the &agonal will split into two parallel "diagonals," separated by Jm in the F2 dimension.

The method is illustratd for a 1.5 mkf sohtion of the N-terminal domain of A- repmsor protein (molecular mass 1 1 m a } in D@, pR 1 1.2. X-Repressor i s a specisc DNA-binding protein from bacteriophage h and provides a model system of biophysiml interest (7). At basic pH the repressor dimer d i s h & into monomers without loss of tertiary structure (8). The domain was purified from an overproducing strain of Esckerichia coli constructed by R. Sauer and co-workers (9). The ceIls were grown in a minimal medium (10) containing alanine "Cenriched in the C, position, resulting in a 60% I3C hbeling of the alanine methy1 groups in the protein (11)- The TI d u e s of the I3C-labe1ed Ala methyl protons were less than 1.0 s, permitiing a relatively short delay ( I .3 s) between scans in the 2D NOE experiment. The 13C-dted NOE spectrum obtained with a mixing time of 350 rns is shown in Fig. 2. Only NOE interactions with the I3C-labeled methyl protons are visible in this spectrum. On the diagonal some

TABLE 1

Phase cycling in the Pulse Scheme of Fig. I

X

-X

X

--x X

--x X

-X

X X

Y Y

-X

--x -Y --Y

X

-X

Y -Y

X

-X

Y --Y

"Data acquired in steps 1, 2, 5 and 6 are stored separately from data acquired in steps 3,4,7 and 8 and data are processed in the standard manner (13) to obtain a 2D absorptive spectrum.

CDMMUNICATIONS 573

weak signals are also observed from protons mupled to natural abundance 13C. Figure 3 compares the boxed region of Fig. 2 with the corcesponding part of a regular NOESY spectrum in which 13C dewupling was employed during both evolution and detection periods. Both spectra were recorded and processed under identical conditions, with the exception of the total number of scans and the delay times between scans. Spectra result from 2 X 128 X 1024 data matriq zero-fded in both dimensions to yield a 1024 X 1024 matrix for the absorptive part of the find spectra. The acquisition times in the bl and t2 dimensions were 25.6 and 102.4 ms, mpedvely. In the tl dimension, 20 Hz exponential line narrowing followed by 40 fxz Gaussian broadening was used. In the t2 dimension, 15 J3z exponential line narrowing followed by 20 Hz Gaussian broadening was used. In the regular NOE spectrum, 32 scans were recorded per bl value, with a deIay time between scans of 2.6 s, resulting in a total meamring time of 3.5 h. In the 13GBdit8d NOE spectnrm, 192 scaas were recorded per tl value, with a 1.3 s delay between scans and a total measuring time of 12 h. The 13C 90" pulse width was 150 ps ( 5 W rf power), and the 13C carrier was positioned in the center of the Ala<, region, 18 ppm downfield from TSP. Figure 3 demonstrates the degree of spectral simplification obtainable with the I3C

+

a 6 q 5 2 0 PPM

FIG. 2. ''CSdited 2D NOE spectrum ofthe Ala- '~ , - labeld A-repressor, morded at 2 5 T , using a 350 ms mixing time. Only protons that have an NOE interaction (direct or via spin diffusion) with the IT- laMd alanine methyl protons give cros wks in this type of spsctrum. The protein was dissolved in a buffer mntaining 100 ml4 K q 10 mM K,HP04 - KOH (pH I 1.2) and 0.1 mM EDTA.

574

a

C O ~ C A T I O N S

b f U 4

B #

I " ' I " ' t " ' I ' 1 . 8 1 .6 1 . q 1 . 2

I t 1.8 1.6 1.4 1 . 2 PPM

FIG. 3. Comparison of (a) an expanded region ofthe spectrum of Fig 2 with (b) the corresponding region of a regular NOE spectrum recordad under similar conditions. Cross peaks due to a unique alanin-e interaction are 1abek.d wlth asterisks. On the basis of the crystal stnrcture the alanim involved are Ah46 (downfield) and Ma-63 (upfield).

labeling approach. Whereas the regular NOE spectrum yields only a few resolvd resonances in the displayed region, the 13C-edited spectrum provides a wealth of re solved cross peaks. The 13Cedi?ed spectrum k asymmetric about the diagonal because only 13Gattached protons. are present in the Fl dimension of the spectrum. Only interactions between 13C-labeled sites give rise to moss peaks that are in Symmetric positions with respect to the diagonal. An example is the NOE interaction indicated by asterisks in Fig. 3a between the methyl protons of a previously vnasSignea alanine and Ala-66 (11). Comparison with the X-ray crystal stnrcture of the N-terminal domain of A-repressor (12) assigns the ht alanine to Ala-63. Additional assignments and implications repding repressor structure- will be presented elsewhere. The difFerences in the relative intensities of the two correlations marked A and B at the top of Fig. 3a and 3b are largely the result of a s m d baseline distortion in Fig. 3b, caused by the nearby mente of the forest of intense NOE cross peaks.

Incorporation of '%- and I5N-labeld amino acids in proteins for which the gene has been cloned i s relatively straightfornard Recording of an edited NOE specbum, as described in this communication, then provides a powerfid method for studying interproton distanm in such molecules, expanding the applicability of the NOE ex- periment to proteins much larger than those studied to date.

COMMUNICATIONS 575

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ACKNOWLEDGMENTS

W e thank Rich Gnfey, JeiTHocb, Laura Jmner, Professor Alfred Redfield, Vladirnir S k l a , and Dennis Torchia for helpful discussions; Professors Martin Kvptus (Harvad Univ.) and Robert Sauer (MIT) for advice and support; and Anna Jeitler-Nilsson and Kathy Hehir for overprducing plasmids and help with protein purification. This work was partly supp01W by grants from the National Instituta of Heatth to M. Karpius (GM-37292) and R. T. Saver (AI- 16892).

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Harbor, NY, 1970. IO. J. MILLER, “A Laboratory Manual in Bacterial Genetics,” Cold Spring Harbor Press, Cold Spring

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