Macromolecules 2002, 35, 2633-2639
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Structural Investigations of Solid Proteins at Natural Abundance Using 2D Multiple-Pulse NMR Jeffery L. White* and Xingwu Wang Department of Chemistry, North Carolina State University, Campus Box 8204, Raleigh, North Carolina 27695-8204 Received November 27, 2001
ABSTRACT: Results from multiple-pulse 2D 1H-13C correlation experiments are described for natural proteins in the solid state. Detailed HETCOR experiments on the complex silk fibroin proteins Samia cynthia ricini (S. c. ricini) and Bombyx mori (B. mori) demonstrate that while the expected dipolarmediated 13C and 1H correlations are observed, additional chemical shift and coupling information involving amide linkages and dilute peptides are also detected indirectly. This is an important result since we show that the 1H chemical shifts for -NH groups in S. c. ricini are not resolved in the CRAMPS experiments, thereby preventing their direct measurement. Chemical shifts via dipolar couplings (twospin and multiple-spin interactions) are also detected for dilute peptides in the silk fibroins, again providing key structure information for these functional residues that is completely absent in the CRAMPS data. Relative dynamics of bulky side groups from dilute amino acid residues are apparent from the 2D multiplepulse data. Differential spin-diffusion behavior is observed in a modified HETCOR experiment for S. c. ricini relative to the B. mori protein, suggesting morphological differences in the arrangement of crystalline segments in the proteins. Information regarding hydrogen bonding and constrained interchain spin pairs is provided based on comparison of cross-peak intensities for several nearest-neighbor C-H spin pairs within the chain and based upon the presence of CO correlations with specific R-hydrogens.
Introduction Structure determination in polypeptides and proteins requires knowledge of local bonding, short-range chain conformation, and long-range geometry or secondary structure. Further, the role of hydrogen bonding in determining secondary structures is a key experimental target in complex protein structure analysis. Solid-state NMR has emerged as an attractive experimental tool in probing chain conformation, hydrogen bonding, and secondary structures in polypeptides, since the native form of the polymer structure may be studied without the complicating effects of dissolution. Recent publications in which NMR has been used to interrogate hydrogen bonding in the solid state have focused on crystalline dipeptides or polypeptides as model systems.1-4 Others have studied macromolecules in which specific isotopic labels have been introduced to probe the spatial arrangement of monomer pairs, torsion angle distributions, and the dynamics of individual side groups.5-9 McDermott and co-workers provided a particularly important demonstration of the power of 2D solids NMR to interrogate structure and hydrogen bonding in model peptides at natural abundance.4 Shoji and co-workers have done extensive multinuclear (13C, 1H, and 15N) NMR work demonstrating the sensitivity of backbone carbonyl (CO), backbone methylene/methine (CH2/CH), and amide chemical shifts to local and overall conformation in model polypeptides.2,3 Recent CRAMPS data suggest that the 1H chemical shifts of both the R and amide hydrogen are sensitive to secondary R-helix or β-sheet structures in homopolypeptides. However, for complex proteins, such chemical shifts may not be resolved even in the CRAMPS experiments. This is particularly true for amide hydrogens. Although some recent work by Shoji and co-workers has demonstrated * To whom all correspondence should be addressed.
the utility of multiple pulse 2D methods for the analysis of silk fibroins with regard to backbone HR shifts, complete structure correlations involving key functional groups (e.g., carbonyl, amide, hydroxyl) were not reported.10 We report experimental results for two different silk fibroins that illustrate the chemical shift, dipolar coupling, hydrogen bonding, and dynamics information that may be obtained for proteins at natural abundance using multiple-pulse 2D techniques. In this article, we present multiple-pulse 1H CRAMPS11,12 and 2D solid-state 1H-13C HETCOR13-15 data for solid, fibrous proteins obtained from the silk worms Samia cynthia ricini (S. c. ricini) and Bombyx mori (B. mori). Silk fibroins are fibrous proteins rich in glycine and alanine (ca. 70-80 mol % total), with a primary structure consisting of the repetitive sequence (Gly-Ala-Gly-Ala-Gly-Ser)n in the B. mori case, and long polyalanine blocks plus glycine-rich blocks in the S. c. ricini protein.8,16 Each protein contains small amounts of tyrosine (Tyr) and valine (Val), and a secondary structure that is best described as a distorted antiparallel β-sheet structure, depending upon whether the fibers exist as the silk I or silk II forms.7,8 The silk II conformational form was investigated in this study. A key difference between the S. c. ricini and B. mori fibroins is in the location of the serine residue; serine resides in the less crystalline glycine-rich blocks of the former while it is primarily found in the repetitive, crystalline sequence of the latter. The structures of the primary amino acid residues for the major constituents in silk fibroin are shown in Scheme 1. The goal of our work on these solid proteins is to determine the level of chemical shift information and dipolar coupling data relevant to structure, hydrogen bonding, and differential dynamics that may be detected at natural abundance using multiple-pulse NMR techniques.
10.1021/ma012067s CCC: $22.00 © 2002 American Chemical Society Published on Web 02/21/2002
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Macromolecules, Vol. 35, No. 7, 2002 Scheme 1
Experimental Section CRAMPS data were obtained with the MREV-8 sequence on a Bruker DSX-300 instrument using the 1H channel of a 4 mm triple-resonance XY-H magic-angle spinning probe. 90° pulse widths were 2 µs with an interpulse spacing of 2 µs. Spinning speeds were controlled at 1.5-2.0 kHz and were chosen for each sample to minimize spurious rotor lines. The chemical shift scaling factor was experimentally determined to be 0.52 by measuring the offset dependence of a confined H2O signal. The 1H chemical shift was referenced to poly(dimethylsiloxane) at 0.15 ppm. For each spectrum, 16 scans were acquired with a repetition time of 10 s. The HETCOR experiment of Burum and Bielecki13 was used for the 2D solidstate heteronuclear correlation results, and 90° pulse widths were 3.2 µs on each channel. Data were collected using one, two, or three windowless isotropic mixing (WIM) cycles. The experimental verification of proper HETCOR performance was done using monoethylfumarate, and carboxylic acid carbon correlations to olefinic and acid protons were observed using both one and two WIM cycles.15 For longer-range dipolar couplings, controlled periods of 1H-1H spin diffusion were introduced prior to the isotropic 1H/13C polarization transfer step. The chemical shift scaling factor in the 1H dimension was measured experimentally to be 0.42 (near the theoretical 0.47 value), and the proton frequency was shifted off-resonance by 4 kHz from the carrier to avoid any zero-frequency artifacts and take advantage of second averaging effects. Spinning speeds of 3.8-4 kHz were used to minimize potential sideband overlap with peaks of interest. These spinning speed periods (250-290 µs) were larger than twice the WIM cycle length, thereby preventing signal elimination or attenuation due to a refocusing of the C-H dipolar interaction at the end of each rotor period. Typically, 1024 scans were taken for each of 64 points in the t1 dimension, and recycle delays of 4 s were used. The total experiment time was typically 36 h. Quadrature detection was maintained in the t1 dimension via use of TPPI. The data were processed with 50 Hz line broadening and zerofilled to 512 points in the t1 dimension prior to Fourier transformation.
Results and Discussion CRAMPS Experiments. High-resolution 1H solidstate NMR experiments using CRAMPS (combined rotation and multiple pulse spectroscopy) is a logical choice for characterization of solid-state hydrogen bonding in proteins based on expected perturbations of proton shifts. However, unambiguous assignments would require resolution of “free” and “bound” hydrogens, i.e., those protons that were or were not involved in hydrogen bonding. This is a difficult task given the relatively
Figure 1. (a) 1H CRAMPS spectra of S. c. ricini (top) and B. mori (bottom) silk fibroins. (b) 13C CP/MAS spectra of S. c. ricini (top) and B. mori (bottom) silk fibroins. Peaks are labeled according to the structures presented in Scheme 1.
poor resolution of CRAMPS for macromolecules. The 300 MHz 1H CRAMPS spectrum for S. c. ricini and B. mori silk fibroins are shown in Figure 1a. Similar to the recent work by Shoji and co-workers,10 resolved Ala β CH3 and R CH/CH2 signals from the Ala and Gly amino acid residues are observed at 1.2 and near 5 ppm, respectively. Since the Gly and Ala residues make up 75% of the protein in each case, the fact that the chemical shifts for HR’s are near 4-5 ppm is consistent with the presence of a β-sheet structure.2 Broad, poorly resolved signals exist in the region from 6 to 10 ppm. The majority of the intensity in this region may be
Macromolecules, Vol. 35, No. 7, 2002
assigned to the NH hydrogens (as they are present in each repeat unit) as well as a small contribution from aromatic hydrogens in the dilute Tyr (tyrosine) units. While assignment of NH and aromatic shifts via deconvolution might be possible in the B. mori case, clearly such shifts are not resolved in the S. c. ricini fibers. Amide 1H signals are expected to be broad due to the dipolar coupling between the I ) 1/2 proton and the quadrupolar 14N nucleus. Further, one might expect that hydrogen bonding both within and between different amino acid segments could lead to inhomogeneous contributions to the line width. The spectral resolution in Figure 1 is not sufficient to identify individual signals from NH groups in any amino acid, much less for backbone hydrogens, aromatic hydrogens, or hydroxyl groups from dilute Ser, Tyr, or Val residues. As such, the available 1H chemical shift information that might be used to follow conformation and hydrogen bonding in different protein structures is limited in the CRAMPS data. 13C CP/MAS spectra are also shown in Figure 1b for reference when viewing the 2D contour plots in subsequent figures. Isotropic Polarization Transfer HETCOR Results on S. c. ricini. Figure 2 shows results from the 2D solid-state HETCOR experiment on the S. c. ricini sample. These data were obtained using 1, 2, or 3 WIM cycles (a, b, and c, respectively) for isotropic polarization transfer.14 The advantage of this experiment is that simultaneous 13C and 1H chemical shift information is obtained along with distance-dependent dipolar coupling data. As such, hydrogen-bonding partners in macromolecules can be identified.17 Carbon signals are only observed for those carbons with proximate hydrogens (
