Conformation-dependent Chemical Shifts Research Articles

Determination of Local Structure of 13C Selectively Labeled 47-mer Peptides as a Model for Gly-Rich Region of Nephila clavipes Dragline Silk Using a Combination of 13C Solid-State NMR and MD Simulation

For the first time, we elucidate the complex structure of the Gly-rich regions in Nephila clavipes dragline silk through synergistic experimental and theoretical studies. First, the 13C selectively labeled 47-mer peptides selected from the glycine (Gly)-rich region of N. clavipes dragline silk were synthesized. The 13C CP/MAS NMR spectra were analyzed to determine the fractions of the conformations of individual Gly and Ala residues through 13C conformation-dependent chemical shifts and peak deconvolution. By comparing the 13C solid-state NMR spectra of several simple model peptides, the presence of 31 helix in the 47-mer peptides was disproved, and the (Ala)6 regions were shown to form β-sheet structure in the staggered arrangement. Although the fraction of β-sheet components tended to increase and the fraction of random coil component decrease toward both chain ends, significant change in the fractions was observed depending on the amino acid position. These results were successfully rationalized throug...

Conformation of Crystalline and Noncrystalline Domains of [3-13C]Ala-, [3-13C]Ser-, and [3-13C]Tyr-Bombyx mori Silk Fibroin in a Hydrated State Studied with 13C DD/MAS NMR

Silk fibroin fiber is a well-known textile, but is also used as biomaterial that is of interest for use in a variety of applications, usually in a hydrated state. Thus, the determination of the hydrated silk fibroin structure is important for understanding the function and in designing novel biomaterials. In this work, 13C dipolar decoupling/magic angle spinning NMR was used to determine the local conformation of [3-13C]Ala-, [3-13C]Ser-, and [3-13C]Tyr -Bombyx mori silk fibroin in a hydrated state. Ser residues are present predominantly in the crystalline domains, Tyr predominantly in the noncrystalline domains and Ala residues in both domains. The fraction of β-sheet and two random coil distributions with fast and slow chain dynamics could be determined for all these residues by 13C conformation-dependent chemical shift. The fraction of β-sheet of these residues in the fiber and the crystalline fraction did not change significantly before and after hydration. The fraction of random coil conformations wi...

Practical use of chemical shift databases for protein solid-state NMR: 2D chemical shift maps and amino-acid assignment with secondary-structure information

We introduce a Python-based program that utilizes the large database of (13)C and (15)N chemical shifts in the Biological Magnetic Resonance Bank to rapidly predict the amino acid type and secondary structure from correlated chemical shifts. The program, called PACSYlite Unified Query (PLUQ), is designed to help assign peaks obtained from 2D (13)C-(13)C, (15)N-(13)C, or 3D (15)N-(13)C-(13)C magic-angle-spinning correlation spectra. We show secondary-structure specific 2D (13)C-(13)C correlation maps of all twenty amino acids, constructed from a chemical shift database of 262,209 residues. The maps reveal interesting conformation-dependent chemical shift distributions and facilitate searching of correlation peaks during amino-acid type assignment. Based on these correlations, PLUQ outputs the most likely amino acid types and the associated secondary structures from inputs of experimental chemical shifts. We test the assignment accuracy using four high-quality protein structures. Based on only the Cα and Cβ chemical shifts, the highest-ranked PLUQ assignments were 40-60 % correct in both the amino-acid type and the secondary structure. For three input chemical shifts (CO-Cα-Cβ or N-Cα-Cβ), the first-ranked assignments were correct for 60 % of the residues, while within the top three predictions, the correct assignments were found for 80 % of the residues. PLUQ and the chemical shift maps are expected to be useful at the first stage of sequential assignment, for combination with automated sequential assignment programs, and for highly disordered proteins for which secondary structure analysis is the main goal of structure determination.

A solid-State NMR Study of Abeta Protofibrils

Aβ(1-40) is the major fibril-forming peptide from Alzheimer's disease, which adopts a highly ordered β-sheet conformation upon aggregation into amyloid fibrils. Several techniques have provided a wealth of structural information on mature Aβ(1-40) fibrils. Yet, the complex formation process of mature fibrils is, from a structural point of view, not well understood. Here we use ssNMR spectroscopy to elucidate the structure of Aβ protofibrils. This analysis is possible since the binding of the antibody B10AP prevents the conversion of these metastable intermediates into mature fibrils. With a set of eight peptides with varying isotope labeling schemes, 30 residues distributed over the entire peptide sequence were covered. 13C CPMAS spectra and 2D correlation experiments were recorded for unambiguous assignment of all carbons. From the conformation dependent chemical shifts we could identify peptide segments of stable secondary structure and evaluated the backbone structure using TALOS. Based on this data, Aβ protofibrils encompass residues 16-22 and 30-36 in β-sheet conformation. Further, three structural regions of the protofibrils present random coil-like chemical shifts, one (residues 23-26) as intermediate segment between the β-strands and two others at the peptide N- and C- terminus. Obviously the structural elements of mature Aβ(1-40) fibrils are already present in protofibrils, but the β-sheet regions apparently elongate during the fibrils conversion. Further information about the dynamics of these regions is provided by measurement of the strength of dipolar couplings, which are converted into an order parameter. Protofibrils show high order parameters (>0.8) within the β-strand regions, while the measured S values are below 0.8 at the termini. We never observed S values below 0.4 that would have indicated very high mobility. Thus, significant structural order exists also within those sequence segments that have chemical shift values corresponding to a random coil.

Structural Characterization of Silk-Based Water-Soluble Peptides (Glu)n(Ala-Gly-Ser-Gly-Ala-Gly)4 (n = 4−8) as a Mimic of Bombyx mori Silk Fibroin by 13C Solid-State NMR

Peptides with a combination of hydrophilic and hydrophobic sequences mimicking the primary structure of Bombyx mori silk fibroin were synthesized and studied in the solid state by NMR using 13C selective labeling coupled with 13C conformation-dependent chemical shifts and 2D solid-state spin-diffusion NMR. The hydrophilic sequence was poly(l-glutamic acid) (E)n, and the hydrophobic one was the consensus sequence of the crystalline fraction of B. mori silk fibroin, (AGSGAG)4. The balance of hydrophilic and hydrophobic characters of the peptide was controlled by changing the relative length, n, of (E)n from 4 to 8. When n = 4 and 5, the structure of the hydrophobic sequence is basically Silk I (the structure of B. mori silk fibroin before spinning in the solid state), and the polyglutamate sequences are random coil. On the other hand, when n = 6−8, the structure of the polyglutamate sequence changes gradually from random coil to β-sheet, and the hydrophobic sequence adopts a mixture of β-sheet and random co...

Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR

Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved 13C and 15N signals of [1- 13C]Tyr-, [ 15N]Pro- and [1- 13C]Val-, [ 15N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1- 13C]Val-, [ 15N]Pro signals to the specific residues in bR. The previous assignments of [1- 13C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent 15N chemical shifts of Pro bonded to Val in the presence of 13C– 15N correlation, although no assignment of peak is feasible for 13C nuclei not bonded to Pro. 13C or 15N spin–lattice relaxation times ( T 1), spin–spin relaxation times under the condition of CP-MAS ( T 2), and cross relaxation times ( T CH and T NH) for 13C and 15N nuclei and carbon or nitrogen-resolved, 1H spin–lattice relaxation times in the rotating flame ( 1H T 1ρ) for the assigned signals were measured in [1- 13C]Val-, [ 15N]Pro-bR. It turned out that V69–P70 in the BC loop in the extracellular side has a rigid β-sheet in spite of longer loop and possesses large amplitude motions as revealed from 13C and 15N conformation-dependent chemical shifts and T 1, T 2, 1H T 1ρ and cross relaxation times. In addition, breakage of the β-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.

Spectral fitting for signal assignment and structural analysis of uniformly 13C-labeled solid proteins by simulated annealing based on chemical shifts and spin dynamics

We describe an approach for the signal assignment and structural analysis with a suite of two-dimensional (13)C-(13)C magic-angle-spinning solid-state NMR spectra of uniformly (13)C-labeled peptides and proteins. We directly fit the calculated spectra to experimental ones by simulated annealing in restrained molecular dynamics program CNS as a function of atomic coordinates. The spectra are calculated from the conformation dependent chemical shift obtained with SHIFTX and the cross-peak intensities computed for recoupled dipolar interactions. This method was applied to a membrane-bound 14-residue peptide, mastoparan-X. The obtained C', C(alpha) and C(beta) chemical shifts agreed with those reported previously at the precisions of 0.2, 0.7 and 0.4 ppm, respectively. This spectral fitting program also provides backbone dihedral angles with a precision of about 50 degrees from the spectra even with resonance overlaps. The restraints on the angles were improved by applying protein database program TALOS to the obtained chemical shifts. The peptide structure provided by these restraints was consistent with the reported structure at the backbone RMSD of about 1 A.

The role of irregular unit, GAAS, on the secondary structure of Bombyx mori silk fibroin studied with 13C CP/MAS NMR and wide-angle X-ray scattering.

Bombyx mori silk fibroin is a fibrous protein whose fiber is extremely strong and tough, although it is produced by the silkworm at room temperature and from an aqueous solution. The primary structure is mainly Ala-Gly alternative copolypeptide, but Gly-Ala-Ala-Ser units appear frequently and periodically. Thus, this study aims at elucidating the role of such Gly-Ala-Ala-Ser units on the secondary structure. The sequential model peptides containing Gly-Ala-Ala-Ser units selected from the primary structure of B. mori silk fibroin were synthesized, and their secondary structure was studied with (13)C CP/MAS NMR and wide-angle X-ray scattering. The (13)C isotope labeling of the peptides and the (13)C conformation-dependent chemical shifts were used for the purpose. The Ala-Ala units take antiparallel beta-sheet structure locally, and the introduction of one Ala-Ala unit in (Ala-Gly)(15) chain promotes dramatical structural changes from silk I (repeated beta-turn type II structure) to silk II (antiparallel beta-sheet structure). Thus, the presence of Ala-Ala units in B. mori silk fibroin chain will be one of the inducing factors of the structural transition for silk fiber formation. The role of Tyr residue in the peptide chain was also studied and clarified to induce "locally nonordered structure."

Use of 13C conformation-dependent chemical shifts to elucidate the local structure of a large protein with homologous domains in solution and solid state

In order to clarify the difference between solution NMR and X-ray diffraction analyses concerning the presence of α-helical structure in protein A, the 13C conformation-dependent chemical shifts of the 13C-labeled carbonyl carbons for selectively labeled protein A were used. In the 13C CP/MAS NMR spectra, the higher-field shifts of the carbonyl carbons of 13C-labeled Thr and Val residues compared with the random coil chemical shifts both in solution and solid state imply the presence of the third helix in the polypeptide chain, in contrast to the crystal structure of Fc-bound B-domain. Thus, a combination of selective isotope labeling and conformation-dependent chemical shifts will be a good Indicator to monitor the local structure of homologous protein in solution and solid state.

NMR study of silk I structure ofBombyx mori silk fibroin with15N- and13C-NMR chemical shift contour plots

The metastable state silk I structures of Bombyx mori silk fibroin in the solid state were studied on the basis of 15N- and 13C-nmr chemical shifts of Ala, Ser, and Gly residues. The 15N cross-polarization magic angle spinning (CP/MAS) nmr spectra of the precipitated fraction after chymotrypsin hydrolysis of B. mori silk fibroin with the silk I and silk II forms were measured to determine the 15N chemical shifts of Gly, Ala, and Ser residues. For comparison, 15N CP/MAS nmr chemical shifts of Ala were measured for [15N] Ala Philosamia cynthia ricini silk fibroin with antiparallel β-sheet and α-helix forms. The 13C CP/MAS nmr chemical shifts of Ala, Ser, and Gly residues of B. mori silk fibroin with the silk I and silk II forms, as well as 13C CP/MAS nmr chemical shifts of Ala residue of P. c. ricini silk fibroin with β-sheet and α-helix forms, are used for the examination of the silk I structure. Both silk I and α-helix peaks are shifted to a lower field than silk II (β-sheet) for the Cα carbons of the Ala residues, while both Cβ carbon peaks are shifted to higher field. However, the silk I peak of the 15N nucleus of the Ala residue is shifted to lower field than the silk II peak, but the α-helix peak is shifted to high field. Thus, the difference in the structure between the silk I and α-helix is reflected in a different manner between the 13C and 15N chemical shifts. The Cα and Cβ chemical shift contour plots for Ala and Ser residues, and the Cα plot for the Gly residue, were prepared from the Protein Data Bank data obtained for 12 proteins and used for discussing the silk I structure quantitatively from the conformation-dependent chemical shifts. The plots reported by Le and Oldfield for 15N chemical shifts were also used for the purpose. All these chemical shift data support Fossey's model (Ala: ϕ = −80°, φ = 150°, Gly: ϕ = −150°, φ = 80°) and do not support Lotz and Keith's model (Ala: ϕ = −104.6°, φ = 112.2°, Gly: ϕ = 79.8°, φ = 49.7° or Ala: ϕ = −124.5°, φ = 88.2°, Gly: ϕ = −49.8°, φ = −76.1°) as the silk I structure. © 1997 John Wiley & Sons, Inc.

Chain conformation in concentrated pectic gels: evidence from 13C NMR

13C NMR spectroscopy is a sensitive method of exploring the conformation of polysaccharides in both solid and gel states, because the positions of the peaks for the two carbons on either side of the glycosidic linkage, C-1 and C-4 for pectins, depend on the chain conformation. This approach was used to determine the conformation of calcium pectate at gel concentrations of ca. 0.3 g cm −3, comparable with those found in vivo. The solid acid, methyl-esterified, and sodium forms of pectate, which are known to have a right-handed, threefold helical ( 3 1 ) conformation, were used to define the spectral features of this conformation. These features were also present in both gel and solid calcium pectate, and were more conspicuous in the solid form. When dilute calcium pectate gels are dried they are known from CD and EXAFS evidence to undergo a conformational transition from the “egg-box” twofold helical ( 2 1 ) conformation to the 3 1 conformation. Calcium guluronate does not undergo this change, nor does calcium pectate in the presence of excess of monovalent cations. Changes in the 13C NMR spectrum marking this transition were observed on drying calcium pectate gels, were prevented by excess of K +, and were not found for guluronate. This enabled the spectral features corresponding to the 2 1 conformation to be identified. The spectral assignments were in agreement with the stereoelectronic theory of conformation-dependent chemical shifts, which also allowed a third set of peaks to be assigned to conformations dispersed between the 3 1 and 2 1 helices. About 70% of the pectate was visible in the gel spectra, the remaining chains being too mobile to be detected. It was concluded that the egg-box form, as dimers or larger aggregates, was the largest single component of the gels studied here, but that substantial quantities of 3 1 and intermediate helical aggregated forms were also present. Intact, pectin-containing plant cell walls also show spectral features characteristic of these components.

Combined use of 13C chemical shift and 1H??13C? heteronuclear NOE data in monitoring a protein NMR structure refinement

A large portion of the 13C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28 degrees C has been determined at natural isotope abundance. Sequence-specific 13C assignments are reported for 100% of the assignable C alpha, 96% of the C beta, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C alpha chemical shifts for regions of regular beta-sheet structure. These assignments also provide the basis for interpreting 1H alpha-13C alpha heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these 1H alpha-13C alpha HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated delta delta C alpha values can be used to monitor the refinement of this protein's structure, particularly for beta-sheet regions. Improved agreement between calculated and observed values of delta delta C alpha is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, delta delta C alpha, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.

Molecular recognition of antigenic polysaccharides: a conformational comparison of capsules from Streptococcus pneumonicæ serogroup 9

Aqueous solution conformations of three antigenic bacterial capsular polysaccharides (CPS) from S. pneumoniæ serogroup 9 were determined using a combination of NMR data (NOE build-up rates and conformation-dependent chemical shifts), simulated annealing, and molecular dynamics simulations. Each polymer adopts a flexible extended ribbon conformation in solution. Conformations of structural elements shared by each PS are indistinguishable. Differences in conformations are minor and localised at the sites of structural variations; there is no evidence of long-range stabilisation of a secondary structure. It is likely that antigenic specificity of Group 9 PS is dominated by local structural variation rather than by conformational differences.

ModAnE: MODelling of ANisotropic effects

Nuclear magnetic resonance (NMR) is a useful tool for 3D-structure eludication. For example, the Nuclear Overhauser Effect (NOE) has been widely used. In this study, we are trying to develop a new complementary modelling method, using the classical [1H]-NMR theory of anisotropic effect. The phenomenon consists of a conformation-dependent chemical shift of an observed hydrogen atom, caused by neighbouring substituents. The ModAnE software includes all mathematical descriptions about the subject. It can be directly addressed as a new module in the SYBYL molecular modelling package. A Monte-Carlo program has been integrated into the ModAnE software in order to allow for conformational refinement. Different classes of molecules have been tested using this Monte-Carlo simulation.

The three‐dimensional structure of guanine‐specific ribonuclease F1 in solution determined by NMR spectroscopy and distance geometry

Two-dimensional 1H-NMR studies have been performed on ribonuclease F1 (RNase F1), which contains 106 amino acid residues. Sequence-specific resonance assignments were accomplished for the backbone protons of 99 amino acid residues and for most of their side-chain protons. The three-dimensional structures were constructed on the basis of 820 interproton-distance restraints derived from NOE, 64 distance restraints for 32 hydrogen bonds and 33 phi torsion-angle restraints. A total of 40 structures were obtained by distance geometry and simulated-annealing calculations. The average root-mean-square deviation (residues 1-106) between the 40 converged structures and the mean structure obtained by averaging their coordinates was 0.116 +/- 0.018 nm for the backbone atoms and 0.182 +/- 0.015 nm for all atoms including the hydrogen atoms. RNase F1 was determined to be an alpha/beta-type protein. A well-defined structure constitutes the core region, which consists of a small N-terminal beta-sheet (beta 1, beta 2) and a central five-stranded beta-sheet (beta 3-beta 7) packed on a long helix. The structure of RNase F1 has been compared with that of RNase T1, which was determined by X-ray crystallography. Both belong to the same family of microbial ribonucleases. The polypeptide backbone fold of RNase F1 is basically identical to that of RNase T1. The conformation-dependent chemical shifts of the C alpha protons are well conserved between RNase F1 and RNase T1. The residues implicated in catalysis are all located on the central beta-sheet in a geometry similar to that of RNase T1.

The 1H‐NMR assignments of the aromatic resonances in complexes of Lactobacillus casei dihydrofolate reductase and the origins of their chemical shifts

All the aromatic proton resonances in the 500-MHz NMR spectra of Lactobacillus casei dihydrofolate reductase have been assigned for several of its complexes with inhibitors. For the complexes with methotrexate and trimethoprim this was achieved by using a combination of NMR techniques in conjunction with a selectively deuterated protein designed to simplify the spectra such that nuclear Overhauser effect (NOE) connections could be detected with greater ease and certainty. By correlating these NOE data with crystal structure data on related complexes it was possible to assign all the aromatic resonances and to extend these assignments to spectra of other complexes of dihydrofolate reductase. The conformation-dependent chemical shifts observed for many of the resonances could be explained qualitatively, but not quantitatively, in terms of ring-current shifts. The discrepancies between calculated ring-current shifts and the observed conformation-dependent shifts could not in general be accounted for satisfactorily in terms of carbonyl-group anisotropic shielding contributions calculated using presently available models. In the case of the H delta 1, delta 2 protons of Phe30 some of the discrepancy probably results from a difference in the conformation of the Phe ring between the solution and crystal states.

Characterization of the proteinase inhibitor IIA from bull seminal plasma by 1H nuclear magnetic resonance : Stability, amide proton exchange and mobility of aromatic residues

The isoinhibitor IIA from bull seminal plasma was investigated in aqueous solution by 1H nuclear magnetic resonance (n.m.r.). The analysis of the 1H n.m.r. data was based on individual resonance assignments, which are described in the following paper. Large conformation-dependent chemical shifts for aliphatic amino acid side-chains, numerous slowly exchanging amide protons and unusual pH titrations of two aromatic residues show that this protein forms a compact, globular conformation. This form of the protein is stable between pH 4 and 12 at 25 degrees C, and between 5 and 50 degrees C at pH 4.9. At temperatures above 50 degrees C there is evidence for an equilibrium between several different conformations, with the rate of exchange between the different species being in the intermediate range on the n.m.r. time-scale. Preliminary data are presented for the individual exchange rates of 18 backbone amide protons. Among the four aromatic rings, Phe10, Phe38 and Tyr16 undergo rapid 180 flips over the entire temperature range, whereas for Tyr32 a temperature-dependent transition from low-frequency to high-frequency flipping motions was observed.

Ring current effects in the conformation dependent NMR chemical shifts of aliphatic protons in the basic pancreatic trypsin inhibitor

Previously, a highly refined crystal structure and energy refined atomic coordinates were obtained for the basic pancreatic trypsin inhibitor, as well as numerous individual resonance assignments in the 1H NMR spectrum. These data were now used to investigate the contributions from the local ring current fields of the aromatic rings to the overall conformation dependent chemical shifts in this globular protein. A program was written which allowed the consideration of certain aspects of internal mobility of the protein, and the different commonly used ring current equations were compared. These studies indicate that ring current shifts are the dominant contribution to the observed conformation dependent chemical shifts of the peripheral aliphatic side chain protons. On the other hand, it appears that ring current shifts do not make dominant contributions to the conformation dependent shifts of the backbone α- and amide protons or the aromatic protons in the inhibitor. On the basis of the empirical calibration with the peripheral aliphatic side chain protons, the Johnson-Bovey ring current equation was selected for an analysis of the ring geometries of two prolines in the inhibitor.

High field 13C NMR studies at 90.5 MHz of the methyl groups in the basic pancreatic trypsin inhibitor

The basic pancreatic trypsin i~ibitor (BPTI) is a well characterized [1,2] small globular protein which functions as an inhibitor of trypsin and other proteases [3]. BFTI was extensively investigated by nuclear magnetic resonance (NMR) techniques [4-g]. In addition to the interest for a better understanding of the structure-function relations in protease-inhibitor systems, these data also provided new insights into general aspects of protein structure and relations between NMR spectral properties and protein conformation. To further pursue these different areas of interest we have started additional investigations using high field 13C NMR at 90.5 MHz. The present paper illustrates the advantages of the high field for the identification of individual resonance lines of protonated carbon atoms, in particular methyl carbons, and reports conformation dependent chemical shifts for numerous methyl 13C resonances in BPTI. While principal aspects of high field 13C nuclear magnetic resonance (NMR) of biological molecules have been much discussed [IO,1 11, relatively few experiments with proteins have so far been reported [ 12141. Much emphasis has actually been on work with extra large sample sizes at the lowest field strength commonly used in high resolution NMR, which were shown to be favorable conditions for studies of non-protonated carbons [15-171. The high field 13C NMR experiments so far mainly included investigations of the field dependence of the spin relaxation times [12,14] and a study of the pH titration of carboxyl groups in lysozyme [ 131. In the

Proton NMR studies of the aromatic residues in the basic pancreatic trypsin inhibitor (BPTI)

The aromatic region of the 1H NMR spectrum of the basic pancreatic trypsin inhibitor (BPTI), which contains the resonances of four tyrosyl and four phenylalanyl residues, was investigated at 360 MHz and 270 MHz. With double resonance experiments, three pairs of coupled tyrosine 3,5- and 2,6-proton resonances could be identified, showing that the rotations of the aromatic rings of these tyrosines about the axis given by the C β-C γ bond are rapid on the NMR time scale. On the other hand, the globular protein conformation is clearly manifested in the chemical shifts of the aromatic protons of the tyrosines. With the available evidence, it appears unlikely that these conformation dependent chemical shifts could be caused by interactions with the ring current fields of neighboring aromatic residues, which emphasizes the importance of alternative mechanisms effective in producing conformation dependent chemical shifts in globular proteins.