Structure Analyses of Alanine Trimer and Tetramer Crystals with

Subscriber access provided by Kaohsiung Medical University

B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Structure Analyses of Alanine Trimer and Tetramer Crystals with Antiparallel and Parallel #-Sheet Structures Using SolidState H Spin Diffusion 2D Correlation NMR Spectroscopy 1

Akira Naito, Shunsuke Kametani, Akihiro Aoki, and Tetsuo Asakura J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b07859 • Publication Date (Web): 20 Sep 2018 Downloaded from http://pubs.acs.org on September 27, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Structure Analyses of Alanine Trimer and Tetramer Crystals with Antiparallel and Parallel β-sheet Structures Using Solid-State

1

H

Spin

Diffusion

2D

Correlation

NMR

Spectroscopy Akira Naito1, Shunsuke Kametani1, Akihiro Aoki1, Tetsuo Asakura1* 1

Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588

*To whom correspondence should be addressed (Tel & Fax, 81-42-383-7733) Email: [email protected]

1

ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Poly-L-alanine (PLA) sequences are key elements of the crystalline domains of spider dragline and wild silkworm silks. In the present work, 1H spin diffusion 2D correlation NMR spectra were observed for selectively deuterated (Ala)3 and (Ala)4 crystals to develop the analytical method for the structure of PLA sequences. The build-up curves of the cross peaks for three kinds of 1H pairs in selectively deuterated (Ala)3 and (Ala)4 crystals were observed to obtain spin diffusion rate constant kj,k from relaxation master equations Pi,j(τm). The kj,k values subsequently lead to effective inter-proton distance reffj,k (obs) values for individual proton-proton pairs which include intra- and inter-molecular contributions. The reffj,k (obs) values were compared to reffj,k (calc) values obtained from the experimentally determined atomic co-ordinates of anti-parallel (AP) β-sheet (Ala)3 and (Ala)4, and parallel (P) β-sheet of (Ala)3 and (Ala)4 crystals. The agreement between the reffj,k (obs) and reffj,k (calc) values was good for AP β-sheet (Ala)3 and (Ala)4 crystals, while poor for P β-sheet (Ala)3 and (Ala)4 crystals. These deviations were obtained from the inter-proton distances of inter-chain contributions due to different packing arrangements. Packing arrangements of PLA region are important when considering the relevant structure and the mechanical properties of silks.

2


Page 2 of 32

Page 3 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. Introduction Spider dragline silks and wild silkworm silks possess superior mechanical properties by combining high modulus, strength, and extensibility. At present, these silks continue to attract the attention of researchers in biology, biochemistry, biophysics, analytical chemistry, polymer technology, textile technology and tissue engineering because of their excellent mechanical properties.1 Poly-L-alanine (PLA) sequences are known as key elements in the structure of the crystalline domains of these silks.2-6

In

addition, PLA also exists in antifreeze proteins7,8 and amyloid proteins.9-12 Thus, the determination of PLA structure is important. The atomic co-ordinates of PLA have been reported for only shorter PLA molecules such as (Ala)3 and (Ala)4 experimentally. The crystal structures for (Ala)3 and (Ala)4 have been determined for antiparallel (AP) β-sheet (Ala)3,13 parallel (P) β-sheet (Ala)3,14 and AP β-sheet (Ala)4

15,16

by X-ray single crystal diffraction method. The

atomic co-ordinate of P β-sheet (Ala)4 crystal has been also determined by using structural constraints obtained from solid-state NMR and refined by molecular dynamics (MD) simulation.17 In addition, PLAs pack into two different arrangements, depending on the length of the PLA sequence. Short sequence (n < 5) of PLAs pack into rectangular arrangement, in which the packing of the adjacent AP β-sheet chains is rectangular.15 On the other hand, longer PLAs (n > 6) pack into a staggered arrangement, in which the packing of the adjacent AP β-sheet chains is staggered.15,18. It is also observed that fractions of β-sheet structure changed by crystallization solvent, temperature variation and application of

mechanical sharing in poly-ala peptides.

19,20

The atomic coordinates of PLA sequence in Samia cynthia ricini silk fibroin fiber packed into a staggered arrangement have been reported.21 However, there are no 3



Page 4 of 32

atomic level structures of AP β-sheet PLA longer than (Ala)4 because it is difficult to prepare large single crystal for use in X-ray single crystal diffraction analysis.15 Thus, it is necessary to develop spectroscopic methods to determine the atomic-resolution structure of AP β-sheet PLA longer than (Ala)4. As 13C homonuclear correlation NMR methods, theoretical and simulation studies of proton driven spin diffusion (PDSD) have been recently improved.22 For example, a combined experiment and theoretical study of PDSD via 2D spectroscopy of a single crystal has been prepared by Suter and Ernst in 1985.23 Additional groups have explored the energy conservation via spatially separated spins,24 or spins with different chemical shifts,25,26 based on the experimental results from single molecules. One merit of the PDSD experiment is that any given peak intensity is not influenced by dipolar truncation.27,28 As a result, information through all of the inter-carbon distances of the sample can be obtained simultaneously.27 Combined with the MD simulations, the PDSD experiment has been recently utilized to refer the structure of proteins by using the NMR determined distance constraints.29 13

C-1H dipolar-assisted rotational resonance (DARR)

solid-state

13

30,31

experiment is a second

C homonuclear correlation NMR experiment that makes use of a 1H radio

frequency (rf) field at the amplitude satisfying the rotary resonance recoupling (R3)

32

condition. Under the R3 condition, the line widths of 13C-1H dipolar recoupled spectra become broadened. As a result of peak broadening, the build-up rate of cross peaks, which reflect the spin-exchange rate between two correlated

13

C nuclei, may be

increased. The build-up of the cross peak intensity as a function of the mixing time (termed a build-up curve) provides the rate constant between the correlated 13C nuclear pair. Consequently, the internuclear distances of the correlated pairs can be determined. 4


Page 5 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Therefore, DARR experiments are useful for detecting weak cross peak signals with long internuclear distances. In our previous work,33 the packing structures of [U-13C] silk fibroin fiber of Samia cynthia ricini (a wild silkworm) and their 34-mer model peptide, GGAGGGYGGDGG(A)12GGAGDGYGAG, with different

13

C-labeled positions have

been examined by DARR. The results showed that the silk fibroin fiber and polypeptide are packed in a staggered arrangement with an AP β-sheet structure. Further, in our previous work, the heavy-atom coordinates of the AP β-sheet (Ala)4 structure, including the intermolecular structure, have been determined by X-ray diffraction with a small single crystal.15 Moreover, the 1H coordinates of the AP β-sheet (Ala)4 peptide have been determined from the 1H chemical shift assignments in a 1H solid-state NMR spectrum by ultrafast magic angle spinning (MAS) and the chemical-shift calculations with the use of Cambridge Serial Total Energy Package.16,34 In further work, the cross peak build-up curves of the DARR spectra of [U-13C] AP β-sheet (Ala)4 were analyzed and determined effective internuclear distances between j and k 13C nuclei, reffj,k (obs).35 The obtained distances were compared with those obtained from a known crystal structure. The best standard deviation (0.244 Å) were obtained for the DARR data obtained without considering zero-quantum line-shape functions in the range from 1.0 to 6.0 Å. As compared with the

13

C DARR experiment in the solid state, the presence of

strong dipolar couplings between protons considerably broadens the spectral resonances of 1H NMR signals, even under MAS. The use of combined rotation and multiple-pulse techniques (CRAMPS) 36 and application of 1H decoupling field tilting the magic angle to the static magnetic field originated by Lee and Goldburg37 (LG) and further modified 5



using flip flop LG 38,39 have been developed to obtain high resolution proton NMR spectra in the solid state. Furthermore, considerable advances in the phase modulated LG field of homonuclear dipolar decoupling have recently made the direct acquisition of highly resolved proton spectra possible.40,41 Recently, high resolution 1H NMR signals and 1H-1H dipolar interaction in the solid state can be obtained by using ultra fast MAS NMR.

5, 16,17,42--44

Proton spin diffusion (PSD) is a ubiquitous process in solids,45

whereby magnetization is exchanged between protons according to a process driven by the internuclear distance dependent dipolar coupling. It has long been recognized that this provides in principle a probe of internuclear distances and therefore structure determination. However, the rate of spin diffusion also depends on the orientation of the internuclear vector in the sample, the details of the anisotropic chemical shifts of the two coupled nuclei, the coupling to other protons, and experimental factors such as the magic angle spinning rate.45 To overcome this problem, recently, 1H spin diffusion with a phenomenological multi-spin kinetic rate matrix approach has been developed and summed over the structure.46,47 The approach is demonstrated with powdered β-L-aspartyl L-alanine, where inter-1H nuclear distances were obtained within the standard deviation of 0.33 Å of the known crystalline coordinates.47 In this paper, we discuss the applicability for distance measurement by 1H spin diffusion 2D correlation NMR spectroscopy in the solid state. Selectively labeled AP β-sheet (Ala)3 ([d4]Ala-[d4]Ala-Ala, [d4]Ala-Ala-[d4]Ala, Ala-[d4]Ala-[d4]Ala) and AP

β-sheet

(Ala)4

([d4]Ala-[d4]Ala-[d4]Ala-Ala,

[d4]Ala-[d4]Ala-Ala-[d4]Ala,

[d4]Ala-Ala-[d4]Ala-[d4]Ala, Ala-[d4]Ala-[d4]Ala-[d4]Ala). These samples show high resolution 1H NMR signals using homonuclear decoupling pulses based on phase modulated Lee-Goldburg (PMLG) pulse sequence

48-50

to resolve each proton signal

6


Page 6 of 32

Page 7 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


belonging to each amino acid residue of selectively deuterated PLAs. The experimentally determined internuclear distance reffj,k (obs) values are compared with the effective internuclear distance reffj,k (calc) values calculated from the experimentally determined atomic co-ordinates. It is also compared the AP β-sheet (Ala)3 and AP β-sheet (Ala)4 packing structures with those of P β-sheet (Ala)3 and P β-sheet (Ala)4 packing structures to inspect the accuracy of these experiments.

2. Experimental Section 2-1. Sample preparation Selectively

labeled

(Ala)3

([2,3,3,3-d4]Ala-[2,3,3,3-d4]Ala-Ala;

dA-dA-A,

[2,3,3,3-d4]Ala-Ala-[2,3,3,3-d4]Ala; dA-A-dA, Ala-[2,3,3,3-d4]Ala-[2,3,3,3-d4]Ala; A-dA-dA)

and

selectively

labeled

(Ala)4

([2,3,3,3-d4]Ala-[2,3,3,3-d4]Ala-[2,3,3,3-d4]Ala-Ala;

dA-dA-dA-A,

[2,3,3,3-d4]Ala-[2,3,3,3-d4]Ala-Ala-[2,3,3,3-d4]Ala;

dA-dA-A-dA,

[2,3,3,3]Ala-Ala-[2,3,3,3-d4]Ala-[2,3,3,3]Ala; dA-A-dA-dA) were synthesized from Fmoc Ala and Fmoc [2,3,3,3-d4]Ala monomers in the solid phase synthesis. 15,17, 35 The purities of these peptides were confirmed by 13C solution NMR and IR to be more than 95 %. The purified peptides were treated with H2O/EtOH = 1:1 to form an AP β-sheet fine crystal15 and was identified as AP β-sheet (Ala)3 and AP β-sheet (Ala)4 peptides.

2-2. Solid-state 1H NMR experiments High-resolution 1H NMR spectra were obtained using phase-modulated Lee-Goldburg (PMLG) experiment.48,49 Using this pulse sequence, isotropic chemical shift is retained, whereas its chemical shift anisotropy and the dipolar interaction should be eliminated 7



during the PMLG homonuclear decoupling. PMLG 1H solid-state NMR spectra were observed in a JEOL ECX400 spectrometer using double resonance MAS probe for the rotor with 4 mm diameter. All the NMR measurements were performed at 25 ºC using temperature controller of the NMR spectrometer. The (Ala)3 and (Ala)4 samples were centered in the rotor by polytetrafluoroethylene spacers. In the 1D experiments, windowed (w)PMLG-3 sequence that consists of three different phase angles (X1=34.6º, X2=103.9º, X3=173.2º) enables one-dimensional acquisition in the PMLG scheme.50 In the 2D 1H-1H correlation experiment (Figure 1), PMLG-3 was used for t1 time and the 1

H magnetization is transferred to the z direction by applying X (π/2) pulse. The 1H

magnetization is turned to the z direction and the spin-diffusion undergoes during mixing time (τm). Consequently, the 1H magnetization is transferred to the y direction and wPMLG-3 was used for t2 time to acquire free induction decay signals. The 90º pulse length of 2.8 µs and PMLG pulse length of 2.4 µs were used. The MAS frequency was set to 15 kHz, the number of scans was 8 and the recycle delay was set to 3 s. In the 2D correlation experiments, size of acquisitions was 64 and 512 for t1 and t2 dimensions, respectively. PMLG-3 and wPMLG-3 sequences were used in the t1 and t2 dimensions, respectively. The mixing times were varied from 0 to 1 ms (0, 0.01, 0.02, 0.04, 0.07, 0.10, 0.13, 0.16, 0.20, 0.70 and 1ms).

8


Page 8 of 32

Page 9 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Pulse sequence used for the 1H spin diffusion 2D correlation NMR experiments. Following X (π/2) pulse, the 1H magnetization evolved during PMLG-3 (t1) (X1 = 34.6º, X2=103.9º, X3=173.2º, X3*=X3+180º, X2*=X2+180º, X1*=X1+180º), and is then transferred to the z-axis. 1H spin exchange takes place during a mixing time τm. Consequently, 1H magnetization again transferred to the y-direction and the FID signals were acquired for t2 time under wPMLG-3 homo-nuclear decoupling, in which windows for samplings are inserted.

2-3. Analysis of 1H spin diffusion 2D correlation NMR spectra To determine the internuclear distances for j and k protons, we use the model of spin diffusion with a phenomenological multi-spin kinetic rate matrix approach. In this model, the rate constant of exchange between two types of spin j and k is given by 47

,

,

(1)

And 35

9



! ∑"

,

# ∑ ,,,

Page 10 of 32

(2)

Where µ0, γ, and ħ are the physical constant of vacuum magnetic permeability, proton magnetogyric ratio, and Plank constant, respectively, and reffj,k is effective internuclear distance between hydrogen nuclei j and k, and rj,k,l,m is the internuclear distance between spin j in molecule l and spin k in molecule m. M is the number of molecules and L is the number of nonequivalent molecules in a system, and sum over exchange between sites j and k in different molecules in the crystalline lattice. A is a phenomenological scaling factor. The peak intensities against mixing time, Pi,j (τm) observed in a two-dimensional exchange spectrum are expressed as relaxation master equation which is given by.47

1 $%, &'" ( )*+,−.'" /%, 0%,

(3)

Where K is an N x N matrix of the rates ki,j of exchange between the N different resonances in the spectrum, where τm is the spin diffusion mixing time, and where M0j,j is the intensity of the j-th peak at τm = 0. We have observed that the 1H T1 values of Ala 1

Hβ are around 0.9 sec 51. Thus, T1 processes were not taken into account the analyses

of the build-up curves. We first evaluate the K matrix using the initial build-up curve of the individual peak heights against the mixing time τm by the relation of Eq. (3). Subsequently, effective internuclear distances reffj,k (obs) are evaluated using Eq. (1) with the scaling factor, A. We then calculate the effective internuclear distances reffj,k (calc) using the values determined from the experimentally determined atomic co-ordinates. These values are compared with reffj,k (obs) and the scaling factor, A was evaluated to get the 10


Page 11 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


best fit between reffj,k (obs) and reffj,k (calc). After the best fit scaling factor A was determined, reffj,k (obs) values are evaluated and finally the standard deviations were evaluated.

Results and Discussion 1D 1H NMR spectra of AP β -sheet (Ala)3 and AP β-sheet (Ala)4 crystals Figure 2 shows the 1H PMLG NMR spectra of AP β-sheet (Ala)3 crystals. Three kinds of protons HCβ (P3), Hcα (P2) and HNH (P1) were resolved in the 1H NMR spectra of dA-dA-A, dA-A-dA and A-dA-dA. It is noted that Hcα signals appeared differently for different carbon position in the backbone. In contrast, HCβ signals appeared in the same position for different Ala positions. It is also noted that the HNH signals showed broad line shapes, indicating large chemical shift difference between the positions of NH and NH3 groups. Similar spectral features were obtained for 1H PMLG NMR spectra of AP β-sheet (Ala)4 crystals. It is important to point out that 1H PMLG NMR signals are good enough to distinguish the HCβ, HCα and HNH signals, however, are not good enough to distinguish inter-residue 1HCβ signals. Therefore, to obtain the 1H-1H distances between the 1H signals for particular Ala residue, deuterated Ala ([2,3,3,3-d4] Ala or simply dA) was used to get A-dA-dA, dA-A-dA and dA-dA-A molecules for (Ala)3 crystals and dA-A-dA-dA, dA-dA-A-dA and dA-dA-dA-A molecules for (Ala)4 crystals. Thus, three kinds of 1H signals for the specific residue can be resolved in the combination of PMLG pulse and selectively deuterated (Ala)3 and (Ala)4 samples.

11



Figure 2.

1

H PMLG NMR spectra of AP β-sheet (Ala)3 crystals of A-A-A, dA-dA-A,

dA-A-dA and A-dA-dA. Window functions were not applied before Fourie transformation. 1H NMR signals of HNH (P1), HCα (P2) and HCβ (P3) are separately appeared.

1

H spin diffusion 2D correlation NMR spectra of AP β -sheet (Ala)3 crystals.

Figure 3 shows 1H spin diffusion 2D correlation NMR spectra of AP β-sheet (Ala)3 crystals of A-dA-dA. Cross peaks for HCβ-HCα (P3P2), HCβ-HNH (P3P1) and HCα-HNH (P2P1) were appeared at the mixing time of 1 ms. These cross peaks are caused by the spin diffusion between intra molecular and inter molecular protons of each group. Deuterated groups do not contribute to the cross peak intensity and thus it was possible 12


Page 12 of 32

Page 13 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


to determine the inter hydrogen nuclear distances for the specific hydrogen in the non-deuterated alanine residues. This provides us to observe many kinds of protons individually using PMLG pulse sequence and selectively deuterated PLA samples. It is, however, important to point out that effective inter-nuclear distances were obtained rather than the specific inter-nuclear distances. Intra molecular inter-nuclear distances contains many information of molecular structure, while inter molecular inter-nuclear distances contains information of molecular packing arrangements. In order to determine the inter-proton distances, time course of the intensity build-up for the inter proton should be determined accurately. As pointed out previously, the inter-proton distances are effective distances which include the inter proton distances of inter and intra molecular contributions. It was, however, not possible to separate the intra-molecular contribution from inter-molecular contribution.

13



Figure 3. 1H spin diffusion 2D correlation NMR spectra of AP β-sheet (Ala)3 crystals taken at τm = 0 ms and τm = 1 ms. Cross peaks of P1P2, P2P3 and P1P3 appeared at τm = 1 ms

14


Page 14 of 32

Page 15 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Analysis of the build-up curve of AP β-sheet (Ala)3 and AP β -sheet (Ala)4 crystals Figure 4 shows the build-up curve of AP β-sheet (Ala)4 crystals of dA-A-dA-dA. It is noted that intensities of the cross peaks increased because of the spin diffusion between two kinds of protons. It is also noted that build up rates are different between each hydrogen nucleus. In contrast, diagonal peaks decrease against the mixing time τm. Particularly, HCα signal intensity decays faster than those of the other protons. In this experiment, only one alanine residue is fully protonated and other alanine residues are deuterated. Therefore, three sets of build-up curves for dA-A-dA-dA, dA-dA-A-dA and dA-dA-dA-A of (Ala)4 crystals were obtained as shown in Figure 4 and Figures S1 and S2. Build up curves for A-dA-dA, dA-A-dA and dA-dA-A of (Ala)3 crystals were also obtained using 1H spin diffusion 2D correlation NMR experiments shown in Figures S3, S4 and S5. Using these build-up curves, inter 1H distances of HNH-HCα (P1P2), HNH-HCβ (P1P3) and HCα-HCβ (P2P3) were evaluated from the initial slopes using Eqs. 1 and 2. Indeed, the effective inter 1H distances reffj,k (obs) were obtained and evaluated values for (Ala)4 are summarized in Table 1 and those for (Ala)3 in Table 2.

15



Figure 4.

Build-up curves for the cross peaks and diagonal peaks of dA-A-dA-dA

molecules in AP β-sheet (Ala)4 crystals.

Correlation of internuclear distances between reffj,k (obs) and reffj,k (calc) Table 1 summarizes inter-proton distances of AP and P β-(Ala)4 crystals. The reff,j,k (obs) values were actually obtained from initial slope curves in this experiment using AP β-sheet (Ala)4 crystals. The reffj,k (calc) values were obtained from the crystal data of AP β-sheet (Ala)4 crystals15 and refined atomic co-ordinates of P β-sheet (Ala)4 crystals that were obtained from solid-state NMR and MD simulation.17 It is noticed that reffj,k (calc) values using X-ray crystallographic data of AP β-sheet (Ala)4 crystal were in good agreement with the reffj,k (obs) values for AP β-sheet (Ala)4 crystals with the standard deviation of 0.128 Å (Figure 5(a)). On the other hand, the reffj,k (calc) values of P β-sheet (Ala)4 crystal were in poor agreement with the reffj,k (obs) values for P 16


Page 16 of 32

Page 17 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


β-sheet (Ala)4 crystal with the standard deviation of 0.270Å (Figure 5(b)). Disagreements appeared in the inter proton distances of inter molecules rather than those of intra molecules. In case of P2P3 (Hα-Hβ) of 4-Ala, rj,k (inter) of P β-sheet (Ala)4 crystal is 3.52 Å and is much longer than rj,k (inter) of 2.85 Å for AP β-sheet (Ala)4 crystals. The reffj,k (obs) values were also obtained from the build-up curves obtained from spin diffusion 2D correlation NMR data of AP β-sheet (Ala)3 crystals (Table 2). These values were compared with those of reffj,k (calc) for AP β-sheet (Ala)3

13

and showed in

good agreement with the standard deviation of 0.101 Å (Figure 6(a)). On the other hand, standard deviation between reffj,k (obs) and reffj,k (calc) for P β-sheet (Ala)3 crystals

14

is

0.171 Å (Figure 6(b)) which is much worse than that for AP β-sheet (Ala)3 crystals. In case of P2P3 (HCα-HCβ) of 3-Ala, rj,k (inter) of P β-sheet (Ala)3 crystals is 3.59 Å and is much longer than rj,k (inter) of 3.02 Å obtained from AP β-sheet (Ala)3 crystals. In our previous

13

C-DARR experiments, inter-carbon distances were evaluated

from the initial slope of the build-up curves for 32 cross peaks in [U-13C]-labeled AP β-sheet (Ala)4 crystals. The standard deviation between reffj,k (obs) and reffj,k (calc) values was a 0.244 Å. In this 1H spin diffusion 2D correlation NMR experiments, totally 9 cross peaks were analyzed using the initial slope curves and the obtained standard deviation was 0.128 Å. This result indicates that the accuracy is better than that in 13

C-DARR experiment, although observed range of distances were 1.81-2.55 Å, which

is much shorter than the cases of

13

C-DARR experiments in which observed ranges

were 1.64-5.66 Å. 35

17



Page 18 of 32

Table 1 Internuclear distances of AP and P β-sheet (Ala)4 crystals. P1, P2 and P3 protons indicate HNH, HCα and HCβ, respectively. rj,k (min) (intra) values indicate minimum internuclear distances of j and k in intra molecules. rj,k (min) (inter) values indicate minimum internuclear distances j and k in inter molecules.

(Ala)4

reffj,k

eff j,k

rj,kj(min)

rj,k(min)

reffi,j

reffi,j

rj,k(min)

rj,k(min)

(obs)

(calc)a)

(intra)

(inter)

(obs)

(calc)b)

(intra)

(inter)

dA-A-dA-dA P1P2 P1P3 P2P3

AP (Å) 2.27 ±0.05 2.16 ±0.06 1.82 ±0.03

2.19

2.32

3.67

2.08

2.77

3.07

2.02

2.45

2.96

dA-dA-A-dA P1P2 P1P3 P2P3

P1P3 P2P3

a) Ref. 15.

2.32 ±0.05 2.21 ±0.06 1.86 ±0.03

2.12

2.30

2.88

1.71

2.78

1.78

2.11

2.47

2.70

AP (Å) 2.27 ±0.05 2.10 ±0.05 1.81 ±0.04

P (Å)

2.10

2.20

3.83

2.08

2.78

2.93

2.04

2.49

2.92

dA-dA-dA-A P1P2

P (Å)

2.24 ±0.05 2.08 ±0.05 1.79 ±0.04

2.10

2.26

2.66

2.01

2.70

2.44

2.06

2.49

2.87

AP(Å) 2.55 ±0.15 2.24 ±0.07 1.91 ±0.04

P (Å)

2.54

2.95

3.29

2.16

2.92

3.17

2.01

2.52

2.85

2.35 ±0.15 2.06 ±0.07 1.76 ±0.04

2.15

2.98

2.51

1.94

2.76

2.50

2.15

2.48

3.52

b) Ref. 17

Table 2 Internuclear distances (Å) of AP and P β-sheet of (Ala)3 crystals. P1, P2 and P3 18


Page 19 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


protons indicate HNH, HCα and HCβ, respectively. rj,k (min) (intra) values indicate minimum internuclear distances of j and k in intra molecules. rj,k (min) (inter) values indicate minimum internuclear distances j and k in inter molecules.

(Ala)3

reffj,k

reffj,k

(obs)

(calc)

A-dA-dA P1P2 P1P3 P2P3

P1P3 P2P3

1.94 ±0.04 1.86 ±0.06 2.03 ±0.05

P1P3 P2P3

a) Ref. 13

(intra)

(inter)

reffj,k (obs)

reffj,k (calc)

2.31

1.81

2.33

2.61

2.07

2.48

3.00

1.97 ±0.04 1.89 ±0.06 2.06 ±0.05

rj,k(min)

(intra)

(inter)

2.33

2.90

1.84

2.50

3.06

2.13

2.45

2.42

P (Å)

2.13

2.23

3.66

2.09

2.78

2.98

1.98

2.45

2.63

2.20 ±0.14 2.10 ±0.11 1.74 ±0.06

2.08

2.27

2.61

2.04

2.87

3.00

1.97

2.44

2.56

AP (Å) 2.52 ±0.13 2.30 ±0.20 1.94 ±0.12

rj,k(min)

1.93

AP (Å) 2.24 ±0.14 2.14 ±0.11 1.77 ±0.06

b)

P (Å)

1.94

dA-dA-A P1P2

a)

Rj,k(min)

AP (Å)

dA-A-dA P1P2

Rj,k(min)

P (Å)

2.57

2.90

3.73

2.17

2.88

3.00

2.04

2.47

3.02

2.31 ±0.13 2.11 ±0.20 1.77 ±0.12

b) Ref. 14

19


2.23

2.92

2.81

1.89

3.06

2.74

2.14

2.49

3.59


Figure 5 Correlation between reffj,k (obs) and reffj,k (calc) in (a) AP β-sheet (Ala)4

Page 20 of 32

15

and

(b) P β-sheet (Ala)4.17 Standard deviation values were evaluated to be 0.128 Å for AP β-sheet (Ala)4 and 0.270 Å for P β-sheet (Ala)4.

20


Page 21 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6 Correlation between reffj,k (obs) and reffj,k (calc) in (a) AP β-sheet (Ala)3 13 and (b) P β-sheet (Ala)3.14 Standard deviation values were evaluated to be 0.101 Å for AP β-sheet (Ala)4 and 0.171 Å for P β-sheet (Ala)4.. 21



Packing arrangements disclosed by inter proton distances reffj,k (obs) Figure 7 shows the molecular packings of AP β-sheet (Ala)4 and P β-sheet (Ala)4 crystals. It is clearly noted that inter proton distances of intra molecules in AP β-sheet structure are not different from those in P β-sheet structure, while those are significantly different for inter molecules (Table 1). Namely these differences clearly reflect the molecular packing structures for the case of solid state samples such as silk fibers. In case of AP and P β-sheet (Ala)4, dA-dA-dA-A crystals, intra molecular distances, P2-P3 (HCβ-HCβ), were 2.52 and 2.48 Å, respectively. In contrast, inter molecular P2-P3 distances were 2.85 and 3.52 Å for AP β-sheet (Ala)4 and P β-sheet (Ala)4 crystals, respectively. In view of the three dimensional structure, the distance between the P2 (4-Ala) and the P3 (4-Ala in the upper β-sheet) of 2.85 Å in AP β-sheet (Ala)4 crystal is shorter than that of the P2 (4-Ala) and P3 (4-Ala in the next β-sheet strand) of 3.52 Å in P β-sheet (Ala)4 crystal (Figure 7). It is of important to point out that inter-proton distance between the P2 (4-Ala) and P3 (4-Ala in the next β-sheet strand) in the case of AP β-sheet (Ala)4 crystal should be much longer than that in the case of P β-sheet (Ala)4 crystal as long as the secondary structures are concern (Figure 7). Because of the inter molecular contribution, reffj,k (obs) of AP β-sheet (Ala)4 was shorter than that of P β-sheet (Ala)4. These results indicate that 1H-1H inter-nuclear distances obtained from 1

H spin diffusion 2D correlation NMR in the solid-state provides the information on

three dimensional molecular packing structure.

22


Page 22 of 32

Page 23 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7 (A) Crystal structures of (A) AP β-sheet (Ala)4 15 Internuclear distances of P2 (4-Ala)-P3(4-Ala in same molecule) is 2.52 Å and P2(4-Ala)-P3(4-Ala in upper β-sheet) is 2.85 Å.

(B) Crystal structure of P β-sheet (Ala)4.17 Internuclear distances of

P2(4-Ala)- P3(4-Ala in same molecule) is 2.48 Å and P2(4-Ala)-P3(4-Ala in next β-strand) is 3.52 Å.

Figure 8 shows the molecular packing of AP β-sheet and P β-sheet (Ala)3 crystals. It is also clearly shown that inter proton distances of inter molecules are significantly different although the inter proton distances of intra molecules are quite similar values as shown in Figures 8 (a) and (b). In case of AP and P β-sheet (Ala)3, dA-dA-A crystals, intra molecular P2-P3 distances were 2.47 and 2.49 Å, respectively. In contrast, inter 23



molecular P2-P3 distances were 3.02 and 3.59 Å for AP and P β-sheet (Ala)3 crystals, respectively. As is the similar as the case of (Ala)4 crystal, the distance P2 (3-Ala)-P3 (3-Ala in upper sheet) of 3.02 Å in AP β-sheet (Ala)3 crystal is shorter than P2 (3-Ala)-P3 (3-Ala in next β-sheet strand) of 3.52 Å in P β-sheet (Ala)3 crystal (Figure 8). Because of the inter molecular contribution, reffj,k (obs) value of AP β-sheet (Ala)3 was significantly shorter than that of P β-sheet (Ala)3, this inter-proton distance information reflects not only single molecular structure and secondary structure of polypeptide but also a three dimensional molecular packing structure.

Figure 8 (A) Crystal structures of (A) AP β-sheet (Ala)313Internuclear distances of P2 (4-Ala)-P3 (4-Ala in same molecule) is 2.52 Å and P2 (4-Ala)-P3 (4-Ala in upper 24


Page 24 of 32

Page 25 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


β-sheet) is 2.85 Å.

(B) Crystal structure of P β-sheet (Ala)3.14 Internuclear distances of

P2(4-Ala)-P3(4-Ala in same molecule) is 2.48 Å and P2(4-Ala)-P3(4-Ala in next β-strand) is 3.52 Å.

Conclusion It is demonstrated that accurate inter-proton distances in the range of 1.8-2.6 Å were evaluated for AP β-sheet (Ala)3 and AP β-sheet (Ala)4 crystals using 1H spin diffusion 2D correlation NMR spectroscopy. By aid of selectively deuterated (Ala)3 and (Ala)4 allowed us to observe the 9 cross peaks of HCα-HCβ (P2-P3), HCα-HNH (P2-P1), HCβ-HNH (P3-P1) for 1-Ala, 2-Ala, and 3-Ala for (Ala)3 and 2-Ala, 3-Ala and 4-Ala for (Ala)4 crystals. Experimentally obtained effective inter proton distances, reffj,k (obs) values were compared with the calculated internuclear distances, reffj,k (calc) values using the experimentally determined atomic co-ordinates and the results showed fairly well correlation. Correlation between reffj,k (obs) and reffj,k (calc) values for the cases of AP β-sheet (Ala)3 and AP β-sheet (Ala)4 showed fairly small standard deviation values of 0.128 and 0.101 Å, respectively. It was noted that inter proton distances obtained from inter molecular contribution provide the information of not only single molecular structure and peptide secondary structure but also three dimensional molecular packing structure in the solid state. It is also important to point out that inter-proton distances are not accurately obtained from X-ray diffraction analysis even in the crystalline state.

ASSOCIATED CONTENT SUPPORTING INFORMATION

25



Figure S1. Build-up curves for dA-dA-A-dA molecules in AP β-sheet (Ala)4 crystals. Figure S2. Build-up curves for dA-dA-dA-A molecules in AP β-sheet (Ala)4 crystals. Figure S3. Build-up curves for A-dA-dA molecules in AP β-sheet (Ala)3 crystals. Figure S4. Build-up curves for dA-A-dA molecules in AP β-sheet (Ala)3 crystals. Figure S5. Build-up curves for dA-dA-A molecules in AP β-sheet (Ala)3 crystals.

AUTHOR INFORMATION Corresponding Authors *(T.A.) Tel & Fax 81-42-383-7733; e-mail [email protected]. ORCID Tetsuo Asakura: 0000-0003-4472-6105 Akira Naito 0000-0003-2443-6135 Notes The authors declare no competing financial interest.

Acknowledgements T.A. acknowledges support by a JSPS KAKENHI, Grant-in-Aid for Scientific Research (A), Grant Number JP26248050 and Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT).

26


Page 26 of 32

Page 27 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


References (1)

Asakura, T.; Miller, T. Biotechnology of Silk; Asakura, T., Miller, T., Eds.; Springer Netherlands: Dordrecht, 2014; Vol. 5.

(2)

Tokareva, O.; Jacobsen, M.; Buehler, M.; Wong, J.; Kaplan, D. L. Structure– function–property–design Interplay in Biopolymers: Spider Silk. Acta Biomater.

(3)

2014, 10, 1612–1626. Sezutsu, H.; Yukuhiro, K. The Complete Nucleotide Sequence of the Eri-silkworm(Samia Cynthia Ricini) Fibroin Gene. J. Insect Biotechnol.

(4)

Sericology 2014, 83, 59–70. Numata, K.; Sato, R.; Yazawa, K.; Hikima, T.; Masunaga, H. Crystal Structure and Physical Properties of Antheraea Yamamai Silk Fibers: Long Poly(alanine) Sequences Are Partially in the Crystalline Region. Polymer (Guildf). 2015, 77, 87–94.

(5)

Asakura, T.; Nishimura, A.; Kametani, S.; Kawanishi, S.; Aoki, A.; Suzuki, F.; Kaji, H.; Naito, A. Refined Crystal Structure of Samia Cynthia Ricini Silk Fibroin Revealed by Solid-State NMR Investigations. Biomacromolecules 2017, 18, 1965–1974.

(6)

Bonev, B.; Grieve, S.; Herberstein, M. E.; Kishore, A. I.; Watts, A.; Separovic, F. Orientational Order of Australian Spider Silks as Determined by Solid-State

(7)

NMR. Biopolymers 2006, 82, 134–143. Cardoso, M. H.; Ribeiro, S. M.; Nolasco, D. O.; de la Fuente-Núñez, C.; Felício, M. R.; Gonçalves, S.; Matos, C. O.; Liao, L. M.; Santos, N. C.; Hancock, R. E. W.; Franco, O. L.; Migliolo, L. A Polyalanine Peptide Derived from Polar Fish

(8)

with Anti-Infectious Activities. Sci. Rep. 2016, 6, 21385. Migliolo, L.; Felício, M. R.; Cardoso, M. H.; Silva, O. N.; Xavier, M.-A. E.; Nolasco, D. O.; de Oliveira, A. S.; Roca-Subira, I.; Vila Estape, J.; Teixeira, L. D.; Freitas, S. M.; Otero-Gonzalez, A. J.; Gonçalves, S.; Santos, N. C.; Franco, O. L. Structural and Functional Evaluation of the Palindromic Alanine-Rich Antimicrobial Peptide Pa-MAP2. Biochim. Biophys. Acta - Biomembr. 2016, 1858, 1488–1498.

(9)

Colletier, J.-P.; Laganowsky, A.; Landau, M.; Zhao, M.; Soriaga, A. B.; Goldschmidt, L.; Flot, D.; Cascio, D.; Sawaya, M. R.; Eisenberg, D. Molecular Basis for Amyloid-β Polymorphism. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 16938–16943.

(10)

Nelson, R.; Sawaya, M. R.; Balbirnie, M.; Madsen, A. Ø.; Riekel, C.; Grothe, R.; 27



Eisenberg, D. Structure of the Cross-β Spine of Amyloid-like Fibrils. Nature (11)

2005, 435, 773–778. Greenwald, J.; Friedmann, M. P.; Riek, R. Amyloid Aggregates Arise from Amino Acid Condensations under Prebiotic Conditions. Angew. Chemie 2016, 128, 11781–11785.

(12)

Polling, S.; Ormsby, A. R.; Wood, R. J.; Lee, K.; Shoubridge, C.; Hughes, J. N.; Thomas, P. Q.; Griffin, M. D. W.; Hill, A. F.; Bowden, Q.; Böcking, T.; Hatters, D. M. Polyalanine Expansions Drive a Shift into α-Helical Clusters without Amyl Pleated Sheet Arrangement in Oid-Fibril Formation. Nat. Struct. Mol. Biol.

2015, 22, 1008–1015. (13) Fawcett, J. K.; Camerman, F. N.; Camerman, A. The Structure of the Tripeptide L-Alanyl-L-Analyl-L-Alanine. Acta Crystallogr., Sect. B: Struct. Cryst. Chem. (14)

1975, 31, 658-665. Hampel, A.; Camerman, N.; Camerman, A. L-Alanyl-L-Analyl-L-Alanine: Parallel Pleated Sheet Arrangement in Unhydratyed Crystal Structure, and Comparisones with the Antiparallel Sheet Structure. Biopolymers 1991, 31, 187-192.

(15)

Asakura, T.; Okonogi, M.; Horiguchi, K.; Aoki, A.; Saitô, H.; Knight, D. P.; Williamson, M. P. Two Different Packing Arrangements of Antiparallel

Polyalanine. Angew. Chem. Int. Ed. Engl. 2012, 51, 1212–1215. (16) Asakura, T.; Yazawa, K.; Horiguchi, K.; Suzuki, F.; Nishiyama, Y.; Nishimura, K.; Kaji, H. Difference in the Structures of Alanine Tri- and Tetra-Peptides with Antiparallel β-Sheet Assessed by X-Ray Diffraction, Solid-State NMR and Chemical Shift Calculations by GIPAW. Biopolymers 2014, 101, 13–20. (17)

Asakura, T.; Horiguchi, K.; Aoki, A.; Tasei, Y.; Naito, A. Parallel β-Sheet Structure of Alanine Tetrapeptide in the Solid State As Studied by Solid-State

(18)

NMR Spectroscopy. J. Phys. Chem. B 2016, 120, 8932–8941. Arnott, S.; Dover, S. D.; Elliot, A. Refined Atomic Co-ordinates for an

Antiparallel Beta-Pleated Sheet. J. Mol. Biol. 1967, 30, 201-208. (19) Lee, D. –K.; Ramamoorthy, A. Determination of the Solid-State Conformations of Polyalanine Using Magic-Angle Spinning NMR Spectroscopy. J. Phys. Chem. (20)

B. 1999, 103, 271-275. Henzer Wildman, K. A.; Lee, D. –K.; Ramamoorthy, A. Determination of

α-Helix and β-Sheet Stability in the Solid State: A Solid-State NMR Investigation ofg Poly(L-Alanine). Biopolymers, 2002, 64, 246-254. (21) Asakura, T.; Nishimura, A.; Kametani, S.; Kawanishi, S.; Aoki, A.; Suzuki F.; 28


Page 28 of 32

Page 29 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Kaji, H.; Naito, A. Refined Crystal Structure of Samia Cynthia Ricini Silk Fibroin Revealed by Solid-State NMR Investigations. Biomacromolecules, 2017, 18, 1965-1974. (22) Dumez, J.-N.; Halse, M. E.; Butler, M. C.; Emsley, L. A First-Principles Description of Proton-Driven Spin Diffusion. Phys. Chem. Chem. Phys. 2012, 14, 86–89. (23) Suter, D.; Ernst, R. R. Spin Diffusion in Resolved Solid-State NMR Spectra.

Phys. Rev. B Cover. ondensed matter Mater. Phys. 1985, 32, 5608–5627. (24) Henrichs, P. M.; Linder, M.; Hewitt, J. M. Dynamics of the 13C Spin‐exchange Process in Solids: A Theoretical and Experimental Study. J. Chem. Phys. 1986, 85, 7077–7086. (25) Kubo, A.; McDowell, C. A. Spectral Spin Diffusion in Polycrystalline Solids under Magic-Angle Spinning. J. Chem. Soc. Faraday Trans. 1 Phys. Chem.

Condens. Phases 1988, 84, 3713–3730. (26) Kubo, A.; McDowell, C. A. 31P Spectral Spin Diffusion in Crystalline Solids. J. Chem. Phys. 1988, 89, 63–70. (27) Grommek, A.; Meier, B. H.; Ernst, M. Distance Information from Proton-Driven Spin Diffusion under MAS. Chem. Phys. Lett. 2006, 427, 404–409. (28) Bayro, M. J.; Huber, M.; Ramachandran, R.; Davenport, T. C.; Meier, B. H.; Ernst, M.; Griffin, R. G. Dipolar Truncation in Magic-Angle Spinning NMR Recoupling Experiments. J. Chem. Phys. 2009, 130, 114506. (29) Egawa, A.; Fujiwara, T.; Mizoguchi, T.; Kakitani, Y.; Koyama, Y.; Akutsu, H. Structure of the Light-Harvesting Bacteriochlorophyll c Assembly in Chlorosomes from Chlorobium Limicola Determined by Solid-State NMR. Proc.

Natl. Acad. Sci. 2007, 104, 790–795. (30) Takegoshi, K.; Nakamura, S.; Terao, T. 13C–1H Dipolar-Assisted Rotational Resonance in Magic-Angle Spinning NMR. Chem. Phys. Lett. 2001, 344, 631– 637. (31) Takegoshi, K.; Nakamura, S.; Terao, T. 13C–1H Dipolar-Driven 13C–13C Recoupling without 13C Rf Irradiation in Nuclear Magnetic Resonance of Rotating Solids. J. Chem. Phys. 2003, 118, 2325–2341. (32) Oas, T. G.; Griffin, R. G.; Levitt, M. H. Rotary Resonance Recoupling of Dipolar Interactions in Solid‐state Nuclear Magnetic Resonance Spectroscopy. J. Chem. Phys. 1988, 89, 692–695. (33) Asakura, T.; Miyazawa, K.; Tasei, Y.; Kametani, S.; Nakazawa, Y.; Aoki, A.; Naito, A. Packing Arrangement of 13C Selectively Labeled Sequence Model 29



Peptides of Samia Cynthia Ricini Silk Fibroin Fibers Studied by Solid-State NMR. Phys. Chem. Chem. Phys. 2017, 19, 13379–13386. (34) Yazawa, K.; Suzuki, F.; Nishiyama, Y.; Ohata, T.; Aoki, A.; Nishimura, K.; Kaji, H.; Shimizu, T.; Asakura, T. Determination of Accurate 1H Positions of an Alanine Tripeptide with Anti-Parallel and Parallel β-Sheet Structures by High Resolution 1H Solid State NMR and GIPAW Chemical Shift Calculation. Chem.

Commun. 2012, 48, 11199–11201. (35) Naito, A.; Okushita, K.; Nishimura, K.; Boutis, G. S.; Aoki, A.; Asakura, T. Quantitative Analysis of Solid-State Homonuclear Correlation Spectra of Antiparallel β-Sheet Alanine Tetramers. J. Phys. Chem. B 2018, 122, 2715–2724. (36) Gerstein, B. C.; Pembleton, R. G.; Wilson, R. C.; Ryan, L. M. High Resolution NMR in Randomly Oriented Solids with Homonuclear Dipolar Broadening: Combined Multiple Pulse NMR and Magic Angle Spinning. J. Chem. Phys. 1977, 66, 361–362. (37)

Lee, M.; Goldburg, W. I. Nuclear-Magnetic-Resonance Line Narrowing by a

(38)

Rotating rf Field. Phys. Rev. A, 1965, 140, 1261-1271. Mehring, M.; Waugh, J. S. Magic Angle NMR Experiment in Solids. Phys. Rev. B,

1972, 5, 3459-3471. (39) Wu, C. H.; Ramamoorthy, A.; Opella, S. J. High-Resolution Heteronuclear Dipolar Solid-State NMR Spectroscopy. J. Magn. Reson. Series A, 1994, 109, 270-272. (40)

Madhu, P. K.; Vinogradov, E.; Vega, S. Multiple-Pulse and Magic-Angle Spinning Aided Double-Quantum Proton Solid-State NMR Spectroscopy. Chem.

Phys. Lett. 2004, 394, 423–428. (41) Lesage, A.; Sakellariou, D.; Hediger, S.; Eléna, B.; Charmont, P.; Steuernagel, S.; Emsley, L. Experimental Aspects of Proton NMR Spectroscopy in Solids Using Phase-Modulated Homonuclear Dipolar Decoupling. J. Magn. Reson. 2003, 163, 105–113. (42) Zhang, R.; Ramamoorthy, A. Dynamics-Based Selective 2D 1H/1H Chemical Shift Correlation Spectroscopy under Ultrafast MAS Conditions. J. Chem. Phys. 2015, 142, 204201. (43) Nishiyama, Y.; Zhang, R.; Ramamoorthy, A. Finite-Pulse Frequency Driven Recoupling with Phase Cycling for 2D 1H/1H Correlation at Ultrafast MAS Frequencies. J. Magn. Reson. 2014, 243, 25-32. (44) Mroue, K. H.; Nishiyama, Y.; Pandey, M. K.; Gong, B.; McNerny, E.; Devid, H. K.; Morris, M. D.; Ramamoorthy, A. Proton-Detected Solid-State NMR 30


Page 30 of 32

Page 31 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Spectroscopy of Bone with Ultrafast Magic Angle Spinning. Sci. Rep. 2015, 5, 11991. (45) Meier, B. H. Polarization Transfer and Spin Diffusion in Solid-State NMR. Adv.

Magn. Opt. Reson 1994, 18, 1–116. (46) Elena, B.; Emsley, L. Powder Crystallography by Proton Solid-State NMR Spectroscopy. J. Am. Chem. Soc. 2005, 127, 9140–9146. (47) Elena, B.; Pintacuda, G.; Mifsud, N.; Emsley, L. Molecular Structure Determination in Powders by NMR Crystallography from Proton Spin Diffusion.

J. Am. Chem. Soc. 2006, 128, 9555–9560. (48) Vinogradov, E.; Madhu, P. K.; Vega, S. High-Resolution Proton Solid-State NMR Spectroscopy by Phase-Modulated Lee–Goldburg Experiment. Chem. Phys.

Lett. 1999, 314, 443–450. (49) Vinogradov, E.; Madhu, P. K.; Vega, S. A Bimodal Floquet Analysis of Phase Modulated Lee–Goldburg High Resolution Proton Magic Angle Spinning NMR Experiments. Chem. Phys. Lett. 2000, 329, 207–214. (50) Vinogradov, E.; Madhu, P. .; Vega, S. Proton Spectroscopy in Solid State Nuclear Magnetic Resonance with Windowed Phase Modulated Lee–Goldburg Decoupling Sequences. Chem. Phys. Lett. 2002, 354, 193–202. (51) Okushita, K.; Asano, A.; Williamson, M. P.; Asakura, T. Local Structure and Dynamics of Serine in the Heterogeneous Structure of the Crystalline Domain of

Bombyx Mori Silk Fibroin in Silk II Form Studied by 2D 13C–13C Homonuclear Correlation NMR and Relaxation Time Observation. Macromolecules 2014, 47, 4308–4316.

31



Table of Contents Graphic

32


Page 32 of 32

Structure Analyses of Alanine Trimer and Tetramer Crystals with

Recommend Documents