Neutron Scattering in Biology - Fitter Gutberlet and Katsaras

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The spectrometer FDS at Los Alamos works in this way, achieving an energy resolution ∆E/Eo of 3–5%.

Biomolecular spectroscopy in the energy transfer regions covered by crystal or filter analyzer instruments is largely limited to the low temperature and low hydration regime, unless the emphasis is on water or ice dynamics “perturbed” by a biomolecular additive. Basic reasons for this are: (i) the scattering kinematics, which necessitates working at relatively high Q (Fig. 19.6) and consequently small mean-square displacements <u2> to give tolerable DW factors, and (ii) the absence of symmetry selection rules. The latter is an asset in studies of small, nearly anhydrous molecules where neutron spectra can provide unique data on optically forbidden modes. In experiments on more complex systems, however, the number of variously broadened excitations together with their overtones and combination lines becomes large and they merge into bands carrying weak, ill-di erentiated features. It is possible, in principle, to introduce contrast selectively by covalent H/D substitution, but at present this is not a realistic option for the sample sizes required. H/D exchange of the water of hydration and of labile groups (mostly OH, some NH) remains as the only way to introduce contrast. This is illustrated in Fig. 19.7 by spectra in the amide A+B region of collagen.

Fig. 19.7. High-frequency TOSCA spectra from collagen fibers: Hydrogenous (open circles) and exchange-deuterated (full circles) samples of aligned fibers (rat tail collagen) at 25 K. Prominent peaks at 3000 (CH, NH) and 3400 cm−1 (OH) are due to stretch modes. Hatched are spectral features “inversely” a ected (i.e., where deuteration leads to enhanced rather than decreased intensities) or shifted due to di erences in e ective oscillator masses

Fig. 19.8. Top: TOSCA spectrum of ACN-h9 at 25 K (solid line), compared with CHARMM simulation (hatched ). Details of ≈35 mode assignments up to 1500 cm−1 and force field parameters are given in [49]. Bottom: TOSCA spectrum of anhydrous glutathione at 30 K (solid line, y-shifted for clarity) compared with a simulation (dotted ) using GAUSSIAN 98 [51]. Note that a full trajectory simulation package (such as CHARMM) is needed to simulate spectra in the acoustic phonon region150 cm−1

Two peptides of biomedical interest are currently being studied using TOSCA in parallel with backscattering spectrometers [51]. Carnosine (CAS, β-alanyl-l-histidine) is an antioxidative and bioregulatory dipeptide, glutathione (GSH, l-(-glutamyl-l-cysteinyl-glycine)) is a ubiquitous tripeptide serving essential cellular functions. Both the CAS and the GSH spectra show numerous well-resolved peaks up to the Amide I and II region. The 100–900 cm−1 region of a TOSCA spectrum from GSH is shown in Fig. 19.8 together with a result from simulations in progress. The spectra obtained provide a good basis for comparison with and refinement of CHARMM simulations of GSH in the context of drug-binding studies involving glutathione S-transferase [52].

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Fig. 19.9. Right: MARI spectra of D2O-hydrated d-phycocyanin binned in eight groups with <Q0> from 0.284 (lowest) to 2.87 ˚A−1 (highest traces) [54]. Distinct Q- dependent bands centered on 3–4 meV (24–32 cm−1) are observed at 100 K. Above the dynamic transition region they merge with rapidly increasing quasi-elastic broadening. Left: Spectral shape of “Boson peak” region with approximate band centroids (dotted lines) for lysozyme (3.80 meV), staphylococcal nuclease (4.03 meV), and bovine serum albumin (3.57 meV), measured using LAM-40 at KEK. Spectra averaged over 0.3 < Q < 2 ˚A−1. Adapted from [42]

in the 1970s [1, 21]. The bands shown in Fig. 19.9 and the way they change across the 200 K region relate to much-discussed questions about the significance of the ubiquitous “Boson peaks” observed in spectra from nearly all biopolymers. Three features of well-resolved data from MARI experiments are of particular interest: (i) substantially higher quasielastic wing intensities (0.5 ω 2 meV) at 240 K relative to 100 K, probably due to enhanced merging of low-frequency α-relaxation processes with a broad distribution of β-type relaxations at somewhat higher ω; (ii) disproportionate intensity increases at low-to-medium Q values relative to the high-Q spectra; (iii) redshift of the Boson peak centroids from 3.3 (100 K) to 2.8 meV (240 K). A more quantitative interpretation of these first results requires data at intermediate temperatures and full MD simulations.

In studies of natural biocomposites like bone or teeth, or of part-synthetic composites developed for medical applications, the dominance of modes due to protonated groups may be exploited to contribute decisive information to the interpretation of optical and NMR spectra. Bone is a dense composite of calcium phosphate crystallites and type I collagen fibers in roughly equal proportions, with about 15% of the volume occupied by living cells in channels and cavities. The structural and spectroscopic properties of variously substituted hydroxyapatite nanocrystals are of central interest here. In IR and Raman spectra, the vibrational modes of OH groups and hydrogen phospate are often masked in the presence of substituent anions, whereas in neutron spectra they show up prominently. Both the OH librational band at 660 cm−1 and

Fig. 19.10. Left: Neutron spectra of micrometer-sized and nanometer-sized hydroxyapatite (HAp) crystalline powders, compared with bovine and rat bone crystallites (y-shifted for clarity) (from [58]). Right: Decomposition of the OH librational band of HAp into components assigned to the monoclinic and the hexagonal form (from [57])

the high-energy bond-stretch region (3000–3600 cm−1), together with overtone and combination bands, provide valuable data relating in particular to questions about the degree of hydroxylation and the extent to which adsorbed water may have “contaminated” certain preparations. These problems are being addressed in current work at ISIS using TOSCA and HET [55], and at IPNS using the chopper spectrometer HRMECS [57] (Fig. 19.10). The bestresolved data come from TOSCA experiments [58]. Shown in Fig. 19.10 is an example of a decomposition of the OH libration band into four components is shown.

19.5.6 Biopolymers

Collagen [59], amylose [60], and DNA [61] are the only natural biopolymers for which wide-range vibrational neutron spectra have been studied. The lowfrequency region typically consists of two broad bands each carrying a number of distinct lines, the first due primarily to dispersive phonon modes and the second to weakly dispersive skeletal deformation modes. In the absence of MD simulations, the semiquantitative interpretation of collagen data from TOSCA and MARI requires comparison with spectra and normal-mode calculations for structurally related triple-helical polypeptides: polyglycine II (PGII), polyproline II (PPII), and (Pro-Pro-Gly)10 (PPG10). Theoretical results for PGII give a maximum due to longitudinal modes at 13 cm−1 in the isolated molecule, but this is shifted upward to 40 cm−1 when H-bonding to neighboring chains is taken into account. The intensity maximum appears to be due to an inflection in the longitudinal mode branch, induced by softening of modes with wavevectors matching the pitch of the crystallographic helix. The 45 cm−1 peak in collagen and PPG10 spectra can be identified with

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Fig. 19.11. Comparison of TOSCA spectra at intermediate and high frequencies (all at 30 K): PPG-h and PPG-d, hydrogenous and deuterated (Pro-Pro-Gly)10; CL-h, collagen fibers oriented at right angles to the scattering plane, “dry” (6% H2O). Spectra shifted by ∆S = 0.05 relative to CL-h (from [59])

this maximum for longitudinal acoustic modes in PGII-like chains that are H-bonded and supercoiled to form the collagen triple helix (Fig. 19.11). The longitudinal acoustic phonon frequency in PGII matches that of its associated interhelical ice shell, suggesting e cient coupling between modes in the latter and the polypeptide backbone.

At higher energy transfers, the modes observed involve predominantly stretching or bending of C–H, N–H and O–H bonds, together with torsional deformations. The Amide V band is one focus of attention here. Triple-helical supercoiling leads to a downward shift of the Amide V mode of the Gly-Pro linkage, together with skeletal deformation and C=O in-plane bending modes of the Pro-Pro linkages. Examples are the various amide modes between 700 and 1550 cm−1, the in-plane and out-of-plane bending modes of aromatic ring hydrogens between 600 and 1600 cm−1, and the methylene and methyl deformations between 950 and 1450 cm−1. These major features of collagen spectra, and the assignment of several other bands and peaks on the basis of optical data and normal-mode calculations for triple-helical polypeptides, have been discussed in considerable detail [59].

19.5.7 Nucleotides and Nucleosides

Low-temperature neutron spectra for dry polycrystalline powder samples of the purines (adenine, guanine), pyrimidines (cytosine, thymine, uracil), and the nucleoside uridine were measured at ISIS using TOSCA [62], whereas similar data for the nucleosides adenosine, guanosine, and cytidine were collected at ILL using the beryllium-filter spectrometer IN1-BeF [62, 63]. Following earlier, less refined simulations, most of the nucleotide and nucleoside spectra were reanalyzed during the past 4 years at the DFT level, by Gaigeot et al. [64] for isolated molecules and by Plazanet et al. [65] for hydrogenbonded unit cells with periodic boundary conditions. In 15 K spectra of the bases, for example, a large number of peaks in the interval 25–200 meV (200– 1600 cm−1) could be assigned to modes involving CH, NH, NH2, and ring deformations (Fig. 19.12). As in the peptide work discussed in Section 19.5.2, neutron spectra at T < 100 K reflect closely the normal modes, and state- of-the-art simulations can provide a wealth of detail on both frequencies and intensities when analyzed in conjunction with crystallographic data. It is an open question, however, to what extent the information gained from anhydrous systems at very low temperatures will be transferable to, and hopefully improve, the empirical potentials of the commonly used biodynamics simulation packages. TOSCA data for peptides [51] and DNA [61] show how features that are well-resolved below 100 K quickly coalesce into broad undi erentiated bands as the temperature is raised, due to the combined e ect of higher DW factors, enhanced overtone and combination lines, and the onset of anharmonic interactions.

Fig. 19.12. Neutron spectra for anhydrous uridine (left) and uracil (right) at 15 K compared with DFT simulations for isolated molecules. Adapted from [64]

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19.6 Neutron Compton Scattering (NCS)

For energy transfers beyond the highest excitations observable by the spectrometers discussed in Section 19.5.2, but below the region of ionizing interactions, neutron–nuclei collisions are dominated by a neutron analogue of the Compton e ect [66]. Neutrons with eV energies “see” target nuclei as almost free and interact with them over distances shorter than 0.1 ˚A. A key concept here is the impulse approximation (IA) according to which the dynamic structure factor S(Q, ω) (e ectively incoherent) consists of a superposition of Gaussians centered on the recoil energy Erec,i = ( Q)2/mi of each atom species i. The relevant partial structure factor Si(Q, ω) can be expressed as an integral over the product of ni(p) and δ(ω + p2/2mi − (p · Q)2/2mi), where p is the nuclear momentum and ni(p) the momentum distribution of nuclei with mass mi. The total intensity of each recoil contribution is proportional to the fraction of nuclei of mass mi in the sample weighted by the scattering cross-section, whilst its width and shape are related analytically to ni(p) along Q. The zero-order interpretation of NCS spectra, valid for Q −→ ∞, is based on the IA. In this limit, Si(Q, ω) is proportional to the neutron Compton profile, Ji(y), i.e., the projection of the ni(p) along the direction of Q. For large but finite Q, deviations from the IA are of great interest since they provide a means to measure two important quantities: ∆2V, the Laplacian of the interatomic potential V, and <F 2>, the mean-square force on the target atom.

At ISIS, the eV spectrometer VESUVIO [67] (formerly called eVS) has been used in several mostly exploratory experiments. Compton profiles have been measured for H and D in ACN crystals (Fig. 19.13) [68], in paracrystals

Fig. 19.13. Left: NCS spectrum from ACN at 2θ = 33◦, showing decomposition into near-elastic (C,O,N), deuterium (D), and proton (H) recoil lines (from [68]). Right: Proton Compton profile analyzed as superposition of pure Gaussian (impulse approximation, dotted line) plus asymmetric component representing the first term of Sears expansion (from [68])