The 13th Symposium on Accelerator Science and Technology, Suita, Osaka, Japan, October 2001

The life science which the 5MW spallation neutron source provides.
Nobuo Niimura
Advanced Science Research Center, Japan Atomic Energy Research Institute,Tokai-mura, Naka-gun, lbaraki-ken, 319-1192, Japan

Abstract
S1ructural biology is one of the most important fields in thelife sciences that will interest hmnan ~ in the 21st centmy.
Neutrons can provide not only the positions of hydrogen atoms
in biological macromolecules but also infunnation on thedynamic molecular motions of hydrogen atoms and watermolecules. The next generation neutron sources, theperfonnance of which is expected to be 100 times better 1hanthat of the existing reactor JRR-3M, will be expected to provide
a breakthrough in the life sciences of the 21st centmy.
1. INTRODUCfiON
It is said that the universe began with the so-called big bang
about 15 billion years ago and the first living cell was bornabout 3.5 billion years ago. As a consequence of evolutionary
processes, a 1remendous nwnber of diverse living cells :6:om
different organisms exist on the ear1h. Human beings, forexample, are constructed from many living cells with a t.o1almunber of proteins of about 100 thousand In the latter half of
20th centmy, X-my single crystal structure analysis has beenused to detennine the three dimensional structures ofbiologicalmacromolecules such as proteins and DNA, to bring many ofthe mysteries of life sciences to light This structural
infunnation conln"butes to the understanding of thephysiological fimction of proteins and DNA This field iscalled structural biology.
S1ructural biology is one of the most important fields in thelife sciences that will interest hmnan ~ in the 21st centmy.
There is already clear evidence that hydrogen atoms and watermolecules arotmd proteins and DNA play a vecy important rolein many physiological fimctions. However, since it is very
hard for X-mys to determine the positions ofhydrogen atoms inprotein molecules, detailed discussion of protonation andhydmtion can be only speculative in many cases.
On the other hand, neutrons can provide not only thepositions of hydrogen atoms in biological macromolecules but
also infonnation on the dynamic molecular motions ofhydrogen atoms and water molecules. Since physiologicalfimctions accompany microscopic motion of atoms at active
sites, neutron inelastic scattering is expected to provide
experimental clues to clarify physiological functions on a

microscopic scale. In principle, these contributions have been
well identified, but there are only a few examples
experimentally detennined at present. Nevertheless, theresults obtained have contributed to the field of the structural
biology.
In the last section we show how the next generation neutron
sources, the performance of which is expected to be 100 times
better 1han that of the existing reactor JRR-3M (20 MW) inJapan Atomic Enetgy Research Institute (JAERI), will be
expected to provide a brealdhrougb in the life sciences of the21st centmy.
2. NEUTRON SCATI'ERING FOR STRUCTURAL

BIOLOGY
A neutron has the following distinctive features of value toinvestigations in structural biology:
1) a neutron can identifY hydrogen in a protein,2) a neutron can distinguish deuterium :6:om hydrogen,3) a neutron can observe dynamics of atoms and molecules,
and4) a neutron does not cause radiation damage to a protein
In order to apply these features to structural biology, single
crystal neutron diffiactome1Iy, small angle neutron scatteringand inelastic neutron scattering experiments are carried out.
3. HYDROGEN AND HYDRATES OF BIOLOGICAL
MACROMOLECULESThe three dimensional structure detenninations of biologicalmacromolecules such as proteins and DNA by X-ray single
crystal structure analyses has helped shed some light on many ofthe mysteries involved in life processes. At the same time, theseresults have clearly suggested that hydrogen atoms and watermolecules around proteins and DNA play a very important rolein many physiological fimctions. However, since it is very hardto detennine the positions of hydrogen atoms in protein
molecules using X-rays alone, a detailed discussion ofprotonation and hydmtion sites can only be speculated upon sofar. In contrast, it is very well-known that neutron diffiaction
provides an experimental method of directly locating hydrogenatoms. In order to cany out this research in Japan Atomic
Enetgy Research Institute (JAERI), two kinds ofdiffiactometers- 10-The 13th Symposium on Accelerator Science and Technology, Suita, Osaka, Japan, October 2001

dedicated for protein ccystallography have been constructed
where technical innovations such as a neutron imaging plate [1]and a monochromator [2] had to be developed By using these
diffiactometer, neutron diffiaction experiments fiom rubredoxin,
myoglobin and hen egg-white lysozyme have been carried out
and the several results ofthese proteins are reported in the paper.
BIX-3

Laue diffiactometry [3] is useful for quick data collection, butsuffer from an upper limit of 2A in resolution because of
background problems. On the other hand, the fixed crystal
method (using a monochromatized neutron beam and an areadetector) provides the best SIN ratio, although this techniqueneeds a large amount of computer storage since the sample
crystal rotates at a small step angle and many frames of datacollected at various positions need to be stored in memory.
More importantly, this method is more time-consuming than theLaue diffiactometry. However, especially in neutron protein
ccystallography, the most important thing is to collect a data set
Wlder the best SIN conditions since the neutron diffraction
technique virtually guarantees the detection of all the hydrogen
atom positions in proteins once data have been collected. Thus,
the monochromatized beam utilization technique is expected tobe the method of choice for collecting high resolution data
( 1.5A or better) although it takes much machine time.
Monochromatized neutron beams represent the oldest andmost classical sources of neutrons, and most of the previous
neutron protein ccystallographic data sets have been collectedusing this type of radiation. Monochromatized neutron
utilization is still channing in its way as discussed incomparison with Laue methods. In this method, amonochromator is indispensable in order to obtain a neutron
beam of a specific wavelength from a 'white radiation' source.
At JAERI, a new type of elastically-bent perfect:Si crystal(EBP-Si) monochromator for a neutron diffiactometer,
dedicated to protein ccystallography, has been developed [2].
The EBP-Si monochromator has been successfully applied to adiffractometer named as BIX-3 [4] at JAERI.

definitively established, it is likely that they function as electrontransfer proteins. Despite the uncertainty of its function in mostspecies, rubredoxins fiom different organisms have been
extensively studied by structural and spectroscopic
methodologies since rubredoxin from the hyperthennophile
Pyrococcus finiosus is extraordinary stable in heat. In order tounderstand the stability, very high resolution X-myccystallography study has been carried out by comparing thestructure between wild and the mutant which is not so stable inheat, but the special differences of backbone structure between
them could not been found. [6] We assume that there might bedifferences in hydrogen bonding and/or hydration structures andas a first step a neutron diffiaction study of the wild type of
rubredoxin with BIX-3. Data were collected at room
temperature up to a resolution of 1.5 A (so far the highest
resolution neutron data set), using wavelength A. =2.35 A. A
single crystal of dimension 2 x 2 x 1 mm, grown via vapor
dilfusion from 3.6 M NaK phosphate, is being used in thisproject. Two sets of data fiom the same crystal, roughly
corresponding to the crystal being mounted along the a and caxes, are being collected and merged. Data collection is by thestep-scan method, with 0.3° intervals in cJ> and exposure
times mnging from 60 to 75 minutes per frame. The
completeness factor of the 1.5 A resolution data set is currently
at 782%. 301 hydrogen atoms and 49 deuteriUill atoms areincluded in the refinement of the structure of rubredoxin. 26
water molecules are also identified. In the present model R
factor and R-free are 0.254 and 0.279, respectively.
Fig.l shows the 12IF0 1-IFcll map ofTyr 10. Because of thehigh resolution, the clear hole is seen in the center of the 6members ring and hydrogen atoms and replaced deuteriUill
atom are clearly seen. The replacement ratio ofhydrogen atoms
by the deuteriUill atoms bound to nitrogen, oxygen and sulfur
atoms has been determined and it is strongly correlated on ASA
and the temperature factors. The results are shown in Fig.2.

T)ir 102fo-fcmap
cr=~2 +2

Rubredoxin

With this BIX-3 a single-crystal neutron diffiaction analysisof the structure of small protein rubredoxin from thehyperthennophile Pyrococcus finiosus is currently under way.
[5] Rubredoxins are small protein containing an iron atom
coordinated by sulfur atoms of four cysteine side chains.
Although the physiological role for rubredoxins have not beenFig.l-11-

The 2IFoi-IFdtnap of'!yr 10 in rubredoxin.

The 13th-Symposium on Accelerator Science and Technology, Suita, Osaka, Japan, October 2001

MYOGLOBIN
HID ExclJange ralml~'llimperature fuclor~

~

ASA~

_·_i[1~~~~~~:"
i,~_. !! ~~t~WL~::::L:{~~:l{~_'::fv''. t~g:~¥i¥I· +~.~~,~5/(,) 1o1p203o=·4o;

-$o·Z

O.S.

<•~> neutron atructure (C C N)
® D posftfoos with oocup. <50%

E~kA~~ ::: [· ~~\~~~~~_J"'~\"'-~1 \;,rj

Fig.2 The replacement ratio, ASA and 1he 1empe:mture fucta ofhydrogen aloo1s by 1he deuteri1Bl1 aloo1s bound 1o nitrogen,oxygen and sulfur a1oo1s in rubredoxin.

#amino acidFig3 The replacement ratio and mean displacement ofhydrogen a1oo1s b)
1he deuterium aloo1s bound 1o nitrogen in myoglobin.

Myoglobin
Myoglobin serves as a ,,model-protein" in biophysics. It is
investigated by many research groups with a wide range ofphysical and biochemical methods. The respiratory heme
protein myoglobin from sperm whale which we have used forthe neutron diffraction experiments consists of 153 amino acids
which form 8 a.-helices..We detennined the neutron structure of
met-myoglobin up to 1.5A resolution. The myoglobin crystal(2.5 x 2.5 x 1.0 mm3) used in this study was soaked indeuterated mother liquor for years. [7] The space group is P21

Bwith the lattice constants a= 64.8A, b = 31.1A, c = 35.0A and f3= 105.8°. Neutron diffraction data were collected at room
temperature at the neutron single crystal diffractometer BOO.
This high resolution data set provides the possibility to analyzehydrogen bridges as well as the extent of exchange of hydrogenatoms by deuteriwn within the protein.
The occupancy of deuteriwn atoms which conesponds toreplacement ratio of hydrogen atoms by the deuteriwn atoms
bound to amide nitrogen atoms has been detennined and it isstrongly correlated on the local position of amide atoms and thetemperature factors as shown in Fig.3 and 4. Fig. 3 shows theoccupancy and mean displacement of atoms ofdeuteriwn atomsof amide, and Fig.4 shows the location of deuteriwn atoms withthe occupancy less than 500/o, and it is clearly localized in thehydrophobic region.

150

10050

E-helix

Fig.4 The locatioo ofdeuterium atoms which has 1he replacement
ratio less than 50"/o in myoglobin.

Hydrogen bonds in a helix
The a -helix in proteins is one of the fimdamental secondary
structure. It is famous that the existence of the helix structure
model proposed by Pauling was firstly verified by the X-mycrystal structure analysis of myoglobin. The helix structure has

been stabilized by the formation of straight N-H·· 0 hydrogenbonds between N-H group in 1he amino acid residue (residue)n and C-O group in residue n+4 as originally proposed byPauling. However, the fonnation of hydrogen bonds has beencharacterized by the discussion of the distance between proton
donor and acceptor and precise structure of hydrogen bonds- 12-

The 13th Symposium on Accelerator Science and Technology, Suita, Osaka, Japan, October 2001

including hydrogen atoms bas remained in dispute, simply
because it is difficult for X-ray ccystallography to identify all
the hydrogen atoms in proteins.
On the other hand, neutron difJ:iaction provides anexperimental method of directly locating hydrogen atoms andthe nature of hydrogen bonds. Neutron diffi:action data fromtetragonal hen egg-white lysozyme (HEWL) [8] andmyoglobin [7] were analyzed and the position of all thehydrogen atoms in both of the protein were identified.
The identification of hydrogen atoms provides us theaccurate infonnation ofhydrogen bonds in proteins. There are
6 a- helices in a HEWL molecule and 8 a- helices in amyoglobin. The ''N-D-0" type inlramolecular hydrogenbonds in the a- helices, which play an important role in thestabili2ation ofthe a- helices ofproteins, were surveyed
4. DYNAMICS OF BIOLOGICAL
MACROMOLECULES
h is expected that most physiological fimctions cotrelateslrongly with the dynamics of biological macromolecules. The
temperature dependence of isotropically-averaged mean-squaredisp1acements of atoms of myoglobin from neutron
experiments was observed. For temperatures below 200 K. themean-square displacement increases linearly with temperalure,in accord with bannonic models for internal dynamics.
Above 200 K there is a 1nmsition above which the mean-squaredisplacement increases more rapidly with temperature. Thisnonlinear behaviour implies an anhatmonicity in thepotential-energy surface. h should be noted that mostphysiological fimctions (such w; the processes mentioned
earlier: hydrolysis, dehydrogenation, etc.) occur most efficiently
at temperatures above 200 K. and not many of them take pJace
below200K.
This predicts that protein dynamics are strongly cotrelatedwith physiological fimctions and INS experiments fromproteins can be expected to proch,Jce useful results.
Dynamics of biological macromolecules are theoreticallytreated by nonnal mode analysis or molecUJar dynamics thatcan provide the dynamic s1ructure :fuctor observed by INS
experiments. The vibratiooal frequency distribution from themolecuJar dynamics of BP11 (bovine pancreatic trypsininhibitor) in solution bas been calculated and ~with·the neutron-derived frequency distnbution (coming fromincoherent scattering) from the BPTI in solution experimentand the good ooincidence between them has been reported
How neutron scattering can provide unique infonnation on theconfigurations and dynamics ofnative and unfolded protein bas
been already reported. However, there is one mystety in theINS data. No big differences in titne-ot:tlight spectra fromBPTI and HEWL are evident except at a large energy transfer
region, which is not so relevant for protein dynamics,
although the molecuJar structures w; well w; physiologicalfimctions of the two proteins are different Why? Is INS
useless for studying protein dynamics? In the INS spectra of·proteins, the incoherent scattering coming from the nuclear
spins of hydrogen atoms in the sample is dominant. Generally
speaking, INS observes the quantity<Rj(O)Rj(t)>, when: Rj(O)and Rj(t) are the position vectors of the i-th atom at 1.9) andthe j-th atom at t=t, respectively. When the i-th and j-th atoms

are different from each other, it is coherent, and when they are
the same, it is incoherent. Coherent or incoherent scattering

provides the dynamical amplitude between different atoms orof an atom itself: respectively. Big differences among proteinsmight not appear in the dynamical amplitudes of the atoms
themselves. If so, coherent scattering must be separated fiumincoherent scattering by measuring neutron spin flip andnon-spin flip processes.
5. TilE NEXr GENERATION NEUTRON SOURCES

AND TilE FUTURE OF NEUTRON STRUCfURAL
BIOLOGY
At present, because ofthe low level ofcurrent neutron fluxes,
it 1akes a prolnbitively long amowlt of time to collect a full
difJ:iaction data set, and/or it is necessary to prepare a largespecimen. An improvement in neutron flux would be
beneficial not only to ew;e the above limitations but also toenable new experiments to be carried out.
The type of proteins that can be measured by neutron single
crys1al diffi'actometry in accordance with increments in incident
neutron flux will be discussed Note 1hat in a diffi:action
experiment the neutron difJ:iaction intensity is proportional tothe crystal volwne, and to the inverse square of the cell volwne.
If the next generation neutron sources project (for example, aseveral-megawatt spallation neutron soun:e) is realised, theneutron soun:e resulting fium the next generation neutron
sources project will cover most of the important proteins to beinvestigated.
To date, neutron structural biology was expected to be
entirely complementmy to X-ray structural biology, but it has
thus fur :fuiled to meet this expectation. The next generation
neutron sources project will surely initiate a new generation in- 13-

The 13th Symposium on Accelerator Science and Technology, Suita, Osaka, Japan, October 2001

neutron structural biology and consequently make a nugoroontn"bution to 1he life sciences.
Acknowledgement
The paper is 1he summary of the results established in ResearchGroup for Neutron in Biology, Advanced Science Research center,
Japan Atomic Energy Research Institute. I greatly appreciate 1hecooperation of all the members in 1he Group.
References[1] NNiimma,et al; NucL Instrum. & Melhods Phys. Res. A349
(1994) 521-525[2] NNiimura, et al; Physica B213&214 (1995) 929-931,I.Tanaka,et al.; J. Appl. Ctyst (1999) 32, 525-529[3] F.Cipriani, et al; J. Neutron Res. 4 (1996) 79-85[4] I.Tanakaet al., J. Appl. Ccyst (to be submitted)[5] I. Tanaka et al., in this volwne.
[6] K.Kurihara et al., in this volwne[7] RBau et al., JBIC 3(1998) 484-493[8] AOstennann et al., Biophys. Chern. (to be submitted)[9] NNiimuraetal., nature struct bio. 4(1997) 909-914- 14-