journal of
`MOLECULAR
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`Page 1 0f 17
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`CSL EXHIBIT 1054
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`Journal of Molecular Biology
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`Editor-in-Chief
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`P. Wright
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`Department of Molecular Biology. Research Institute ol't-icripps Clinic
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`Assistant Editor
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`J. Karn
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`MR1] Laboratory of Molecular Biology
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`Hills Road. Cambridge CB2 ZQH. UK.
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`Founding Editor
`Sir John Kendrew
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`Consulting Editor
`Sydney Brenner
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`Editors
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`I'. ( 'linmhon. Laliorutoire ile ('iénétique Moléculuire ties Eucaryotes ilu (FNRS. Institut de Chimie Biologique.
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`Fuenlté de Médecine. ll Rue Human”. 67085 Strasbourg Cedex. France.
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`M. Golf-simian, Institute of Cam-er Research. College of Physicians lit Surgeons of (.‘olumbin University.
`701 W. lfiBth Street. New York. NY [0032. U.S.A.
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`P.
`i-‘on Hipppl, institute of Moleeuiar Biology. University of Oregon. Eugene. UH 974034229. UHA.
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`R. Huber. Max—Plunek—luotitut. l'ijr Biochemie. 8033 Martinsrietl hei Miinchen. Germany.
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`A. King. MRC Laboratory of Molecular Biology. Hills Road. Cambridge CB? 2QH. UK.
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`Associate Editors
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`f”. R. Con-for. Human Genome Center. Donner Laboratory. [Alwrenre Berkeley Laboratory. University of California.
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`Berkeley. CA 94720.
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`.-'\".—H. Chen. The Rockefeller University. I230 York Avenue. New York. NY H1121. [Le-LA.
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`F. E. ("o/ten. Department of l’ha-rmareutieal (ihenith-ry. School of Pharmacy. University of (.‘olil'ornia. San Francisco.
`CA 94143-0446. U.H.A.
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`h. J. Drffnéiin'r. Rosenstiel Basic Medical Sciences Research l."-enter, Brandeis University. Waltham. MA 02254. USA.
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`A. R. Fens-ht. University (.Themicul Laboratory. Cambridge University. Lenufield Road. Cambridge (‘32 ll‘IW. LLK.
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`ll".
`.4. Hendrirknon. Department. of Biochemistry (it Molecular Biophysics. College of Physicians (it Surgeons of
`Columbia University. 630 West. 168th Street. New York. NY 10032. U.S.A.
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`lnutitute de (Sienetique et Microbiologie. Bdtiment 4119. Université de Paris XI. 9l405 Uri-say Cedex ()5.
`1.”. Holland.
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`France.
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`H. Honig. Department of Biochemistry uh Molecular Biophysics. College of Physicians at Surgeons of Columbia
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`University. 630 l’i'est 168th Street. New York. NY 10032. U.b‘.A.
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`l'. Luzsoli. Centre llt‘ Genet-lone Moléeuluire. (IT-entre National de. la Recherche Scientifioue. 91 (iif~survactte. France.
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`J. L. Mandel, Luboratoire de Génetique Moleeuluire dos Eucaryoics du UNRS.
`lnstitut de ('fhiniie Biologique.
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`Faculté dc Médcrine. ll Rue Humann. 67085 Strasbourg Cede-x. France.
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`H. Matthew». institute of Molecular Biology. University of Oregon. Eugene. OR 97403~ I229. UJ‘SA.
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`J. H. Miller. Depart-ment- of Microbiology. University of California. 405 Hilgurd Avenue. Los Angeles. CA 90024. USA.
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`M. F. Moody. School of Pharmacy. University of' London. 29.39 Brunswick Square. London WClN lAX. UK.
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`1". Richmond. Institut fiir Molekulurbiologie und Biophysik. Eidgeniissiurhe Teelmisohe Hoehschulr. Hiinggerherg.
`(.‘H 8093 Zurich. Switzerland.
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`H. Schleif. Biology Department. Johns Hopkins University. Charles 5: 34th Streets. Baltimore. MD 2I2l8. USA.
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`.V. L. h'lrmbcrg. (.‘rntral Research 5; Development Department. E. I. do Pont Nemours 3;. Company. Wilmington.
`DE 19898. USA.
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`K. R. Yanmmoto. Department of Biochemistry and Biophysics. School of Medicine. University of California.
`San Francisco. CA 94143—0448. USA.
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`l'nnngido. I'lepurtment ol' Binpl‘lyaieu. Faculty ol’ Science. Kyoto University. Saliyo-Ku. Kyoto 606. Japan.
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`JOURNAL OF MOLECULAR. BIOLOGY: ISSN 0022—2336. Volumes 211‘216.
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`Page 2 of 17
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`Page 2 of 17
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`‘I'l
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`Journal of Molecular Biology
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`Volume 214, Number 3
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`Contents
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`Communications
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`Strategy for Analysing the (.‘o—operat-ivit..\,-' of
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`lntramoleculnr lnteratctiims in Peptides and
`Proteins
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`A. Horovitz and A. R. Fersht
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`til 3—617
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`Novel (JAB-AA R-ecept:.u' o: Subunit. is Expressed
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`only in ('erel'aellar Granule (‘ells
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`K. Kato
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`(Z'rystals ol'o—Momorcharin. A New
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`liillostnne-inatel-iruting Protein
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`Calcium Binding Sites in Tomato Bushy Stunt
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`Virus Visualized 1]}? Lane (‘rystallography
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`The Longest. Regular Polypeptide 3,0 Helix in.
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`A tom it- Resolution
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`('Trysiallizution and Prelimiiuu‘y X-ray Study of
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`AaH l'l‘z. an insect—specific Toxin from the
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`Scorpion Andmrtonus oustmlis Hector
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`New ('I'ystalline Forms of Neuroaminitlase of
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`Type H Human Influenza Virus
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`(_'r_vsta|li2alion and Preliminary X-ray Studies of
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`11-Serine Dehydrutast- from Escherichia ml!
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`Z. Feng, W. W. Li, H. W. Yeung,
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`S.-z. Chen, Y.-p. Wang, X.-y. Lin,
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`Y.-c. Dong and J.-h. Wang
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`J. W. Campbell, I. J. Clifton,
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`T. J. Greenhough, J. Hajdu,
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`S. C. Harrison, R. C. Liddington and
`A. K. Shrive
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`V. Pavone, E. Di Blasio, A. Santini,
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`E. Benedetti, C. Pedone, C. Toniolo
`and M. Crisma
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`C. Abergel, E. Loret, H. Rochat and
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`J. C. Fontecilla-Camps
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`Y.
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`in, M. Luo, W. G. Laver,
`G.
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`M. Air, C. D. Smith and
`R.
`G. Webster
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`0bmolova, A. Tepliakov,
`G.
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`E.
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`Harutyunyan, G. Wahler and
`K.
`D. Schnackerz
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`Articles
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`Expression and Function of the.
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`Bacteriophage T-l
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`il'f-‘u‘il'r (hole of
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`L. K. Derr and K. N. Kreuzer
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`ljl 9—bit
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`625—626
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`633—fi35
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`637—638
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`639—640
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`64 l —-li42
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`Levanuse (lperon of Barium sum-tilts [neludes a
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`Fructose—specific Pliospholra-nsi'ernse System
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`Regulating the Expression of the. (lperon
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`Establishment ofdr Now DNA MeLhyIat-ion
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`Patterns. Transcription Factor Binding and
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`Deoxycylidine Mcthyltttion at (7110: and Non-(‘pG
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`Segue-macs in an Integrated Adenovirus Promoter
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`Deletion ol‘ Sites for Initiation of DNA Synthesis
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`in the. Origin of Broad Host-range Plasmid R1162
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`Myosin Step Size. Estimatirm From Slow Sliding
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`Movement. of Act-in Over Low Densities ol‘ Heavy
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`Meromyosin
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`Determining Local Conformational Variations in
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`DNA. Nuclear Magnetic Resonance Structures of
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`the DNA Duplexes tl((TG('-l_"TAAT(‘G} and
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`(“CCTV-Al‘GCGLH Generated Using
`Hack—calculation of the Nuclear (lverhauser Eli'eet
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`Spectra, a Distance Geometry Algorithm and
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`Constrained Molecular Dyna-mics
`Three-dimensional Structure of the Mammalian
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`Cytoplasmic Ribosome
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`Page 3 0f 17
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`I. Martin-Verstraete, M. Débarbouille,
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`A. Klier and G. Rapoport
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`M. Toth, U. Muller and W. Doerfler
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`673—683
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`{543—656
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`657-6“
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`H. Zhou and R. J. Meyer
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`T. Q. P. Uyeda, S. J. Kron and
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`J. A. Spudich
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`W. J. Metzler, C. Wang,
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`D. B. Kitchen, R. M. Levy and
`A. Pardi
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`685 - 697
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`699-110
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`Til—T36
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`A. Verschoor and J. Frank
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`731' 749
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`Page 3 of 17
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`Two-domain Strut-tum of the Native and Reactive
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`(fentrr {'leavvd Farms of (‘T lnhihimr of Human
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`Complement by Neutron Scattering
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`Three-dimensional Structure of Human
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`|l”Cd,IMeLnIlothiunein-E in Saint-ion Determined
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`by Nuclear Magnetic Resonant? Spectroscopy
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`Amide Proton hm. hangs in Human
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`Motallnthionein-fi Measured by Nutlear Magnetic
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`Resonance Spec trouc.npy
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`I’hyuuhilipmtein Mot-hyhtinn. Effect of the y-N-
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`Methylaspamginu Residue on Energy Transfer in
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`Phyunuyaniu and the Phycuhilisome
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`Author Index
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`S. J Perkins, K. F. Smith
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`S. Amatayakul, D. Ashford,
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`T. W Rademacher. R. A. Dwek,
`P. J. Lachmann and R A. Harrison
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`B. A. Messerle. A. Schéifl‘er, M. Vafiék
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`J. H. R. Ka'gi and K. Wtithrich
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`B. A. Messerle, M. Boa, A. Schifi'cr,
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`M. Vafik. J. H. R. Kfigi and
`K. Wiithrich
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`R. V. Swanson and A. N. Glazer
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`751—763
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`Tfi5—T?9
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`781—?86
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`797-798
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`(fowr HZuNfrrdimL' [‘nnfnrmatirm of an RNA Pseutlnknnt. I‘ugliai at ”-1.. J. MOI. Biol. 214. 437—453.
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`(Jr-igimtitm by BPU _-' Whitefriflw Md. Tmtbridgw Wells,
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`and printed and bound in Great Britain. by HIV '( ' When-tom: Ltd. £de?“
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`This journal is printed (m. art-Mfrs? pap”
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`Page 4 of 17
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`Page 4 of 17
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`J. Mal. Biol. “9910214. 751—763
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`Two-domain Structure of the Native and
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`Reactive Centre Cleaved Forms of CT Inhibitor of
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`Human Complement by Neutron Scattering
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`Stephen J. PerkinsT, Kathryn F. Smith
`
`Departments of Biochemistry and Chemistry
`and Protein. and Molecular Biology
`Royal Free Hospital School of Medicine
`Rowland Hill Street. London N W3 2PF, U .K .
`
`Supavadee Amatayakul, David Ashford, Thomas W. Rademacher
`Raymond A. Dwek
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`(llgcobiology Unit. Department of Biochemistry
`University of Oxford. South Parks Road , Oxford 0X1 SQI'. I'.K.
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`Peter J. Lachmann and Richard A. Harrison
`
`M RC Molecular Immunopathotogy U nit. M RC Centre.
`Hills Road. Cambridge (9'82 ZQH. U .K .
`
`( Receired 14 November 1989: accepted 20 April 1990)
`
`The CT inhibitor component of human complement is a member of the serpin superfamily.
`and controls (‘1 activation. Carbohydrate analyses showed that there are seven O—linked
`oligosaccharides in Cl inhibitor. Together with six N-linked complex-type. oligosaccharides.
`the carbohydrate content is therefore 26% by weight and the molecular weight (M,) is
`calculated as 71.100. Neutron scattering gives an M, of 76.000 (:4000) and a matchpoint. of
`418 to 423% zHZO.
`in agreement with this carbohydrate and amino acid composition.
`Guinier plots to determine the radius of gyration R6 were biphasic. Neutron contrast
`variation of CT inhibitor in HZO—ZH ZO mixtures gave an overall radius of gyration R5 at
`infinite contrast of 4-85 nm. from analyses at low Q. and a cross-sectional R6 of I-43 nm. The
`reactive centre cleaved form of CT inhibitor has the. same M, and structure as the native
`molecule. The. length of Cl inhibitor. 16 to 19 nm. is far greater than that of the putative
`serpin domain. This is attributed to an elongated structure for the carbohydrate-rich
`ll3-residue N-terminal domain. The radial inhomogeneity of scattering density. at. is large
`at 59 x l0” from the RC, data and 28 x10" from the cross-sectional analysis. and this is
`accounted for by the high oligosaccharide content of CT inhibitor. The scattering data were
`modelled using small spheres. A two-domain structure of length [8 nm based on two distinct
`scattering densities accounted for all the contrast variation data. One domain is based on
`the crystal structure of at, antitrypsin (7 nm x 3 nm x 3 nm). The other corresponds to an
`extended heavily glycosylatcd N-tcrminal domain of length l5 nm. whose long axis is close
`to the longest axis of the serpin domain. Calculation of the sedimentation coefficient 320. w
`for Cl inhibitor using the hydrodynamic sphere approach showed that a two-domain head-
`and-tail structure with an M of 71.000 and longest axis of 16 to 19 nm successfullv
`reproduced the 82., w of 37 S. Possible roles of the N-terminal domainIn the function of (‘1
`inhibitor are discussed.
`
`TAuthor to whom correspondence should be. addressed. in the Department of Biochemistry and Chemistry. Royal
`Free Hospital School of Medicine. Rowland Hill Street. London NW3 2P1“. UK
`75]
`
`0022— ’ ‘ti/90/l' 75l—oflilitlti..00
`age
`
`(C I990 Arudrmic Press Limited
`
`

`

`-..I ('5!to
`
`
`
`S. J. Perkins et al.
`
`
`
`
`
`
`1. Introduction
`
`
`
`
`
`
`
`
`
`
`
`The complement system comprises a family of
`
`
`
`
`
`
`plasma and cell—surface glyeoproteins that operate
`
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`
`
`
`
`
`
`
`as a cascade and play a. major
`role in immune
`
`
`
`
`
`
`defence mechanisms. The classical pathway of
`
`
`
`
`
`
`
`complement activation is initiated by the first: com—
`
`
`
`
`
`
`
`
`(11. which reacts largely with immune
`ponent.
`
`
`
`
`
`
`
`
`complexes of lg.“ or 1:50 inlInnuoglobnlins bound to
`
`
`
`
`
`
`foreign material
`((‘ooper.
`I985; Reid.
`I986:
`Hchumakcr cl at" IQST). The unrestrained activation
`
`
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`of (.‘I would eel-use excessive consumption of (7-2 and
`
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`
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`(‘4. thus (‘1 is regulated by (.‘T inhibitor (Harpel.
`
`
`
`
`
`
`
`
`lQ'ifi: Him .5: Reboot.
`liltil: (Iooper
`I985: Davis.
`
`
`
`
`
`
`
`liltiti). This protease inhibitor. present at about-
`
`
`
`
`
`
`
`
`I'I’Etltngtml
`in plasma. forms stable l2! complexes
`
`
`
`
`
`
`
`
`
`with the (‘ll' and Ii'is subcompmuénts of CT to
`
`
`
`
`
`prevent
`further complement activation.
`(‘omplex
`
`
`
`
`
`
`formation is
`characterized by
`the irreversibic
`
`
`
`
`
`
`
`cleavage of the peptide bond at Arg444—Tbr445 in
`
`
`
`
`
`
`
`
`
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`the reactive centre of (‘l inhibitor (Halvcsen of rd.
`
`
`
`
`
`
`liltifi). Conformational changes after cleavage lead
`
`
`
`
`
`
`
`
`
`to a much more stable protein structure (Bruch c!
`
`
`
`
`
`
`
`
`mt. ltlHH: l’emberton Pt rd. 1939}. While f‘l inhibitor
`
`
`
`
`
`
`
`
`
`
`
`is the only known inhibitor of ('lr and {.‘Ts.
`it. is also
`
`
`
`
`
`
`a critical regulatory component of the coagulatitm.
`
`
`
`
`
`
`librinolytic and kinin—releasing systems. and its
`
`
`
`
`
`or
`inactivation
`"non—productive"
`by
`absence
`
`
`
`
`
`
`
`cleavage of the Arg-tct-‘t-Thr-‘t-l-S peptide bond is
`
`
`
`
`
`
`
`important in certain disease states (Davis. 1988].
`
`
`
`
`
`
`Compositional stut'lies of ("l inhibitor are required
`to understand the mechanism of control of ('l
`
`
`
`
`
`
`
`
`
`activation in serum. (.‘l inhibitor is a I'uember of the
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`serpin supertamily (scrine protease hihibitor) of
`
`
`
`
`serum inhibitors (Davis et
`til" 1986: Buck et at.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Its H, has been est-inmt-ed as 93.00” to
`ltititi].
`
`
`
`
`
`”6.00“ by gel electrophoresis and ultracentri—
`
`
`
`
`
`
`
`
`
`
`fugation analyses (l’ensky p! at. itlfil: Hu-upt at at.
`ltl'r'tl: R-elnml H at. lili'i: Nilsson &. Wiman. 1982:
`
`
`
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`
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`Harrison. till-$3}. However. the sequence shows that
`
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`
`
`
`
`the mature protein M, is only 52.300 and it has been
`
`
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`
`
`suggested that this implies a carbohydrate content
`
`
`
`
`
`
`
`
`
`
`
`as high as 49'?“ (Bock e! at" l98ti}. This is not.
`
`
`
`
`
`suppm'ted
`previous carbohydrate
`analyses.
`by
`
`
`
`
`
`
`
`
`
`which found a total of 33 ‘53 to 350i: by mass (Haupt
`
`
`
`
`
`
`
`
`
`
`ct rd” ltl'i'll; Harrison. 1933}. The stI'Lu-ture of the
`
`
`
`
`
`
`1N-linked and {ii-linked oligosaeeharides have been
`
`determined. but not their [tidal amounts (Ht-rocker P!
`
`
`
`
`
`
`
`
`
`
`
`
`at... I985}. Accordingly. further carbirihydrate analy-
`
`
`
`
`
`
`
`
`ses were performed in order to define the composi-
`tion of UT inhibitor.
`
`
`
`
`
`
`
`
`
`
`
`Structural data are required also to undemt-aud
`the function oft'l inhibit-or. Hedimei'itatimi data for
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`inhibitor give.
`s30... values mostly at. 3'75
`('l
`
`
`
`
`
`
`
`
`
`(Schultze rt (11.. 1962: Haupt. c! at... 19?“; Reboot M
`
`
`
`
`
`
`
`
`rd. 1977: (lhesne c! ”L. 198:!) from which highly
`
`
`
`
`
`
`
`t-‘It'll'lgtlatfll structtm-‘s have. been deduced ((ldermatt
`
`
`
`
`
`
`
`(11.. 1981: Perkins.
`[985]. Electron microscopy
`at
`
`
`
`
`
`
`
`suggested that. (‘l inhibitor has an elongated two-
`
`
`domain ‘"Iu-ad-amt-tail" structure (thiermatt e! at”
`
`
`
`
`
`
`
`
`
`lilBl }. and Inicrocalol'imetry experiment-s supported
`
`
`this lwo-donmiu structure (Lennick et at.
`I985).
`
`
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`
`
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`
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`
`
`
`
`
`The crystal
`st-ructln't-- of the homologous serpiu
`
`Page 6 0f 17
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`Oil-antitr_\'psin can he. used to model the L‘-t-erminai
`
`
`
`
`
`
`
`365 amino acid residues (Loberniaun rt rd. 1934-.
`
`
`
`
`
`
`
`
`Bock e! at” [986: Harrison. 1989}. which probably
`
`
`
`
`
`
`
`
`corresponds to the “twat!" of ('l
`inhibitor. The
`
`
`
`
`
`
`the
`therefore
`heavily
`glycos__vlatcd
`is
`"tail”
`N-tcrmiual
`Il3 amino acid
`residues. with
`a
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`presumed length of anywhere between '36 um and
`53nm. The structure of the. N-terminal domain is
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`poorly understood:
`indeed.
`the functional
`t‘Ul'lt-‘rlt-
`
`
`
`
`
`
`
`nuances of this domain are wholly unknown (Davis.
`H388).
`
`
`
`
`
`
`
`Neutron scattering is an efi'ectivc multiparameter
`
`
`
`
`
`
`
`tool for structural studies of glycoproteins (Perkins
`
`
`
`
`pt at" l985: Smith et at. 1990). Contrast variation in
`
`
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`
`
`
`
`
`
`
`
`Hzl l—EHZU mixtures will identity the overall struc—
`
`
`
`
`
`
`
`ture under conditions close to physiological.
`in
`
`
`
`
`
`
`
`which the internal arrangemii-nt of protein and
`
`
`
`
`
`
`
`
`carbohydrate can he allowed for (Perkins. IQSSH. b).
`The conformational transitirm between the native
`
`
`
`
`
`
`
`
`
`
`
`
`
`and reactive centre cleaved proteins can be moni—
`tored. The two—domain model for (.‘l inhibitor can
`
`
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`
`
`
`
`
`
`
`
`
`be compared quantitatively with the crystal struc—
`
`
`
`
`
`
`
`ture of Gil-antitrypsin (Lt'ibermann at at” IQRJ-t
`
`
`
`
`
`
`
`
`Smith P! m'..
`I990}. where molecular models for (‘l
`
`
`
`
`
`
`
`
`inhibitor can be developed. Possible roles for the
`
`
`
`
`
`
`heavily glycosylated N-ierminal domain for
`the
`function of Cl inhibitor are discussed.
`
`
`
`
`
`
`
`
`
`
`
`
`
`2. Materials and Methods
`
`
`
`
`
`
`
`(aj Preparations of the rudim- and reaction renlrr- cleared
`
`
`
`
`
`
`forms of t 'l'
`inhir‘rflor
`
`
`
`
`
`
`
`
`
`(‘l
`plasma
`from human
`inhibitor was prepared
`l..:1clnnann (Hit-iii]
`according to the method of Harrison 6’:
`
`
`
`
`
`
`
`
`
`and either used fresh or stored frozen at —Tt|"t" until
`
`
`
`
`
`
`
`
`
`
`required. if frozen. samples were prepared for |.-:ratt.ei'ill,t_lr
`
`
`
`
`
`
`
`
`studies by gel filtration on Hepbarnse tilt in 12 vasndium
`
`
`
`
`
`
`
`
`
`
`phosphate. 2th} rim-Nat”. I mM-El'lTA. pH Tell. The peak
`
`
`
`
`
`
`
`
`
`fractiolds) at about
`ltl mgfml were then dialysed agaii'lst-
`
`
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`
`
`
`
`
`
`the same buffer. filtered through a (N? am filter (Colman)
`
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`
`
`
`
`
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`
`
`into sterile glass or plastic vials. and held at 4"1.‘ until
`
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`
`
`
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`
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`
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`
`
`
`
`required for analysis. Two [towns of ("'T inhibitor were used
`in analysis. The native form showed a single band on
`
`
`
`
`
`
`
`
`
`
`tiI)Stpolyaciylumide gel electrophoresis (Laemrnli. I970].
`
`
`
`
`
`and was fully active against (‘Is and plasn‘lin. The split
`
`
`
`
`
`
`
`
`
`
`form oft 'T inhibitor was generated subsequent to isolation
`
`
`
`
`
`
`
`
`by the action of an unidentified prot-cztsets]. (tel electro—
`
`
`
`
`
`
`
`
`liquid
`197th]
`phoresis
`(Laeuuuli.
`and
`high-pressure
`
`
`
`
`
`
`r-.hromatography analyses of this slmwed it to have been
`
`
`
`
`
`
`
`
`
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`
`
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`
`
`
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`
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`
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`split at 2 sites. one at or close to the reactive centre
`
`
`
`
`
`
`
`
`
`
`
`exposed loop, and the other close to the amino terminus of
`the pron-in. generating fragments indistinguislmble from
`
`
`
`
`
`
`
`those generated by Pseudomrmm (imagines-n elastase
`
`
`
`
`
`
`
`
`
`
`
`
`
`(l’embertou N m'” 1939]. The sites of cleavage were
`
`confirmed by amino-terminal sequence analysis (per-
`
`
`
`
`
`formed by Ilr I.. Paektuan at
`the Protein Sequencing
`
`
`
`
`
`
`
`
`
`Facility. Department of Bim-hcmistry. University of
`
`
`
`
`
`
`
`Cambridge} ot' the cleaved material both before and after
`
`
`
`
`
`
`
`
`
`dialysis against scattering buffers. This indicated that
`
`
`
`
`
`
`
`cleavage had occurred bet ween tier-HI and Valli-12 in the
`
`
`
`
`
`
`
`
`
`reactive cent-re and, in appmximately equimolar amounts.
`
`
`
`
`
`
`
`either bet-warm Metal and Leu3‘2 or between la‘u32 and
`
`
`
`
`
`
`
`
`
`
`Phe33 in the :uninn—termiiml region. While the (‘-terminal
`
`
`
`
`
`
`
`peptide. containing the. reactive cent-re residues. was fully
`
`
`
`
`
`
`
`
`retained in the dialysed cleaved molecule. partial (up to
`
`
`
`
`
`
`
`
`
`30‘5“} loss of the amino—terminal 3|f32 residues may have
`
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`
`
`Page 6 of 17
`
`

`

`
`
`
`Solution. Structure of ("f Inhibitor
`
`
`
`
`753
`
`the
`occurred. although the nature of the residues at.
`
`
`
`
`
`
`
`
`
`amino terminus of the protein (N- PleC-Hnin-LTfi-fi-S-S}
`
`
`
`
`
`
`make this difficult.
`to quantify precisely. Samples were
`
`
`
`
`
`
`
`
`manalysed by gel electrophoresis suhsequent to solution
`
`
`
`
`
`
`
`scattering studies. and no dil’r‘erenccs lieforc or after the
`
`
`
`
`
`
`
`
`
`scattering experiments were detected. In addition. native
`
`
`
`
`
`
`
`
`(1T inhibit-or after the scattering experiments had an unal—
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`tered ka‘t'ifit' activity against active—site titrated plasmin.
`
`
`
`[ii]
`
`
`('Ffrhtli’lyfl’t’fll‘f" unofyxio of f '7 inhibitor
`
`
`
`oligov
`the
`release. of N—linked
`for
`The methods
`
`
`
`
`
`
`
`saccharidea by hydrazinol‘vsis and their subsequent analy~
`
`
`
`
`
`
`sis have been dcscrilu-c'! (Ashi'ord c! m"..
`I‘Inuymc
`I987).
`
`
`
`
`
`
`
`
`
`digestion
`of
`oligosaucharidcs with
`ncuraniinidasc
`
`
`
`
`
`
`
`(A rlfimlmricr urcafur'icnxi and fi-N-arctylhcmisaminidasc
`
`
`
`(Jack hean} have also been tlescl'ilici'l (I’arekh c! m'.. 1987}.
`
`
`
`
`
`
`
`
`
`{'l-linkcd oiigosaccharidcs were rclca-scd and reduced l.|_\'
`
`
`
`
`
`
`
`alkaiine sodium liorohydride treatment using a modifica-
`
`
`
`
`
`
`
`tion of the method described In: Mizuorhi ct m". [I980].
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Typically. 2'5: mg of (TI inhibitor was treated with 2'30 ,ul
`
`of' ”'6 .‘I-Hfldllllll
`lsHllJorohydride liftitlmt‘ifmmol. New
`
`
`
`
`
`
`
`England Nuclear} iI'I {HIS M-sodillm hydroxide For so h at.
`
`
`
`
`
`
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`
`
`
`
`
`:‘jl.l"'(_' with shaking. 'l‘lu- mixture was corded to 4'i't' and
`
`
`
`
`
`
`
`I‘lui of" acetic onhydride was added. After
`llimin. an
`
`
`
`
`
`
`
`
`
`
`
`additional
`lfliil of' acetic auhydridc was added and thc
`
`
`
`
`
`
`
`
`
`reaction allowed to [um-cod at room t-cinpcrat-uw For a
`
`
`
`
`
`
`
`
`
`lllI min. This addition and incubation was
`further
`
`
`
`
`
`
`
`
`repeated twice more. The reaction mixture was adjusted
`
`
`
`
`
`
`
`
`to approximately pH ti liy dropwise addition of I M-acetic
`
`
`
`
`
`
`
`
`
`acid. and then applicd to a
`lml column of DUWI‘X
`
`
`
`
`
`
`
`
`
`
`
`AHSHX l2 (H‘
`form. Bio-Rad}. The coiunm was cilitod
`
`
`
`
`
`
`
`
`
`with 5 column volumes ul'distillcd watt-r. Thc. cluatc was
`
`
`
`
`
`
`
`
`
`
`tiltcrcd through a 0-5 pm 'l‘cllou filter and evaporated to
`
`
`
`
`
`
`
`
`
`dryness. Borate was mmoved IJ'Y repeated evaporation
`
`
`
`
`
`
`
`then
`[fix] with incthanol
`(iiihlul). The sample was
`
`
`
`
`
`
`
`
`
`
`snhjeoled to descending paper chromatography for 4H h
`
`
`
`
`
`
`
`
`with butan-l-oliethanolll'water (4 :
`I
`: l. by vol}.
`
`
`
`
`
`
`For quantification of the nlllnher olil—linked chains on
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`("i
`inhibit-or.
`lactose was added prior to the. alkaline
`imrohydride.‘
`trcatmcnt. After isolation of the» reduccc'l.
`
`
`
`
`
`
`
`radio—labelled oligosaccharides
`from paper chromato-
`
`
`
`
`
`they were treated with neuraminidaac and
`graphy.
`
`
`
`
`
`
`
`suhjcctcd to high—voltage eioctmplmresis in
`[.iyridinc;
`
`
`
`
`
`
`acetic iuridi’watcr (3:1 :378. by vol.) hufier. pH 5-4 at
`
`
`
`
`
`
`
`
`
`
`
`Bi] V cm"I
`for 45 min. The. neutral oligosaccharidcs
`
`
`
`
`
`
`
`
`remaining at
`the origin were. eluted with water and
`
`
`
`
`
`
`
`
`
`applied to a Bio-Gel Put high-resolution gel
`fiItrat-ion
`
`
`
`
`
`
`
`
`system. Radioactivc fractions eluting from I to 5 glucose
`
`
`
`
`
`
`
`
`
`by
`comparison with
`internal
`isomalto-
`the
`units.
`
`
`
`
`
`
`oligosacrharide standards. were pooled Urge-their. Lactito]
`
`
`
`
`
`
`
`
`
`
`
`
`
`was separated from reduced ('l-glycuns by high—voltagc
`
`
`Plect-mphorcsis in horntc hufi'er {Ashitirrl e! n.3,. 1937]. The
`
`
`
`
`
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`radil'iactive areas were eluted from the elm‘trophm'etoA
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`gram and quantified by liquid scintillation counting.
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`A portion of the Bio-t.lel
`I’-~l pool. was also subjected to
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`the procedure
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`reducing—torn:inal moiiosaccimridc
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`determination (Aahford cl' oh. I987). The. radioactive areas
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`the resulting clectrophoretograln corresponding to
`of
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`glucit-ol and N-acct_\'lgaiartosaminit-oi \verc quantified hy
`integration of the raditmctivc [malts detected by the linear
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`analyser. f‘orrcctions in the quantification were made for
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`trace contaminants in the lart-itol and for background.
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`ll'l' Nt'm’mu duh: ciiflcr'iinn um} rmm'yscs
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`Neutron data acre rollcctml on Instrument. DI? at thc
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`(Iuinicr
`Institut-‘Laue-Idlngeviu.
`(lrvnohlc.
`radius of
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`duration Ru data wcrc hosed on a sample to doti-ctor
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`Page 7 0f 17
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`tlifit'ance ol‘ 346111 and neutron wavelengths of 1‘38?) to
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`I-395 nm or
`I'tiUil Inn. corresponding to a
`t.)
`range
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`((3:43: sin {if}. whore 20 is the scattering anglc} ol‘ III-(I33
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`to li-486om'l. Data at.
`larger Q were obtained with a
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`sample to detcctor distance of" 140:3). wavelengths of
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`H301 to 1-004 um. and main beam to detector angles ofU "
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`and Iii-89° to give. 0 ranges ofIi-l 3 to Hill nm " and trial to
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`3-6 run".
`Instrument 011 with a sample to detector
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`distance of “Hi to and u‘m‘elel'lgth of I'll“ not was used to
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`obtain a lower Q rangc of 0-0019 to (1-221) nm' 1. Samples
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`warc dialysed at 6°C with stirring int-0 ”man-sodium
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`phosphate. 20(lmM-Nat‘l.
`l mat-EDTA {pH 7-1}
`in 0'10.
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`80% or 100%, 2H 20 solutions with 4 changes over 36 to
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`48 h). All neutron data were. recorded at. 201'. (Hint-en—
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`trations were measured using an absorption coetficicnt.
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`. $3me of 3-6 (Harrison. 1983; Salvesen cl rd.
`i985).
`Data reduction was hased on standard Grenoble- software
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`((ihosh. 198]}. and the final analyses in London were
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`hased on SII'TPL {Perkins it. Sim. 198d}. Statistical anal .V‘
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`aei-‘i were l'mi'for'med using MINITAB (version til) on a
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`microcomputer {Ryan at at. I985].
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`the t'iuinier equation gives the radius of
`At small Q.
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`gyration It"; and the forward scattering at act-o scat-owing
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`angle [{0} {(iuinier & Fouruet. 1955}:
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`In HQ) = ln tffll—Réqlig
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`In a given solute-‘solrent contrast. HG measures the
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`degree ol’ ciongation of a glycoprotcin. If this struct-urc is
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`sufficiently elongated, the [fa of t-hc cross—sectional struc-
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`turc 33:5 and the cross—sectional
`intensity at zero angle
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`[HQ] x Q1040 are obtained from:
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`In mm x Q] = In [1in x Ola .u — Hisczo.
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`The matchpoini
`is ohtaincd l'rorn a graph of JIflHfl'TS!
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`against
`”=0 2H20 [7"!
`is sample transmission. i
`is sample
`thickncss, r is <.-.<.ui<.-cnt-ration}. and from t-hc (.‘I‘tiHfi-Ht-‘t'ljtlllill
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`analyses using JIHQ) ijq flui'c’l‘st. Experiment-a] match—
`points can be compared with the amino acid acqucncc on
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`the basis of. the unhydratiul shape of arr—VT.
`the use of
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`crystallographic volumes {unhydrutedL and thc lti‘i-D
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`non-exchange of the main-chain amide protons (Perkins.
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`1986). The contrast. variation analysis of the arrangement
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`of ¢.-.a.i-lioh_\;ti1-at.c and protein is based on t-hc Htohrmann
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`equation {Ibel & Stuhrimtnn. 1975):
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`3%; = Ré_c+atfi x Ap‘ I —,86 xAp'2
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`”is = His—Eta“ x AP- 1 *lgxs x AP. 2.
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`where Ref and Rxsc are the radii of gyration of the
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`macromolecules and its cross-section at
`infinite contrast.
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`inhomo-
`are and 1x5; Incasurc thc correspondin
`radial
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`geneity of scattering densities. and tip '
`is thc reciprocal
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`solvent—solute contra-st dilTert-noe. The terms in Be and
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`if“ measurc the. displacement of the centre of scattering
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`densityr as the contrast. is varied. From the term in )3. the
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`diatamre A hut-ween the centres ol‘ 2 components of
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`distim'i scattering densities ,01 and p2 and volumes 1'. and
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`I}. respectively. can he calculated; however. 3 parameter-
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`weightcd least—squares fitting of the data in Fig.3{hi
`showed that this was not measurablc.
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`The neutmn-scattering curve HQ} in reciprocal space
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`can be transformed into real space P(r) by use of the
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`indirect- t-ransi'ornmtion procedure (ITI’) incihod [Glat-tcr.
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`1982]. This requircd the full scattering curvc to 3-6 um ' ‘.
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`It. offcis an alternativc calculation of the R6 and the
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`length L for ('I inhibit-or. The 75 experimental HQ] data
`points [in 3 suhdomains to correspond to thc 3 HI?
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`configurations} were heat
`tittcd using It} to 15 cubic
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`splines, which were transliirmed into Itll points of tho
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`Page 7 of 17
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`

`

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`T54 H. J. Perkins et a].
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`Pirl
`function based on n maximum length of dilnm.
`llesnn-aring ol‘ the neutron curve was performed on the
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`hilt-tits of u [BIT wavelength nprend of I00” full~width to
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`hall—tmuimum. The slit width and length corrections were
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`to he t‘tllilll and toured on a beam divergence of
`set
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`Will l2 rm] nt Instrument DIT.
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`{d} Mule-Hing of Hu- ufrmi'nrr- of {'17 inhibitor
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`Modelling of the I'IelllrlIII-st‘atlei‘lttg eurves was based
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`on the dry volume of the gl‘t'eoprotein [(‘hothia.
`[975:
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`Perkins.
`lilt'llil. and used IJehye simulations based on
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