Ministerio da Ciencia e Tecnologia / MCT lK)iS fiUlConselho Nacional de Desenvolvimento Cienti'fico e Tecnologico / CNPq ' u ' " ° ^ ~ ~

Laboratorio Nacional de Luz Sincrotron / LNLS

BR9736533

Workshop: "Structural Molecular Biology UsingSynchrotron Radiation"

LNLS, Campinas-SP, July 10-12, 1995

Abstracts

Labcratbno Nacional de Luz SincrotronCaixa Postal 6192. 13081-970, Campinas. SP. Brasil

VOi

Index

LNLS: past, present, future 5C.E.T. Gongalves da Silva

Optical system of the LNLS beamline for protein crystallography 6A. Craievich

Conformational heterogeneity of tryptophan in peptides in solution 7Amando S. Ito

The role of NifA protein on nif transcription regulation 8L.M.P. Passaglia, J. Frazzon, C. Vedoy, L.F. Revers, R.D. Sperhacke,I.S. Schrank

Structural and functional studies on brain Myosin-V 9Roy E. Larson

Protein structure determination by 2D 1H NMR 10Carlos Bloch Jr.

Protein dynamics and folding investigated by frequency domainfluorescence spectroscopy 11Sergio T. Ferreira

Beta lactoglobulin: unknown well-known protein 12/. Polikarpov, S. Brownlow and I. Sawyer

Some like it hot: novel trends in X-ray structure determination 13/. Polikarpov

The molecular basic for Ca2x / Mg2+ discrimination in EF-hands 14Fernando C. Reinach

The crystal structure of a lectin from the seeds of Cratylia Mollis 15G.A. Tavares, I. Caracelli, R. Burger, L.C.B.B. Coelho, M.T.S. Correia andG. Oliva

Preliminary crystal data of an oc-amylase inhibitor from triticum aestivum(WHEAT) '. 16Jorge lelek, Elza louko Ida, Ignez Caracelli and Glaucius Oliva

Expression in E. coli, purification and crystallization of the Trypanosomacruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase 17D.H.F. Souza, R. Burger. V.M. Barbosa, W. Perussi de Jesus, R. Garrat,A.P.U. Araujo, V. Hannaert and G. Oliva

The structure of carbonic anhydrase from bovine ehythrocytes at 2.5 Aresolution 18Regiane Burger. Wanda Draghetta, Glaucius Oliva & Ignez Caracelli

Structural studies of Ô-endotoxin from BACILLUS THURINGIENSIS 19B.G. Guimarães, M.V.F. Lemos. O.M.N. Arantes, R.C. Garrat, J. lulek, G. Oliva

Spectropolarimetric analysis of secondary structure changes in humanserun albumin (HSA) 20N.B.S. Cretelli, W.D.P. Jesusand Y.P. Mascarenhas

X-ray diffraction data collection to 2.8 Á resolution and determination of non-crystallografic symmetry of crystals of a neutrophil migration-inducing lectin(KM+) of seeds from Artocarpus integrifolia 21P.S.L Oliveira, R.C. Garrat. Y.P. Mascarenhas, L.M. Beltramini, G. Oliva, M.C.Roque-Barreira and I. Harvey

Quantificação das estruturas secundárias de proteínas em amostrasamorfas por espectroscopia de infravermelho 22Lucimara A. Forato, Rubens B. Filho, Luiz A. Colnago

Crystal structure of recombinant triosephosphate isomerase from Bacillusstearothermophilus complexed with 2-phosphoglicolate at 2.8 Á resolution....23Luis F. Delboni, Shekhar C. Mande, Françoise Rentier-Deirue,Véronique Mainfroid, Joseph A. Matial and Win G.J. Hol

An integrated study of snake venom phospholipase A2 homologues fromBothrops species 24FU. Ward

Accessing the structural determinants for polipeptide bifunctionality: thelysine degrading act iv i t ies of the lys ine-ketoglutaratereductase/saccharopine dehydrogenase enzyme 25Germano Cord Neto and Paulo Arruda

Structural aspects of DNA binding activity of the BZIP transcriptionalactivator opaque 2 26Adilson Leite, José Andres Yunes, Michel Vincentz, André L. Vettore,Germano Cord Neto, Eugen Gander, Goran Neshich and Paulo Arruda

Protein structure analysis at atomic resolution 27Hans D. Bartunik and Alexandre N. Popov

Macromolecular crystallography using synchrotron radiation at DESY 28Hans. D. Bartunik

X-ray structure analysis of the catalytic and allosteric sites of glucosamine-6-phosphate deaminase from Escherichia coli K12 29Marcos R. M. Fontes, E. Horjales. R.C. Garrat, M.M. Altamirano.M.L. Calcagno and G. Oliva

The refined crystal structure of hold-D-glyceraldehyde-3-phosphatedehydrogenase (GAPHD) from duck muscle at 2.7 Â resolution 30Valma Martins Barbosa. Richard Garrat and Glaucius Oliva

Refinement and temperature factor analysis of the R-state M 5, C118, 239Sdouble-mutant of the enzyme glucosamine-6-phophate deaminase fromEscherichia co//K12 31Rodrigo, N.R. Pereira, R.C. Garrat, E. Horjales, M. Calcagno, M. Altamirano& G. Oliva

Stations for non-crystalline diffraction and protein crystallography at theSRS, CCL Daresbury Laboratory 32Peter F. Lindley

Applications of synchrotron radiation to structural biology 33Peter F. Lindley

Protein crystallography in Brazil 34Glaucius Oliva, Richard Garrat and Eduardo E. Castellano

LNLS: PAST, PRESENT, FUTURE

C.E.T. Goncalves da SilvaLaboratories Nacional de Luz Si'ncrotron /CNPq and Institute de Ffsica/UNICAMP

Caixa Postal 6192, 13081-970 Campinas, SP

In this talk we will give a brief review of LNLS, its history, scope

achievements, and plans for the future. LNLS is a National Laboratory, under

construction in Campinas. Sao Paulo, Brazil, since 1986, supervised by the

Conselho Nacional de Desenvolvimento Cientffico e Tecnologico (CNPq). It was

set up with three main objectives: to develop particle accelerator technologies, by

designing and building the Brazilian synchrotron light source, as much as possible

with local industry; to stimulate materials research using synchrotron light, creating

a community of external users and developing scientific instrumentation; and to

operate as the first large scale National Laboratory dedicated to materials research

in Brazil. LNLS is well on the way to achieve most of these goals - the storage ring

is expected to come into operation in 1996, marking the official debut of LNLS as a

research laboratory. LNLS wants to attract users from the life sciences, from ail

areas which could benefit from its light source, recognizing the tremendous

importance of biological research, both for increasing our knowledge about living

beings and for practical applications. Of particular interest to LNLS are programs

which can be of interest to public health issues, agriculture, and animal husbandry.

A protein crystallography beam line is already being funded by LNLS/CNPq and

FAPESP (the Sao Paulo state research funding agency). Beam lines being

installed at LNLS may also be used by the life sciences, such as small angle X-ray

scattering, X-ray fluorescence, and EXAFS. Other projects for biological research,

which require the construction of further beam lines, are in preliminary stages of

discussion.

IlllllllliilBR9736534

OPTICAL SYSTEM OF THE LNLS BEAML1NE FOR PROTEIN

CRYSTALLOGRAPHY

A. CraievichLaboratorio Nacional de Luz Sincrotron /CNPq and Instituto de Ffsica/USP

Caixa Postal 6192, 13081-970 Campinas, SP

The protein crystallography beamline of LNLS(1>2) will be composed of: i) afront-end, ii) a mirror for vertical focusing, iii) a bent Si (or Ge) single crystal formonochromatization and horizontal focusing, iv) two slit systems and v) an imagingplate detector. The front-end includes an X-ray shutter, a fast vacuum valve, a slowvacuum valve and a y-ray stopper. The mirror is composed of a float glasssubstrate in which gold is deposited; it will be elastically bent to produce a nearlycylindrical shape with horizontal axis. A 125 n.m Be window separates the ultrahigh vacuum volume (front-end and mirror) from the monochromator. Before (up-stream) the monochromator, a four slit system is placed. The monochromator is anasymmetrically cut triangular Si (or Ge) single-crystal which will be elastically bentby means of a wire driven by an external motor. The two relevant rotations of the

crystal (6 and q>) will also be driven externally. This will allow for high vacuuminside the monochromator (~10-8 Torr). A secondary X-ray shutter for themonochromatic beam and a second four slit system are located downstream justafter the monochromator. The final part of the workstation is an imaging platesystem which includes a collimator just before the sample, an ionization chamberfor monitoring the incident beam, a goniometer, an optical device for samplecontering, a beam-stopper and a two-dimensional detector (imaging plate) fordiffraction data recording. The basic idea for the design of this workstation was tomaximize the flux, within wavelengths ranging from 1.0 to 1.5 A. Themonochromator accepts horizontally up to 10 mrad. Using a (111) Si reflection, weexpect to have an X-ray beam, through the sample, with the following features:

E (GeV)

1.151.37

l(mA)

300100

1.4. 10 -4

1.4. 10'4

Number of photons(X=1.5A)

2 . 1010

1011

Number of photon(X=1.0A)

1091010

Size of the beam atthe sample

- 1 x 1 mm2

- 1 x 1 mm2

These values correspond to Si crystals; using Ge single crystals the number ofphotons is increased by a factor of about 2. The funds for the imaging plate systemand most of the components of the beamline were provided by FAPESP (SaoPaulo State funding agency), as a result of an application which includes a numberof research proposals from Brazilian biologists and crystallographers(2).

References

0) "LNLS beamlines. workstations and related instrumentation", LNLS Scientific Departament, LNLSNT 01/94.(2) "The construction of a protein crystallography experimental workstation at the Laboratorio Nacionalde Luz Sincrotron-LNLS, Projet FAPESP n9 94/0711-1. E. E. Castellano (Coordinator).

BR9736535

CONFORMATIONAL HETEROGENEITY OF TRYPTOPHAN IN

PEPTIDES IN SOLUTION

Amando S. ItoInstituto de Ffsica da Universidade de Sao PauloCaixa Postal 66318, 05389-970 Sao Paulo, SP

Time-resolved fluorescence spectroscopy has been claimed as a valuable

tool for the elucidation of structural features and segmental dynamics of peptides

and proteins, using the tryptophan residue as an intrinsic fluorescent probe. A

considerable amount of spectroscopic data reveals that many proteins and

peptides containing a single tryptophan residue show multiexponential decay of

fluorescence. One model for the interpretation of this observation is the so called

rotamer model, in which the Trp residue is supposed to adopt three distinct

rotameric conformations in the macromolecule. The interaction of the Trp side

chain with different structural elements (peptide backbone or other side chains)

would then lead to the different experimentally observed decay times. A system,

composed of melanotropin peptides (a-melanocyte stimulating hormone and

analogs) in aqueous solution and in the presence of lipid vesicles, was studied

under the above assumptions. We discuss in this presentation the relationship

between spectroscopic results obtained by time-resolved fluorescence

spectroscopy and conformational modeling performed for the peptides. Different

conformational families, dependent on the rotameric state of Trp, were obtained for

each peptide. These agree with the heterogeneous decay of fluorescence of Trp.

Furthermore, the conformations show different affinities to the lipid phase in the

interaction with lipid vesicles. The results are correlated with the different biological

activity observed for the native hormone and its analogs.

7

THE ROLE OF NifA PROTEIN ON nif TRANSCRIPTION REGULATION

L.M.P. Passaglia, J. Frazzon, C. Vedoy, L.F. ReversR.D. Sperhacke, I.S. Schrank

The positive control protein NifA from Azospirillum brasilense belongs to a

family of proteins that activate transcription by the o 54 containing holoenzyme form

of RNA polymerase. NifA binds to an upstream activation sequence (UAS) that is

typically located more than 100 bp upstream of the transcriptional start site and

contacts promoter-bound RNA polymerase by means of a DNA loop. The

Azospirillum brasilense n/7H promoter is positively controlled by the NifA protein

bound to the UAS. Two overlapping UAS located at -191 and -182 were identified

with the consensus TGT-N10-ACA motif. The role of the two UAS of A. brasilense

n/7H promoter was examined by introducing base substitutions in the NifA binding

sites. Both the promoter down phenotype of a mutation in UAS2 and increased

activation when UAS1 was mutated, reveal that the integrity of the UAS2 is

required for the efficient activation of nifH promoter. This typical NifA binding site

may represent a region interacting with two NifA dimers.

8

l \ •,

STRUCTURAL AND FUNCTIONAL STUDIES ON BRAIN

MYOSIN-V

Roy E. LarsonDepartamento de Bioqui'mica

FMRP/USPAv. Bandeirantes, 3900, 14049-900 Ribeirao Preto, SP

Novel myosin motors may play important roles in actin-based cell motility.

Myosin-V refers to a recently described class of unconventional myosins found in

neuronal and glial cells, melanocytes and other vertebrate cells, as well as in

primitive eukaryotic cells such as yeast. Our laboratory has been investigating the

domain structure of brain myosin-V (BM-V) which is a dimeric unconventional

myosin composed of three major structural and functional domains: 1) a motor-

head domain containing the ATP-hydrolysis and actin binding sites responsible for

the mechanochemical transduction, 2) a neck region containing multiple

calmodulin and light chain binding sites (6 IQ motifs) which acts as the regulatory

switch for the mechanochemical activity, 3) and a unique tail with coiled-coil and

globular regions having an. as yet, unknown function. We are using a number of

molecular approaches to investigate the structural-functional relationships between

these domains generated by limited proteolysis, b) expression in bacteria of

segments of BM-V corresponding to biologically important regions of the molecule,

c) analysis of a synthesized peptide corresponding to a calmodulin-binding IQ

motif, and d) immunolocalization in cells and subcellular fractions utilizing

monospecific, affinity-purified antibodies. In this presentation I will review recent

results using these approaches which are permitting us to refine our molecular

model of this exotic myosin. Financial support from FAPESP, CAPES and CNPq.

BR9736536

PROTEIN STRUCTURE DETERMINATION BY 2D 1H NMR

Carlos Bloch Jr.Centra Brasileiro de Sequenciamento de Protei'nas

Laborat6rio de Bioquimica, UnB70910-900 Brasilia, DF

Since the last decade, the solution structures of several small proteins (less

than 20 kDa) have been determined by high-resolution nuclear magnetic

resonance experiments, and independently of diffraction data. This advance has

been possible because of improvements in instrumentation and methodology,

including the use of very intense, homogeneous magnetic fields, new pulse

techniques and computer-based methods for interpreting the NMR data. Nuclear

magnetic resonance basics applied to proteins, as well as main features of the

methods available to determine tridimensional structures, are going to be

presented. An application example on the 3D structure elucidation of an antifungal

y-thionin from sorghum seeds will also be discussed.

10

BR9736537

PROTEIN DYNAMICS AND FOLDING INVESTIGATED BY FREQUENCY

DOMAIN FLUORESCENCE SPECTROSCOPY

Sergio T. FerreiraDepartamento de Bioquimica Medica

Instituto de Ciencias Biomedicas/UFRJ21944-590 Rio de Janeiro, RJ

Protein dynamics occurs over a broad time range extending from a few

picoseconds to seconds. In recent years a number of reports have appeared on the

use of time-resolved fluorescence spectroscopy to follow conformational motions of

the protein matrix and on the correlations between protein dynamics and biological

function. Advantages of fluorescence spectroscopy over other experimental

techniques include the relative simplicity and high sensibility, allowing experiments

to be carried out with sample concentrations approaching physiological values.

Synchrotron radiation or pulsed lasers have both been effectively used as light

sources for time-resolved fluorescence measurements. Frequency-domain protein

fluorescence techniques take advantage of the harmonic content of synchrotron

radiation or pulsed lasers in order to obtain information on fluorescence lifetimes,

rotational diffusion or excited-state reactions. In this communication, basic aspects

involved in the application of synchrotron or laser light sources to frequency-

domain fluorometry will be reviewed. Applications of these methodologies in the

investigation of nanosecond dynamics of the protein matrix and in the detection of

intermediate conformational states along the folding/unfolding pathway will be

discussed.

11

BR9736538

BETA LACTOGLOBULIN: UNKNOWN WELL-KNOWN PROTEIN

I. Polikarpov, S. Brownlow and I. SawyerBiochemistry Department

Edinburgh University, Edinburgh, Scotland

Bovine beta lactoglobulin (BLG) is a small globular protein of the lipocalinfamily. When exposed to pH's in the region 6 to 9, it undergoes a conformationalchange (termed the Tanford transition). At each side of the transition, it crystallizesin a different lattice form. Lattice Y and Z crystals are grown in the region of the pH7.8 and lattice X crystals grow at pH's below 6.9. The protein fold of these crystalforms has been solvedf1-3]. However, structure refinement has proved problematic.We used data from a crystal of spacegroup PI (lattice X) with the cell parametersa=37.8 A b=49.6 A c=56.6 A a=123.4° 3=97.3° 7=103,70° and dimer per unit cell.The structure has been resolved with molecular replacement and refined usingXPLOR to an R-factor of 27.7% (free R-factor 31.5%) against data between 8 to 1.8A resolution with a 2a cutoff level. The structure contained 65 water molecules.Several of the loop regions were not in density and further attempts to build theminto the density including ncs averaging with RAVE and/or to reduce R-f and R-freewere unsuccessful. At that stage, we removed all the water, which increased R-factor to 30.4% (R-free to 32.6%) and modified electron density using the programIPCOR. As the result, the electron density maps became clearer and the R-factordropped to 26.2% (R-free to 28.5%) against all the data between 15 to 1.8 A. Aftervisual inspection of the map, one loop (residues 108-115) was rebuilt, severalresidues at the C- terminus have been inserted and a number of sidechains havebeen placed into density. This model was then subjected to several cycles ofpositional and individual B-factor refinement. The R-factor of the model against allthe data between 15 to 1.8 A in an initial uncorrected dataset equals 20.0% (R-free=25.8%). Another crystal form of the BLG (lattice Y) was successfully refined atthe same time in an independent way. This belongs to a spacegroup C222, withthe cell parameters a=82.4 A b=55.4 A c=66.4 A and a monomer per asymmetricunit. The model was refined with PROLSQ. The water molecules have beeninserted with use of ARP. The current R-factor of the structure against all the databetween 14 to 2.03 A equals 18.0% (R-free = 28.3%). At present, the modelcontains all the 162 residues, except six residues at the C- terminus (residues 157-162). Comparison of these two structures sheds some light on the nature of Tanfordtransition. The non-crystallographic dimer of the low pll model (lattice X) can besuperimposed with the crystallographic dimer of the lattice Y by a rigid bodyrotation of one of the monomers of the dimer by 11°. This breakes a number ofhydrogen bonds on the monomer-monomer interface and forces a carboxyl groupto be exposed to the solvent.

References

l1l Papiz. M.Z.. Sawyer. L Eliopoulos. E.E. et al (1986) Nature 324, 383-385£] Yewdall, S.J. (1988) PhD Thesis. Astbury Dept. of Biophysics. University of Leeds[3] Monaki, H.L. et al (1987) J. Mol. Biol. 197, 695-706

12

BR9736539

SOME LIKE IT HOT: NOVEL TRENDS IN X-RAY

STRUCTURE DETERMINATION

I. PolikarpovBiochemistry Department

Edinburgh University, Edinburgh, Scotland

X-ray structure determination depends a lot on the quality of experimental

information available: structure amplitudes and experimentally derived MIR and

MAD phases. It also depends on the software used in data reduction, scaling,

model building, refinement and subsequent visualization of the model coordinates.

Protein crystallography utilizes the tremendous hardware advances and increased

CPU performance of modern computers and takes full advantage of sophisticated

algorithms relating to data manipulation. In this presentation, the author's

experience with novel developments in density modification, X-ray crystallographic

structure refinement and validation procedures including R-free, automatic

refinement, phase combination, ncs averaging, solvent flattening and flipping are

reviewed. Possible data collection strategies as applied to the LNLS protein

crystallography beamline are discussed.

13

THE MOLECULAR BASIC FOR Ca*+ / Mg2+ DISCRIMINATION

IN EF-HANDS

Fernando C. ReinachDepartamento de Bioqufmica, Instituto de Qufmica/USP

Caixa Postal 20780, 01498-970 Sao Paulo, SP

A large number of biochemical processes are regulated by calcium. Whileintracellular free calcium levels fluctuate between 1 and 105M, magnesiumconcentrations are kept constant at high levels (~2mM). Regulatory proteins mustbe capable of detecting these small changes in calcium concentrations in thepresence of a thousand-fold molar excess of magnesium. We investigated themolecular basis for this discrimination. The EF-hand Ca2+ binding motif isresponsible for this discrimination, in most regulatory. It consists of a twelve amino-acid binding loop flanked by two perpendicular a-helices. Four copies of this motifare found in calmodulin and troponin-C, two in the parvalbumins and one in themyosin light chains. EF-hands are either calcium specific (Ca2+ sites) or capable ofbinding calcium and magnesium (Ca2+/ Mg2+ sites). Due to the high concentrationof Mg2+ in the cytoplasm, Ca2+/Mg2+ sites are thought to be occupied by Mg2+ "invivo" and involved in stabilizing the structure of proteins while the Ca2+ specificsites perform the regulatory functions. We used a chicken smooth muscle myosinregulatory light chain (RLC) containing a single high-affinity Ca2+/Mg2+ site toinvestigate the determinants of ion specificity. Using site-directed mutagenesis weincreased the specificity of the site, converting this Ca2+/Mg2+ site into a Ca2+

specific site. The replacement of the aspartic acid present in the 12th position (-zcoordinating position) with a glutamic acid increases calcium affinity and abolishesMg2 binding. A possible explanation for this result is that restrictions on the ability ofthis side-chain (at the -z coordinating position) to change conformation,contributing one (for Mg2+ binding) or two coordinations (for Ca^+ binding) may bethe structural basis of metal specificity in EF-hands.

14

BR9736540

THE CRYSTAL STRUCTURE OF A LECTIN FROM THE SEEDSOF CRATYLIA MOLLIS

G.A. Tavares'12\ I. Caracelli'1'' R. BurgeK1', LC.B.B. Coelho'3)M.T.S. Correia<3> and G. Oliva*1'

(1"FSC/USP - Caixa Postal 369, 13560-970 Sao Carlos SP(2'IQSC/USP, Sao Carlos

<3>Depta de Bioquimica - CCB UFP 50730 Recife - PE

Cratyiia mollis lectin is a saccharide-binding protein similar from

concanavalin A. Although the biological role of plant lectins is still unknown, their

specific saccharide-binding properties make them an ideal object for the study of

protein-saccharide interactions. The lectin crystals were obtained by hanging drop

vapor diffusion method, using the following conditions: 12 mg/ml lectin, 0.15 M

methyl-a-D-mannopyranoside, 0.01 M sodium acetate pH 4.5, 0.15 M NaCI, 9%

polyethylene glycol 6000 and 0.01 M NaN3 at 20cC. The crystals were grown for

periods of a week to a month. The average crystal size was 0.2 x 0.2 x 0.15 mm. X-

ray diffraction data, collected at 4°C using a computer-linked image plate area

detector R-AXIS II from RIGAKU, show that the lectin crystallizes in the

orthorhombic crystal system, space group I222 with cell parameter a = 63.3 A, b =

77.4 A and c = 105.0 A, containing a monomer per asymmetric unit. A dataset was

collected up to 2.46 A resolution and the merging of all equivalent reflections

results in a data completeness of about 90% with Rmer = 10.8%. The structure was

solved by molecular replacement (MR) using the package AMoRe (Navaza, 1993)

with concanavalin A as model structure. The model currently being refined contains

237 amino acids, one Ca ion, one transitional-metal ion, probably Mn, and methyl-

a-D-mannopyranoside bound to the lectin. The single molecule in the asymmetric

unit is a part of a tetramer with 222 symmetry, with subunits generated by

crystallographic symmetry operations.

15

BR9736541

PRELIMINARY CRYSTAL DATA OF AN a-AMYLASE INHIBITOR FROM

Triticum aestivum (WHEAT)

Jorge lelek<1^3', Elza louko lda<4>, Iqnez Caracelli<1>and Glaucius OlivaC)

(•"Instituto de Física de São Carlos, USP, Depts de InformáticaCaixa Postal 369,13560-970 São Carlos, SP

(^Instituto de Química de São Carlos, USP, Dep8 de Físico-Química, São Carlos, SP(^Universidade Estadual de Ponta Grossa, Dept8 de Química, Ponta Grossa, PR

<4>Universidade Estadual de Londrina,Dept9 de Tecnologia em Alimentos e Medicamentos, Londrina, PR

Wide interest has been devoted to the study of a-amylase inhibitors, not onlyfor their obvious nutritional role, but also because of the many potentialapplications in medicine, e.g., the use in controlling disorders such as obesity anddiabetes. This application development can be strongly aided by theunderstanding of the structural mechanism of inhibition, which depends on theknowledge of the three dimensional structure of the inhibitor and its complex withthe target enzyme (Whitaker, J.R., 79th An. Meet. Am. Oil Chem. Soc). This workaims to crystallize the inhibitor and proceed with the three dimensional structuredetermination by X-ray crystallography. Trigonal prismatic crystals have beenobtained in a 100mM Tris/HCI buffer at pH 7.3, 100mM MgCI2 and 14 to 20% ofPEG 20000 (w/V) as the precipitant, with the hanging drop vapour diffusiontechnique. Crystallization is complete after 2 weeks with typical crystal size of 0.2 x0.2 x 0.4 mm. The crystals belong to the trigonal system, with unit cell dimensionsa=b=79.44Á, c=60.62Á. The Laue group and systematic absences are compatiblewith space groups P3i or P32. With conventional X-ray sources, the crystals diffractto 2.0Á resolution and are suitable for high-resolution structure determination. Anative diffraction dataset was collected on an RAXIS-IIC image plate area detector,with 90% completeness at 2.1Â resolution with Rmerge=8.9%. As no significanthomology was found to any other structure at the Brookhaven databank, anextensive search for suitable heavy atom derivatives is underway, aiming to solvethe structure by the Multiple Isomorphous Replacement technique. This work hasreceived financial support from CAPES, CNPq, FINEP and FAPESP.

16

BR9736542EXPRESSION IN E. coli, PURIFICATION AND CRYSTALLIZATION OF

THE Trypanosoma cruzi GLYCOSOMAL GLYCERALDEHYDE-3-

PHOSPHATE DEHYDROGENASE

D.H.F. Souza<1>, R. Burger<1>, V.M. Barbosa*1', W. Perussi de Jesus'1',R. Garrat<1>, A.P.U. Araujo<1>, V. Hannaert'21 and G. Oliva<1>

<1> IF/USP, Caixa Postal 369, 13560-970 Sao Carlos, SP<2> Unit for Tropical Diseases, International Institute of Cellular and Molecular Pathology (ICP),

Ave Hippocrate 74, B-1200 Brussels, Belgium

With the aim of obtaining new drugs against Chagas' disease by exploiting

peculiarities of the trypanosomal biochemical machinery, we started a project to

study the structure of enzymes involved in glycolysis, a key mechanism for the

generation of metabolic energy for the parasite. We describe here the expression

in E. coli, purification and crystallization of the T. cruzi glycosomal Glyceraldehyde-

3-Phosphate Dehydrogenase. The T. cruzi gene was cloned into the plasmid

pET3A as described by Hannaert et a/0) and significant expression levels of fully

active enzyme were obtained in E. coli. The enzyme was purified by ammonium

sulfate precipitation followed by affinity and ion-exchange columns. The purity of

the enzyme was analyzed by SDS-PAGE and the yield from 1 liter of culture is 14

mg of the enzyme. Crystals of diffraction power suitable for crystallographic studies

have been obtained by the vapour diffusion technique in hanging drops, at 4°C.

Protein concentration was 9 mg/ml in 25 mM Tris pH 7.8, 0.5 M Ammonium Sulfate.

2mM NAD, 1mM Azide, 1mM EDTA and 1 mM DTT, and reservoir solutions had

12-18% PEG 8000, 0.2 M Calcium Acetate and 0.1 M Cacodylate, at pH 6.0. 6.5

and 7.0. The crystals grow as clusters of plates which were very difficult to

separate into single crystals. Initial X-ray diffraction experiments show that the

crystals diffract to 2.5 A resolution. Experiments are being conducted to improve

crystal quality and morphology, that could lead to a complete structure

determination at high resolution. Acknowledgments: PADCT/SBIO, CNPq, FAPESP

and FINEP.

Reference

i 1 ) Hannaert. V. and Michels, P.A.M. "Glycosomal glyceraldehyde dehydrogenase of Trypanosomabrucei and Trypancsoma cruzi: expression in E. coli. purification and characterization of the enzymes"Protein and Purification, submitted 1994.

17

BR9736543

THE STRUCTURE OF CARBONIC ANHYDRASE FROM BOVINE

ERYTHROCYTES AT 2.5 A RESOLUTION

Regiane Burger, Wanda Draghetta, Glaucius Oliva & Ignez Caracelli

Laboratorio de CristalografiaInstitute* de Ffsica de Sao Carlos/USP

Caixa Postal 369 -13560-970 Sao Carlos, SP

Carbonic Anhydrase is a zinc-containing enzyme, that catalyses the

reversible hydration of carbon dioxide to bicarbonate. Seven different isoenzymes

have been characterized in mammals. It has a single polypeptide chain with one

zinc ion per molecule and a molecular weight of about 30 KDa. The protein was

provided under lyophilized form and stored at 4°C. The crystallization experiments

were conducted at 18°C, and the crystals were obtained by the hanging drop

technique, in which the reservoir solution had 53-56% ammonium sulfate and 50

mM TRIS/HC1 buffer, pH ranging from 7.75 to 8.00. The drop contained 2.5 \i\ of

protein solution (15 mg/ml) and 7.5 jil of the reservoir solution. The data collection

was obtained using a computer-linked image plate area detector from Rigaku. The

enzyme crystallizes in the monoclinic space group C2, with a=127.80A, b=48.74 A,

c=98.73A and p=122.5°. A complete diffraction dataset is currently being collected

to approximately 2.5 A resolution. The structure was solved by molecular

replacement (MR) using the package AMoRe (Navaza, 1994) with the human

erythrocytes enzyme as search model. The structure of the Bovine Carbonic

Anhydrase is being refined using the refinement programa XPLOR. The zinc ion is

tetracoordinated with a water molecule bound in addition to the three histidine

residues. Acknowledgments are due to: Prof. Dr. Arnaldo Simoes of Departamento

de Bioqui'mica of Faculdade de Medicina de Ribeirao Preto, USP, for providing the

purified protein & FAPESP, FINEP, CNPq & CAPES for financial support.

18

BR9736544

STRUCTURAL STUDIES OF Ô-ENDOTOXIN FROM BACILLUS

THURINGIENSIS

B.G. Guimarães(t», M.V.F. Lemos'2', O.M.N. Arantes'3',R.C. Garrat<1', J. lulekí1-4-5». G Oliva.'1»

(1) IFSC/USP, Departamento de Física e InformáticaCP. 369 -13560-970, São Carlos, SP

(2) Departamento de Biologia Aplicada AgropecuáriaAJNESP, Jaboticabal, SP(3) Departamento de Biologia Geral

Universidade Estadual de Londrina. Londrina. PR(4) Instituto de Química de São Carios/USP, Departamento de Físico-Química

São Carlos, SP(5) Departamento de Química, Universidade Estadual de Ponta Grossa

Ponta Grossa, PR

The 5-endotoxins are a family of crystal proteins produced by a soil

bacterium, Bacillus thuringiensis. The study of these proteins has been of great

interest due to their highly specific activity against insects of the orders

Lepidoptera, Diptera and Coleoptera. Thus, the 5-endotoxins have been used for

more than two decades as biological insecticides to control agricultural pests and,

more recently, insect vectors of some diseases. The knowledge of their

tridimensional structures is very important to understand their mechanism of action

and their high specificity. The structure of only one protein of the d-endotoxins

family, named CrylllA, toxic to insects in the order Coleoptera (beetle toxin)(1^ has

been reported. Our work aims at the determination of the crystallographic structure

by X-ray diffraction of 5-endotoxin CrylC, specific against insects in the order of

Lepidoptera (butterflies). A comparison between the two structures may lead to

important conclusions about the reasons of the specificity and would allow the

planning of mutants with more efficient activity. The CrylC gene was cloned into an

adequate vector and expressed in a B.t. strain depleted of endogenous 5-

endotoxins. After cell culture and sporulation, the crystals of CrylC are separated by

ultra-centrifugation in sacharose. We have determined the best conditions of

solubilization and activation of the crystal protein and started its purification by

Chromatographie methods. Financial Support: CNPq. FAPESP, FINEP.

Reference

<1^ Li. J.. Carroll. J.. Ellar. D.J. Nature 353. 815-821 (1991).

19

SPECTROPOLARIMETRIC ANALYSIS OF SECONDARY STRUCTURECHANGES IN HUMAN SERUN ALBUMIN (HSA)

N.B.S. Cretelli, W. DP. Jesus and Y.P. Mascarenhas

DFI/IFSC/USPCaixa Postal 369 -13560-970 Sao Carlos, SP

In a previous communication (Reuniao Anual da SBBq, 1994) we presented

some results of secondary structure changes in HSA as detected by Circular

Dicroism (CD) analyzed using the SSE program. Here we are presenting an

extended study in a wider pH range and analyzed using the more powerful

program CCA (Perczel, A. and Fasman, G.D. Convex Constraint Analysis.

Analytical Biochemistry 203, 1992). Changes in the secondary structure of HSA

were analyzed in relation to changes in the pH of the solution (from 2.5 to 9.0) and

also adding methanol in varying concentration (0, 5, 10, 20, 30%, v/v) as a co-

solvent. The observed structural changes with pH in the absence of methanol are

in agreement with the ones related in the literature (Foster, J.S. 1977, "Albumin

Structure Function and Uses". Pergamon, Oxford.) indicating a higher content in oc-

helix at pH's 6.0 and 6.5 (65%, 66%). The co-solvent action is in general more

pronounced at higher concentrations of methanol when the low helical content

(from about 40% to 50%) is increased to about 50 to 60%. Financial support:

FAPESP, CNPq, FINEP, CAPES.

20

BR9736545

X-RAY DIFFRACTION DATA COLLECTION TO 2.8 Á RESOLUTION AND

DETERMINATION OF NON-CRYSTALLOGRAFIC SYMMETRY OF

CRYSTALS OF A NEUTROPHIL MIGRATION-INDUCING LECTIN (KM+)

OF SEEDS FROM Artocarpus integrifolia.

P.S.L. Oliveira, R.C. Garrat, Y.P. Mascarenhas, LM. Beltramini, G. Oliva*1'M.C. Roque-Barreira*2' and I. Harvey'3'

<1> Instituto de Física de São Carlos/USPCaixa Postal 369 13560-970 São Carlos, SP

'2>Faculdade de Medicina de Ribeirão Preto/USP, Ribeirão Preto, SP(3)Daresbury Laboratory, Warrington, UK

In the course of the last years, the interest on lectin studies has greatly

increased with the perception that they have the capability to serve as mediators of

cell recognition in many biological systems. The understanding of how lectins bind

sugars at atomic level is essential to explain how these proteins act in the cellular

recognition processes. In this aspect, the determination of three-dimensional

structures by high resolution X-ray crystallography for many lectins has helped to

elucidate important features about the specificity of these proteins. In the present

work we report the preliminary crystallographic studies for the crystals of Neutrophil

Migration-Inducing Lectin (KM+ Lectin), an oc-D-mannose specific lectin, isolated by

Santos de Oliveira et ali. (1994). A complete native dataset up to 2.8 Á resolution

was achieved by merging the data collected at laboratory X-ray source *S. Carlos.

Brazil) and synchrotron radiation (Photon Factory, Japan). The analysis of the self-

rotation function shows the presence of non-crystallographic symmetry elements

(2-fold axes). Efforts have been made aiming at the growth of better quality crystals

that diffract to higher resolution and that are more resilient to experiments of heavy

atom soaking. These steps are essential for the determination of the three-

dimensional structure of KM+ by X-ray crystallography. Acknowledgments are due

to FAPESP, CAPES.CNPq and PADCT.

21

QUANTIFICAÇÃO DAS ESTRUTURAS SECUNDÁRIAS DE

PROTEÍNAS EM AMOSTRAS AMORFAS POR ESPECTROSCOPIA

DE INFRAVERMELHO

Lucimara A. Forato*1', Rubens B. Filho*12», Luiz A. CoInagoO-2)

<1>lnstituto de Química de São CarlosDepartamento de Físico-Química/USP

^'Centro Nacional de Pesquisa e Desenvolvimento de Instrumentação AgropecuáriaEMBRAPA - CNPDIA

As limitações práticas encontradas na determinação da estrutura

tridimensional de proteínas com alta resolução por Raios X e RMN, têm estimulado

o desenvolvimento e aperfeiçoamento de métodos de baixa resolução como a

espectroscopia de Infravermelho com Transformada de Fourier. Esta técnica

fornece informações sobre as proporções das estruturas secundárias nas

proteínas sem no entanto estabelecer a localização desses elementos estruturais,

como no dicroismo circular (DC). As principais vantagens do Infravermelho com

relação ao DC são que os equipamentos são mais comumente encontrados em

laboratórios, as amostras podem ser preparadas em solução aquosa, em

membranas e em fase sólida amorfa ou cristalina. As informações das estruturas

secundárias obtidas por FTIR são decorrentes de suas diferentes freqüências de

absorção, principalmente na banda de amida I (vibração de estiramento da

ligação C=O do grupo peptídico), na região de 1600 a 1700 cm"1. Neste trabalho

estamos desenvolvendo a técnica de FTIR aplicada a proteínas preparadas sob a

forma de pastilhas de KBr. Os espectros foram processados por um programa que

permitiu convoluir os mesmos com uma função filtro, proporcionando assim melhor

resolução da banda de amida I. Em seguida foi feita a deconvolução do sinal e as

áreas dos picos individuais foram determinadas por um programa de "fitting". Com

a análise dessas áreas é possível fazer uma estimativa semi-quantitativa das

estruturas secundárias. No momento estamos estudando alguns métodos de

quantificação: método dos mínimos quadrados, análise fatorial e regressão linear

múltipla e método de reconhecimento padrão. Apoio: EMBRAPA, CNPq.

22

BR9736546

CRYSTAL STRUCTURE OF RECOMBINANT TRIOSEPHOSPHATE

ISOMERASE FROM Bacillus stearothermophilus COMPLEXED WITH 2-

PHOSPHOGLICOLATE AT 2.8 A RESOLUTION

Luis F. DelboniC), ShekharC. Mande(^), Francoise Rentier-Delrue^), Veronique Mainfroid(3),

Joseph A. Matial(3) and Wim G.J.Ho|(2)

(1>lnstituto de Fi'sica de Sao Carlos/USPCaixa Postal 369, 13560-970 Sao Carlos, SP

i2)Department of Biological Structure, MS-20, School of MedicineUniversity of Washington, Seattle, WA, 98195, USA

l31Laboratorie de Biologie Moleculaire et de Genie GenetiqueUniversite de Liege, B6, Sart Tilman, 4000, Liege, Belgium

The crystal structure of the recombinant thermostable triosephosphate

isomerase (TIM) form Bacillus stearothermophilus in complex with the transition

state analogue 2PG was determined by X-ray crystallography to a resolution of 2.8

A. The structure was solved by Molecular Replacement with XPLOR using the

structure ofT. bruceiTM as search model, which shares 40.7% sequence identity

with Bacillus stearothemophilus TIM. The model was refined to a R-factor of 17.6%

for reflections from 8.0 to 2.8 A with good geometry. In general, increased

thermostability can be achieved in several ways by proteins. Global factors, such

as improved hydrophobic interactions, hydrogen bonding and electrostatic

interactions may stabilize not only the tertiary but also the quaternary structure.

Also some very localized mutations improve thermostability in some instances. In

this work we compare the thermostable TIM structure reported here with five other

TIM structures already described (human, chicken, yeast, T. brucei, e E. coli). It

appears that elucidating the cause of the greater thermostability of Bacillus

stearothermophilus TIM is not trivial but improved hydrophobic interactions in the

dimer interface, as well as the increased number of proline, may be significant

factors. Supported by CNPq, FAPESP, WHO/TDR, Howard Hughes Medical

Center.

23

BR9736547

AN INTEGRATED STUDY OF SNAKE VENOM PHOSPHOLIPASE A2

HOMOLOGUES FROM Bothrops SPECIES

R.J. WardDepartamento de Ffsica,

IBILCE/UNESP, Sao Jose do Rio PretoRua Cristovao Colombo, 2265, 15054-000 S.J. Rio Preto

Due to their availability and ease of purification, snake venom

phospholipases A2 (PLA2) have been extensively investigated. These 14 kD

proteins catalyse the hydrolysis of lipids in plasma membranes, leading to loss of

bilayer integrity and cell death. Snake venom PLA2s show high primary sequence

homology to mammalian small cytoplasmic PLA2s, whose activity regulates the

supply of lipid precursors for eicosanoid and leukotrine production. Therefore,

disorders in PLA2 activity can lead to disease such as arthritis. All catalyticaily

active PLA2s have a fully conserved Asp at position 49 (Asp49), which is essential

for the formation of a co-factor complex with Ca2+. The venom from a number of

viperid snakes, including those from Bothrops species, contain a PLA2 homologue

showing a Asp49=>Lys49 mutation, a substitution resulting in the loss of Ca2+

blinding, and the lack of catalytic activity. These PLA2 homologues, however, still

disrupt both synthetic and biological membranes by a poorly understood Ca2+

independent mechanism of action. We are combining X-ray crystallographic,

molecular biology and spectroscopic techniques to understand this novel

mechanism of action in more detail. Using bothropstoxin I from B. jararacussiu as a

model system, our initial results indicate that dimerization plays a crucial role in the

activity of the protein. Furthermore, our studies reveal detail of PLA2/lipid

interactions, information which is relevant in the further understanding of the

mechanism of action of the catalyticaily active PLA2s.

24

ACCESSING THE STRUCTURAL DETERMINANTS FOR POLIPEPTIDEBIFUNCTIONALITY: THE LYSINE DEGRADING ACTIVITIES OF THE

LYSINE-KETOGLUTARATE REDUCTASE/SACCHAROPINEDEHYDROGENASE ENZYME

Germano Cord Neto and Paulo ArrudaDepartamento de Genetica e Evolucao

Institute) de Biologia and CBMEG/UNICAMP13083-970 Campinas, SP

The control of lysine concentration in maize seeds is accomplished by fine-

tuned processes envolved in its synthesis, deposition and degradation. Lysine

degradation is carried out by a bifunctional polipetide bearing the Lysine-

ketoglutarate Reductase (LKR) and Saccharopine Dehydrogenase (SDH)

activities. The first step, performed by LKR, condenses lysine and oc-keto-glutarate

into saccharopine. Saccharopine is subsequently hydrolised to a-amino adipic-5-

semialdehyde and glutamic acid by the SDH activity. The LKR/SDH model has

several features that may became attractive to access structural determinants of

polipeptide bifuncionality. Such enzymatic activities exist in plants, mammals and

yeast. The active form of this enzyme can be as diverse as a dimer in maize, a

tetramer in human and bovine or two separated polipeptides ("monofuncional", in

opposition) in yeast. The LKR and SDR activities from the bifuncional polipeptides

in maize and mammals can be separated by tryptic digestion in two distinct

polipeptides. The maize enzyme has another important feature contrasting with the

animal enzymes: The LKR activity, but not the SDH, is modulated by Ca2+. In

opposition, the bovine or yeast enzyme are not affected by Ca"2 levels. This shall

provide us with insightful information concerning the structural changes that are

required for insertion of a calcium binding domain into the polipeptide. Recently,

the maize enzyme has been purified in our laboratory, opening an opportunity to

access the gene. The cloning of this gene and the expression of the protein shall

provide us the chance to start on the crystallization and structural analysis of the

enzyme.

25

IIBR9736548

STRUCTURAL ASPECTS OF ONA BINDING ACTIVITY OF THE BZIP

TRANSCRIPTIONAL ACTIVATOR OPAQUE 2

Adilson Leite<1', Jose Andres Yunes'1', Michel Vincentz<2\ Andre L. Vettore'1',Germando Cord Neto<1\ Eugen Gander*2', Goran Neshich<2> and Paulo Arruda<1>

<1>CBMEG, UNICAMP13083-970 Campinas, SP

(2)Laborat6rio de Biologia Molecular, Centro Nacional de Recursos Geneticos e Biotecnologia(CENARGEN/EMBRAPA), SAIN Parque Rural

70770- Brasilia, DF

The Opaque 2 protein (02) is a member of the basic domain/leucine zipper(bZIP) class of transcriptional activators. Studies on this protein and its functionhave been recently intensified and it became one of the best characterized plantbZIP transactivating factors. The DNA binding activity of bZIP proteins derives fromtwo connected properties; direct interaction with DNA and dimerization, which arefunctions of the basic region and leucine zipper, respectively. The two leucinezippers are packed in a coiled-coil region for dimerization and the basic regions ofthe dimer fit into the major groove of the half sites of the target DNA. 02 proteinrecognizes two different types of binding site: one based on the AGGT core, andthe other presenting some similarity to the GCN4-binding site. In the promoter ofthe 2S albumin gene (BE2S1) of the Brazil nut we have identified three differentbinding sites, two of them being F1 (TCCACGTCGA) and F3 (TCCACGTAGA),ACGT core-based, and the third F2 (GCCACCTCAT) a GCN4-like. Molecularmodeling showed that an asparagine and a serine in the 02 DNA binding domainmake different base specific contacts with each half-site of F1, F2, F3 and otherbinding sites. It is quite likely that these two residues may have contributed to the02 binding specificity. X-ray structure of the GCN4 bZIP motif bound to DNA targetsites has shown that the stability of the dimer results from side-by-side packing ofleucines and nonpolar residues in positions (a) and (b) the heptad repeat, as wellas a limited number of intra- and interhelical salt bridges. Differently from most bZIPproteins that have leucine zippers composed of three to five heptameric repeats, 02and related proteins contain seven of such repeats. It is noteworthy that in theseproteins, the leucine of the fifth heptad repeat has been substituted by an alanineresidue. In the homodimer, the presence of alanine, a small residue, could producepacking defects in the interface, but in the 02 and related proteins, this effect seemsto be compensated by the near presence of larger hydrophobic residues such astyrosine and phenilanine. The presence of the alanine and the large residuescould also stand for an important determinant of dimerization specificity. Supportedby: FAPESP, CNPq, CAPES, FINEP

26

BR9736549

PROTEIN STRUCTURE ANALYSIS AT ATOMIC RESOLUTION

Hans D. Bartunik and Alexandre N. PopovMax-Planck Society, AG Proteindynamik

MPG-ASMB c/o DESY, Hamburg, Germany

We have carried out protein structure analysis on the basis of diffraction dataextending to atomic resolution. Such extreme resolution may be achieved withhighly ordered crystals using synchrotron radiation and area detectors with lowintrinsic noise and broad dynamical range. For a number of different medium-sizedprotein structures, we were able to measure structure factor amplitudes up to 0.8 Aresolution (i.e.. to the present geometrical diffraction limit) At a resolutionapproaching 1.0 A, a sufficient number of independent reflection intensities may bemeasured for significantly softening or even omitting the restraints that are neededin conventional refinement procedures. Furthermore, anisotropic temperaturefactors may be refined. Atomic resolution studies thus may yield an essentiallyunbiased picture of the protein conformation, and information on (time and spaceaveraged) intra- and intermolecular motions. In particular, we extended theanalysis of two crystal structures of bovine pancreatic trypsin in differentorthorhombic crystal forms (Marquartf1) et al., 1984; BartuniM2) et al., 1989) to 1.1 Aresolution. Subsequent to conventional refinement with X-PLOR, anisotropicrefinement with SHELXL-93 led to electron density maps of very high contrast(crystallographic R=8.5%). As a consequence, atoms could be located in densityeven if their temperature factors were high or occupancies were low. Thus,alternate conformations became visible for about 15% of all protein residues, mostof them being located on the surface of the protein. These alternate conformationsappear to be correlated within regions of 10-15 A diameter. Molecular packingeffects may be separated out by comparing both structures. A great number ofsolvent molecules couJd be located in density. These include ca. 350 watermolecules and several sulphates. Many solvent molecules form clusters with (up tomore than 100) other solvents within hydrogen-bonding distance. Analysis atatomic resolution provides a basis for critical assessment of the accuracy in proteinstructure determination with conventional (isotropic) refinement procedures. Suchcomparisons with a-priori structural information potentially may lead substantiallyfurther than investigations at lower resolution like, e.g., the recent study of theaccuracy and reliability in protein crystallography based on high-resolution (1.3-1.6A) structural analyses of poplar plastocyanin (Guss<3) et al., 1992; Fields^ et al.,1994).

References

(1> Marquart. M. Waiter. J., Deisenhofer. J., Bode. W. and Huber, R. (1983), Acta Cryst. B39. 480-490.W Bartunik, H.D.. Summers. LJ . and Bartsch, H.H. (1989). J. Mol. Biol. 210, 813-828(3) Guss, J.M.. Bartunik. H.D. and Freeman. H.C. (1992). Acta Cryst. B48, 790-811.W Fields, B.A.. Bartsch. H.H. Bartunik, H.D., Cordes. F.. Guss. J.M. and Freeman, H.C. (1994). ActaCryst. D50. 709-730.

27

BR9736550

MACROMOLECULAR CRYSTALLOGRAPHY USING

SYNCHROTRON RADIATION AT DESY

Hans D. BartunikMax-Planck Society, AG Proteindynamik

MPG-ASMB c/o DESY, Hamburg, Germany

DESY has played a pioneering role in biological structural analysis usingsynchrotron radiation diffraction through a number of experimental developmentsand applications. The muscle fibre diffraction patterns recorded at the DESYsynchrotron from 1970 on by the group of Ken Holmes represented the firstapplication of synchrotron radiation to biological structural analysis. At the sametime, double-focusing X-ray optics were developed. A few years later, the firstprotein diffraction patterns were recorded for test purposes. From 1978 on, thestorage ring DORIS was the first facility where protein crystallographic datacollection was carried out (at the EMBL Outstation) in a routine way by a rapidlygrowing number of users. Since 1993, DORIS has been operated as a dedicatedsource. It provides good conditions for monochromatic and white-beam studies atphoton energies up to about 20 keV. Presently, four beamlines are predominantlyused for protein crystallography. These include two wiggler beamlines run by theMax-Planck Society (BW6) and by the EMBL Outstation (BM7). Recently, anundulator has been installed at PETRA. This station, which will be partly availableto protein crystallography, provides higher brilliance at photon energies > 20 keVthan the ESRF. A possible future development of a free-electron laser emitting inthe hard X-ray range is under study. Such a source would provide coherentradiation, a small cross-section, and a gigantic photon flux per pulse of 100 fsduration. Examples of outstanding applications include the first new proteinstructure, kallikrein, solved on the basis of synchrotron radiation data (Bode et al.,1982), the first high-resolution structure of a membrane protein, a reaction centre(Deisenhofer et al., 1985), the Laue diffraction analysis of an intermediate of rasp21 (Schlichting et al., 1990), and the structure of a proteasome (Lowe et al.,1995). The crystal structure analysis of intact ribosomal particles (Yonath andcoworkers) represents the most challenging application which is presentlypursued. Methodological developments at DESY have been carried out primarilyfor low-temperature crystallography, time-resolved diffraction, high-molecularweight studies, and structural analysis at true atomic resolution. Experimentalphasing by resonant (MAD) techniques has been successfully employed sinceDORIS has become dedicated. Future developments may include the use of veryshort wavelengths at PETRA, and ns time-resolved studies exploiting thefavourable time-structure of DORIS.

28

BR9736551

X-RAY STRUCTURE ANALYSIS OF THE CATALYTIC AND

ALLOSTERIC SITES OF GLUCOSAMINE-6-PHOSPHATE DEAMINASE

FROM Escherichia coli K12

Marcos R.M. Fontes«12', E. Horjalesí2-3', R.C. Garratt«2', M.M. Altamirano'3'M.L. Calcagno*3' and G.OIiva'2'

<1>Dept9 de Biofísica, Instituto de Biociências/UNESPCaixa Postal 510, 18618-000 Botucatu, SP

<2>lnstituto de Física de São Carlos/USPCaixa Postal 369, 13560-970 São Carlos, SP<3'Depts de Bioquímica, Facultad de Medicina

Universidad Nacional Autônoma de México

Glucosamine-6-phosphate deaminase is a hexameric enzyme (266

residues per monomer) that catalyses the reversible conversion of D-glucosamine-

6-phosphate (GlcN6P) into Fructose-6-phosphate and ammonia. The GlcN6P

deaminase exhibits ah intense homotropic cooperativity towards GlcN6P

(substract) which is allosterically modulated by the activator N-acetyl-D-

glucosamine 6-phosphate. Biochemical studies showed that 2-deoxy-2-

aminoglucitol 6-phosphate is a competitive inhibitor of the GIN6P deaminase. To

increase our understanding about the catalytic and allosteric sites, we studied

these sites using X-ray crystallographic techniques. The enzyme was initially

incubated with activator (and inhibitor) and subsequently, this solution was utilized

for growing crystals by the vapour diffusion method using Na/K phosphate and

sodium acetate (NaAc) as précipitants. The crystals obtained had maximum

dimensions of 0.5x0.4x0.2 mm. The data collection was performed using an image-

plate detector RAXIS-II (RIGAKU CO). The space group is R32 and cell parameters

a=b=125, c=223 Á for both crystals types. The crystals are isomorphous with native

ones (the native structure of GlcN6P deaminase was already determined by us)

and using difference Fourier techniques, it was possible to locate the inhibitor and

activator binding sites. The refinements were made using the XPLOR program

(Brunger. 1992). There are six catalytic and six allosteric sites (one catalytic and

one allosteric per monomer). The allosteric site is located between two monomers.

On the basis of analyses of the contacts of the inhibitor with the enzyme, a catalytic

mechanism is proposed for the isomerisation-deamination of GlcN6P deaminase.

This work has received partial support from PADCT-SBIO, CNPq, FAPESP and

FINEP.

29

THE REFINED CRYSTAL STRUCTURE OF HOLO-D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPHD)

FROM DUCK MUSCLE AT 2.7 A RESOLUTION

Valma Martins Barbosa, Richard Garrat and Glaucius OlivaInstittuto de Fi'sica de Sao Carios/USP

Caixa Postal 369, 13560-970 Sao Carlos, SP

GAPDH is an enzyme composed of four chemically identical subunits,which catalyses the NAD+ dependent oxidative phosphorylation of D-glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. The structural basis of itsreaction mechanism has been extensively studied because of its biologicalimportance and interesting properties regarding cooperativity of NAD binding. TheGAPDH's from different species are highly conserved, an indicator of the specificrelevance of their biological role. Holo-GAPDH from duck muscle crystallizes in themonoclinic space group P2i with a=68.93 A, b=159.47 A, c=77,76 A and p=97,68°,containing two functional tetramers (mol. wt=144 kD) per unit cell. The structurewas solved by molecular replacement (MR) using the package AMoRe (Navaza,1993) with the human enzyme as model structure. The self-rotation functionconfirmed three molecular non-crystallographic dyad axes in an approximate 222arrangement. The refinement of the model was carried out in various steps ofsimulated annealing with the package X-PLOR, restraining the model with the non-crystallographic symmetry relations. The final cycles of refinement resulted in an Rfactor of 20,1% for 39416 reflections (Rfree=26,3%). The final model includes all332 residues for each of the four monomers, 4 NAD+ molecules, 8 sulphate ionsand 163 water molecules. The structure has been analyzed with respect tomolecular symmetry, cofactor binding, intersubunit contacts and active sitegeometry. The refined model shows the four subunits similar apart from localdeviations of some side chains, mainly due to packing contacts. Each monomerhas one bound NAD+ molecule. The functional phosphate binding sites areoccupied by sulphate ions, similarly to the structure of lobster and Bacillusstearothermophilus GAPDH, and the stability of anion binding can be understoodfrom the structure conformations of their ligands. The analysis has revealed thepresence of several internal salt-bridges stabilizing the structure. Several orderedwater molecules have been located, and play an important role in the structuralintegrity of the protein. This work has received support from: PADCT/SBIO, CNPq,FINEP and FAPESP.

30

REFINEMENT AND TEMPERATURE FACTOR ANALYSIS OF THE R-

STATE, M 5, C118, 239S DOUBLE-MUTANT OF THE ENZYME

GLUCOSAMINE-6-PHOPHATE DEAMINASE FROM

Escherichia coli K12

Rodrigo, N.R. Pereira*1', R.C. Garrat<1>, E. Horjales'2»M. Calcagno*2) M. Altamirano<2>& G. Oliva'1'

<1'lnstituto of Phisics of São Carlos/USPCaixa Postal 369, 13560-970 São Carlos, SP

<2'Universidad Nacional Autonoma de Mexico/UNAMCiudad de Mexico, Mexico

Glucosamine-6-phosphate deaminase has a key role in the catabolicmetabolism of the amino sugars D-glucosamine-6-phosphate and N-acctyl-D-glucosamine-6-phosphate in Eschericha coli. This is an allosteric enzyme thatcatalyses the reversible conversion of glucosamine-6-phosphate to fructose-6-phosphate and ammonium. It is a homohexamer and presents intense homotropiccooperativity towards its substrate and has N-acetyl-glucosamine-6-phosphate asallosteric activator. The amino acid sequence presents 4 cysteine residues (C118,C219, C228, C239). Biochemical data shows that the C118 and C239 areimportant for the catalysis and allosteric transition. We have crystallized andcollected a data set for the C118, C239S double-mutant of the enzymeglucosamine-6-phosphate deaminase in both states, T and R. Preliminary modelswere obtained using initial phases of the native enzyme due to the fact that themutant crystals were isomorfous in relation to the native. So far, the refinement andthe temperature factor analysis has been carried out for R state mutant. Thisrefinement was accomplished with the X-PLOR program using simulated annealingand testing three weighting schemes (Wa) between the crystallographic andstereochemical terms. During this testing phase we tried to refine the atomic B-factors using initial values of 15.0 Á (type I) and native structure B-factors asstarting values (type II). The best refinement scheme was that with weight of 80%and native initial B-factors. The final R-factor was 20.3% without water molecules.The temperature factor analysis was performed separately for the two monomers (A

and B) in the asymmetric unit. The specific analysis of the Ser118-Oy and Ser239-Oy B-factors showed low values in both monomers and were comparable to thelowest values of the native Ser-Oy B-factors. In addition to these results, the mutantand native structure had no large differences (Ca-superposition rms=0.24 Â) andthe electron density for the Se 118-Oy and Ser239-Oy was also similar to therespective Cys118-Sy and Cys239-Sy in the native structure. Further experimentsare being conducted to unambiguously determine the effective nature of the proteinmaterial present in our crystals. We gratefully acknowledge the financial support ofFAPESP. CNPq and FINEP.

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BR9736552

STATIONS FOR NON-CRYSTALLINE DIFFRACTION AND PROTEIN

CRYSTALLOGRAPHY AT THE SRS, CCL DARESBURY LABORATORY

Peter F. LindleyCCL-Daresbury Laboratory, Warrington, Cheshire WA4 4AD, UK

This lecture will briefly describe the main features of the X-ray beamlines atCCL Daresbury Laboratory used for Macromolecular Crystallography (PX) andNon-crystalline Diffraction (NCD) experiments. Further stations at the SRS permitEXAFS measurements of biological samples containing metal centres, time-resolved fluorescence and circular dichroism measurements and confocalmicroscopy. PX operations involve three stations, two of which, 9.5 and 9.6, derivetheir radiation from a 3-pole wiggler magnet operating at a magnetic field strengthof 5 tesla; the third station 7.2, receives radiation from a 1.2 tesla dipole magnetand is also used for fibre diffraction experiments. Station 9.5 is a dual purposestation designed for focussed Laue experiments and rapidly tunablemonochromatic applications including multi-wavelength anomalous dispersionmeasurements. The principal optical components are a platinum coated fusedquartz toroidal mirror and water cooled channel cut Si(111) double crystalmonochromator. The sample stage is a Nonius 3-circle goniometer for precisecrystal orientation and the station is equipped with a 30 cm diameter Mar-Researchimage plate detector with the option of an Arndt-Wonacott camera and X-ray filmLaue work if appropriate. Station 9.6 is mainly used as a fixed wavelength station

with A. = 0.9 A. The optical components are a platinum coated fused cylindricalcurved quartz mirror which provides 1:1 vertical focussing and a bent triangularSi(111) monochromator. The station is equipped with a 30 cm Mar-Researchimage plate detector. The optical components of station 7.2 are essentially thesame as 9.6, but the wavelength is selected at 1.49 A. All three stations areequipped with Oxford Cryostream cooling devices enabling data collection attemperatures down to 120 K. NCD operations also involve three stations, 2.1 and8.2 receiving radiation from 1.2 tesla dipole magnets, and a new station 16.1,accepting some 11 mrads of radiation from a 3-pole wiggler magnet operating at 6tesla: all three stations are essentially fixed wavelength with A, = 1.5 A. The NCDstations have horizontally focussing bent triangular monochromators as their firstelements and these are followed by cylindrical curved mirrors. The stations have avariety of sample stages to enable a wide range of studies including X-ray solutionscattering measurements, muscle diffraction measurements, and polymer studies.Considerable resources have been invested in developing multi-wire gas detectorsin conjunction with fast data acquisition systems which can be used for time-resolved measurements on the millesecond time scale. A recent development hasbeen the use of two detectors for simultaneous measurement of both the small andwide angle X-ray diffractions patterns.

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BR9736553

APPLICATIONS OF SYNCHROTRON RADIATION TO

STRUCTURAL BIOLOGY

Peter F. LindleyCCL-Daresbury Laboratory, Warrington, Cheshire WA4 4AD, UK

Synchrotron radiation provides a source of high intensity, highly collimated,wavelength tunable X-radiation which is becoming increasingly important instudies of structure-function relationships in biological macromolecules. Where it ispossible to grow single crystals giving rise to well-defined diffraction patterns, themaximum amount of information can be obtained at the atomic and molecular levelusing macromolecular crystallographic (PX) techniques. These studies give rise tospatially and time-averaged models which underpin much of modern molecularbiology. Recent examples include: F-i ATPasc from bovine mitochondria, anenzyme central transfer in cells; avian 5-crystallin, an example of a "high-jacked"enzyme used for structural purposes so that the avian lens can accommodate overlarge distances; human ceruloplasmin, a copper containing enzyme which isthought to play a key role in the release of iron from cells; and the B800-850 light-harvesting complex from hotosynthetic bacteria, an essential component of thephotosynthetic pathway. None of these large, weakly diffracting problems couldhave been solved without the use of synchrotron radiation. A recent developmenthas been data collection at cryogenic temperatures leading to minimal radiationdamage and near atomic resolution. Thus, the structure of a 21 kDa bovine eyelens protein, y-crystallin, has been elucidated to 1.2 A resolution, revealing specificside chain disorder and 80% of the water structure. However, many importantproblems are not accessible through the single crystal route and in these casesnon-crystalline diffraction (NCD) methods can provide the only feasible way ofobtaining important structural information. They are also highly suited to studyingchanges in molecular conformation in solution. Thus, important insights into thestructures of macromolecular complexes such as plasminogen and low-densitylipoproteins have been obtained from-ray solution scattering measurements. PXmethods are also severely limited with regard to time-resolved measurements,whereas NCD techniques using detector technology developed at the SRS. canreadily make measurements on the millisecond time scale. Such technology hasbeen used to investigate the mechanism of muscle contraction, and the assemblyof micro tubules and chlathrin cages. Biological spectroscopy (BS) can also beused to provide important information on molecular flexibility and mechanisms ofmacromolecular folding. X-ray absorption spectroscopy (EXAFS) can be usefullyused to probe the character of metal centres in metalloproteins under a variety ofdifferent chemical conditions, particularly if PX models are a available for a givenstate. Synchrotron radiation has become an essential tool with wide-rangingapplications in the field of structural biology.

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PROTEIN CRYSTALLOGRAPHY IN BRAZIL

Glaucius Oliva, Richard Garrat and Eduardo E. CastellanoInstitute of Physics of Sao Carlos/USP

Caixa Postal 369,13560-970 Sao Carlos, SP

The design and characteristics of the storage ring of LNLS are the

determining factors in the design of the dedicated protein crystallography

beamline. The solution adopted aims at the maximization of the monochromatic

photon flux at the crystalline sample, at wavelengths in the range 1 -» 1.5 A. This

station will ultimately require a wiggler, and a vertical focusing mirror and single-

crystal curved monochromator as main optical elements. The detector chosen is an

Imaging-Plate based system, the large radius MAR-Research equipment. The

construction of the station is being supported by FAPESP. The projects on

macromolecular structure determination being conducted at the Laboratory of

Protein Crystallography, Institute of Physics of Sao Carlos, as well as from other

laboratories (e.g. IBILCE-UNESP, S.J. Rio Preto), are currently dependent on

diffraction data collected at lab sources or synchrotron facilities abroad, and will

greatly benefit from the PX-station of LNLS. As an example, the structure of the

enzyme Glucosamine-6-Phosphate deaminase from E. coli, solved at Sao Carlos,

will be presented.

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