MPSA abstracts

Page 1

s of Protein Chemistry, Vol. J3, No. 5, July t994

l Oth International Conference on Methods in Protein Structure Analysis (September 8-13, 1994, Snowbird, Utah)

ABSTRACTS

Special Issue Editors: M. Zouhair Atassi Ettore Appella

515

0027-8033/94/0700 0515507.00/0 �9 1994 Plenum Publishing Corporation

Page 2

Page 3

Journal of Protein Chemistry, Vol. 13, No. 5, July 1994

MPSA Abstract Listing

A1. Mahmoud Aminlari A2. Thomas Asquith and

Katherine Sarlo B1. Jerome M. Bailey, Oanh Tu,

Gilbert Issai, Alice Ha, and John E. Shively

B2. Alexander W. Bell, Nicole C. Baur, John J. M. Bergeron, Wei-Jia Ou, David Y. Thomas, Katherine Cianflone, Allain Baldo, Maxwell T. Hincke, Richard L. Momparler, Josde Lalibert6, David M. P. Thomson, and M. Sutherland

B3. Vladimir Besada, Javier Gonzalez, Gabriel Padron, Hilda Garay, Osvaldo Reyes, Toshifumi Takao, and Yasutsugu Shimonishi

B4. Rainer Bischoff, Dominique Roecklin, Bernadette Bouchon, Klaus Klarskov, and Alain Van Dorsselaer

B5. Patricia G. Brake, Anne Pacitti, Terry Higgins, Panos Stevis, John Malinowski, Sue McElhiney, James Huang, and Christine Vestal

B6. John M. Brewer, Vani S. Sangadala, Robert L. Robson, Claiborne C. V. Glover, Michael J. Holland, and Lukasz Lebioda

B7. Scott D. Buckel, Tracy Stevenson, and Joseph A. Loo

C1. Martin Caffrey, Jin Wang, Carmichael J. A. Wallace, and Ian Clark-Lewis

C2. C. A. Carothers Carraway, J. Huang, Y. Li, S.-H. Juang, A. Gallo, B. J. Mayer, and K. L. Carraway

Probing the active site of rhodanase with disulfide reagents Uses of gel electrophoresis to monitor detergent enzymes: application to safety assessments C-terminal sequence analysis of polypeptides containing C-terminal proline

Exploiting automated protein sequence analysis: in situ cleavage and resequencing

Study of a rearrangement at the C-terminus of peptides during the collision activated dissociation experiments

Analysis of the microheterogeneity of recombinant Schistosoma mansoni antigen rSmp28 reveals N-formyl-alanine as a new post-translational modification in Saccharomyces cerevisiae

Characterization of a recombinant cytosolic domain of CD45 phosphatase

Preparation and characterization of the site-directed E211Q mutant of yeast enolase 1

Direct mass spectrometric analyses for protein chemistry studies

Structure characterization of membrane bound and surface adsorbed protein

p58, A membrane-and microfilament-associated gag-like protein from ascites tumor cell microvilli, binds SrC SH3 domain through a poly-proline motif

517 0027-8033/94/0700-0517507.00/0 �9 1994 Plenum Publishing Corporation

Page 4

518 MPSA Abstract Listing

C3. Patrick L. Coleman and Daniel Sarpong

D1. David W. Deerfield, Amanda Holland-Minkley, John D. Hempel, and Hugh B. Nicholas, Jr.

D2. Nancy D. Denslow, Leroy C. Folmar, and Craig V. Sullivan

D3. James D. Dixon, Jonathan P. Mark, Christopher P. Elicone, Simin D. Maleknia, Brian F. McGuinness, Fred. E. Regnier, and Noubar B. Afeyan

D4. Julia M. Dolence and C. Dale Poulter

El. Tsezi Egorov, Alexander Musolyamov, Yves Popineau, Jens Andersen, and Peter Roepstorff

F1. Roberto J. Falkenstein, Mirtha J. Biscoglio de Jimenez Bonino, and Clara Pefia

G1. D. L. Gauggel, T. N. Asquith, R. J. Isfort, N. S. Miller, and D. B. Cody

G2. Michael F. Giblin, Tuck C. Wong, and Thomas P. Quinn

G3. Gregory A. Grant and Mark W. Crankshaw

G4. Scott Griffith, Steve Schroeder, and Thomas Quinn

G5. F. Guinet, Y. Petillot, J. M. Chapsal, J. Dubayle, F. Greco, O. Barge, E. Forest, and C. Valentin

H1. Frederick M. Hahn, Jonathan A. Baker, and C. Dale Poulter

H2. Torben Halkier and Arne Agerlin Olsen

H3. Mitsuru Haniu, William C. Kenney, and Michael F. Rohde

H4. James G. Harman, Eun Ju Lee, Joel Glasgow, Sew Fen Lew, and Ali O. Belduz

Direct determination of immobilized protein concentration

Conformational flexibility of the Gly-Gly dipeptide within protein structures

A highly conserved N-terminal sequence for teleost vitellogenins

Rapid fingerprinting of proteins with immobilized protease columns

Protein, farnesyi transferase. Evidence for an electrophilic substitution mechanism. Disulphide mapping of prolamins

Conformational comparison in the snake toxin family

Mapping and characterization of proteins associated with morphological transformation in the SHE cell assay

Solution structure of a cyclic c~-MSH analogue--initial NMR studies

Identification of modified PTH-amino acids in protein sequence analysis Crystallization of a thermal stable trypsin inhibitor from Phaseolus lunatus

Characterization of recombinant porin by mass spectrometry

Insights into the structure and active-site architecture of IPP:DMAPP isomerase

Counting cysteine and cystine residues in proteins and peptides using MALDI-TOF mass spectrometry Detection of di-PTH-cys for assignment of disulfide linkages

Mutagenesis of the cyclic AMP receptor protein (CRP) of Escherichia coli

Page 5

MPSA Abstract Listing 519

Reed J. Harris H5.

H6. Reed J. Harris, Michael S. Molony, Lene H. Keyt, and Shiaw-Lin Wu

H7. David H. Hawke, Jacqueline Tso, Sherrell Early, and Chad Miller

H8. G. Thomas Hayman and Jan A. Miernyk

H9. Ulf Hellman, Christer Wernstedt, and Jorge Gdfiez

H10. Daniel Hess, Ralph Studer, and Peter E. Hunziker

Hl l . Hisashi Hirano, Yoshihiro Watanabe, Sergei F. Barbashov, Setsuko Komatsu, Andrew M. Hemmings, Masaru Miyagi, and Susumu Tsunasawa

H12. Reuben E. Huber, Nathan J. Roth, and Michael T. Gaunt

J1. Paul JeniS, Thierry Mini, Suzette Moes, and Martin Horst

J2. Kenji Jinnai, Tetsuo Ashizawa, and M. Zouhair Atassi

J3. Anders H. Johnsen, Hanne Jensen, and Jens F. Rehfeld

K1. Masaharu Kamo, Takao Kawakami, Norifumi Miyatake, and Akira Tsugita

K2. J. N. Keen, P. F. Zagalsky, and J. B. C. Findlay

K3. Regine Kraft, Susanne Kostka, and Enno Hartmann

K4. Henry C. Krutzsch and John K. Inman

M1. Claudia Machalinski and Mirtha Biscoglio de Jimdnez Bonino

M2. Donald K. McRorie, Gregg R. Dieckmann, Susan Heilman, William F. DeGrado, and Vincent L. Pecoraro

M3. Chad G. Miller, James Kenny, Julie Sahakian, David H. Hawke, and Jacqueline Tso

Evaluating protein modification sites observed by N-terminal sequence analysis Detection of low levels of hydroxylysine in non-collagenous proteins

Chemical examination of the diphenylphosphoroisothiocyanatidate/trimethylsilnolate method for protein carboxyl-terminal degradations Aptase and molecular chaperone activities of the maize endoplasmic reticulum-resident Stress-70 protein Micropreparation for sequence analysis of peptides of proteins obtained from old archived polyacrylamide gels by in situ trypsin digestion Identification and characterization of metal protein complexes of crude cell extracts by mass-spectrometry The structure and function of plant leginsulin

Probing the Mg 2+ binding site of/3-galactosidase (E. coli)

In-gel reduction and alkylation of proteins and enzymatic digestion: application to the sequence analysis of proteins separated by 1-D gel electrophoresis Analysis of the extracellular organization of the u-chain of mouse acetylcholine receptor on live muscle cells

Probing of the phylogeny of the CCK/gastrin family by antisera directed against the conserved common C-terminus Separation and characterization of proieins with two electrophoresis

An investigation of the bathochromic shift phenomenon in biology

Microsequencing of proteins of the endoplasmic reticulum (ER) membrane N-isopropyliodoacetamide in the reduction and alkylation of proteins: Use in microsequence analysis Determination of the primary structure of a new milk-clotting protease by microsequencing techniques

A pH dependence on the association properties of model coiled coil peptides

C-terminal protein sequence analysis using the Hewlett-Packard C-terminal protein sequencing system

Page 6

520 MPSA Abstract Listing

M4. Jeffrey A. Moore and C. Dale Poulter

M5. Mary B. Moyer and William A. Burkhart

M6. Tatyana Muranova and Lubov Makova

N1. Hugh Nicholas, John Hempel, Amy Hinich, David Deerfield, Joseph Behrmann, and Alex Ropelewski

N2. Lori Nixon and Leonard Maneri

N3. Kerry Nugent and Ken Stoney

N4. Kerry Nugent and John Wieser

O1. Hiroshi Ohguro, Krzysztof Palczewski, Kenneth A. Walsh, and Richard S. Johnson

P1. Leonard C. Packman, Carl Webster, and John Gray

P2. G. Padr6n, V. Morera, L. J. Gonzfilez, Y. T~mbara, V. Besada, R. Villalonga, G. Chinea, O. Reyes, H. Garay, R. Bringas, and C. Nazfibal

P3. Bruce P. Parkinson, Kent A. Yamada and Anne Randolph

P4. Anthony Pisano, Nicole H. Packer, John W. Redmond, Keith L. Williams, and Andrew A. Gooley

R1. Hanne H. Rasmussen, Ejvind MCrtz, Matthias Mann, Peter Roepstorff, and Julio E. Celis

R2. Lone K. Rasmussen, Esben S. SCrensen, Torben E. Petersen, Jcrgen Gliemann, and Poul Henning Jensen

R3. Staffan Renlund, Henrik Wadensten, Annika Persson, Per Persson, Agneta Johansson, and Per-Olof Edlund

R4. Donald J. Rose

S1. Ragna Sack and Peter E. Hunziker

Escherichia coli DMAPP-tRNA transferase expression and characterization Alternative reverse-phase supports for direct electroelution and in situ digestion on the Hewlett-Packard sequencing column A convenient procedure for determination of carboxyterminal amino acids of proteins immobilized on the membrane Quantitative evaluation of different multiple alignment programs

Degradative modifications of rhIL-lra (recombinant human interleukin receptor antagonist) Automated preparation of complex biological samples prior to protein or peptide sequence analysis A new PTH amino acid separation system to improve performance at the low picomole level for older sequencers Arrestin topographic study using selective chemical modifications and hydrogen-deuterium exchange analyzed by mass spectrometry

The use of maldron mass spectrometry and amino acid sequence analysis in characterizing small amounts on N-blocked protein Characterization of antigenic determinants of P64K a Neisseria rneningitidis membrane protein

A straightforward method for amino-terminal sequencing of gamma-carboxyglutamic acid using standard Edman chemistry

Characterization of individual N- and O- linked glycosylation sites using Edman degradation

Identification of proteins recorded in human 2-D gel protein databases by mass spectrometric peptide mapping

Localization of transglutaminase reactive glutamine and lysine residues in Alzheimer amyloid/3A4 peptide: Cross-linking to extracellular matrix proteins and c~2M receptor

Different forms of apolipoprotein A and alcohol dehydrogenase studied by electrospray LC/MS

Automated two-dimensional electrophoresis-liquid chromatography for the micropreparative isolation of proteins Determination of cysteine by amino acid analysis: Online oxidation and hydrolysis

Page 7

MPSA Abstract Listing 521

$2. Julie Sahakian, Alex Apffel, Chad Miller, and Rodney L. Levine

$3. Kazuyasu Sakaguchi, Nicola Zambrano, Marc S. Lewis, Eric T. Baldwin, Bruce A. Shapiro, John W. Erickson, James G. Omichinski, G. Marius Clore, Angela M. Gronenborn, and Ettore Appella

$4. Werner Schr/Sder and Irmgard Moser

$5. Werner Schr/3der, Werner Pansegrau, and Erich Lanka

$6. Richard J. Simpson, James Eddes, Hong Ji, Gavin E. Reid, and Robert L. Moritz

$7. Esben S. SCrensen, Lone K. Rasmussen, Poul Henning Jensen, Peter Hcjrup, and Torben E. Petersen

$8. David W. Speicher, David F. Reim, and Kaye D. Speicher

$9. B. R. Srinivasa and S. P. Barde

$10. William G. Stirtan and C. Dale Poulter

Sll. Alyona Sukhanova, Sergey Vorob'ev, Alexander Gabibov, and Igor Bronstein

T1. Kenji Tanaka, Kuniko Einaga, Minoru Tsukada, Jonathan F. Tait, and Kazuo Fujikawa

T2. Akira Tsugita, Masaharu Kamo, Keiji Takamoto, and Kazuo Satake

V1. Ilya A. Vakser V2. V. V. Velikodvorskaia, A.

G. Gabibov, and A. G. Rabinkov

V3. Tennie Videler, Michael Osborne, Geoffrey Moore, Richard James, and Colin Kleanthous

V4. Nikolie Vtyurin W1. Jane H. Walent, Richard

Bessen, Dick Marsh, and Ronald L. Niece

Identification of an oxidative modification of glutamine synthetase using electrospray LC/MS and Edman sequencing techniques

Characterization of a binding site of the human immunodeficiency virus type 1 RNA for the nucleocapsid protein P7

Functional and protein analytical studies on the major outer membrane protein of Campylobacter jejuni Relaxase (TraI) of IncP o~ Plasmid RP4 catalyzes a site-specific cleaving-joining reaction of single-stranded DNA High-speed chromatographic separation of proteins and peptides: Application to rapid peptide mapping of in-gel digested proteins

Identification of posttranslational modifications in bovine osteopontin: localization of 28 phosphorylation sites, 30-glycosylation sites and 2 transglutaminase reactive glutamines

Integration of MALDI mass spectrometry with in situ protease digestions on high retention PVDF membranes to obtain internal sequences at maximum sensitivity A thiol mediated autolytic cleavage of homocysteinyl prolyl bond

Characterization of purified yeast protein geranylgeranyltransferase type I using a continuous fluorescence assay The DNA-topoisomerase I. Analysis by limited proteolysis of domain structure and conformational changes

Preparation and characterization of a conjugate of annexin V and the B-chain of urokinase

A novel C-terminal sequencing method using perfluoroacyl anhydrides

Protein docking in the absence of detailed molecular structures Acetyl-CoA-carboxylase: registration of active polymer complex

Structure determination by NMR of the nuclease inhibitor protein IM8

Local tight packing of hydrophobic groups in beta-structure Identification of distinct proteinase K cleavage sites in two strains of scrapie PRP by chemical isolation of unique sequence by orthophtalaldehyde and by comparison of N-terminal sequence maps of PRP fragments

Page 8

52Z MPSA Abstract Listing

W2. Jane H. Walent, Francis H. C. Tsao, and Ronald L. Niece

W3. Hong Wang and C. Dale Poulter

W4. Scot R. Weinberger, Lynn M. Chakel, Alex Apffel, Julie Sahakian, and Chad G. Miller

W5. Ewald M. W0ndrak, Alan R. Kimmel, and John M. Louis

Identification of a cleavage-sensitive region in lipocortin 1 by isolation of unique N-terminal sequence by orthophtalaldehyde

Purification and general characterization of dimethylallyl tryptophan synthase A rapid screening strategy for the identification of modified peptide fragments of glutamine synthetase using the Hewlett-Packard MALDI-TOF system

Processing of the mature HIV-1 nucleocapsid protein by the viral protease

Page 9

PR

OB

ING

TH

E A

CT

IVE

SIT

E O

F R

HO

DA

NE

SE

WIT

H D

ISU

LF

IDE

RE

AG

EN

TS

M

ahm

oud

Am

inla

ri,

Dep

artm

ent o

f B

ioch

emis

try,

Sch

ool o

f V

eter

inar

y M

edic

ine,

Shi

raz

Uni

vers

ity,

Shi

raz,

713

65

Iran

The

pur

pose

of

pres

ent i

nves

tiga

tion

was

to

obta

in a

n in

sigh

t int

o th

e ch

emic

al p

rope

rtie

s of

the

act

ive

site

of

the

enzy

me

rhod

anes

e (t

hios

ulfa

te:

cyan

ide

sulf

urtr

ansf

eras

e).

Thi

s en

zym

e is

wid

ely

dist

ribu

ted

in n

atur

e, f

rom

bac

teri

a to

man

. Se

vera

l fu

ncti

ons,

inc

ludi

ng

cyan

ide

deto

xifi

cati

on a

nd s

ynth

esis

of

iron

-sul

fur

cent

ers,

hav

e be

en p

ropo

sed

for

this

en

zym

e. D

iffe

rent

dis

ulfi

des

wit

h di

ffer

ent c

hem

ical

pro

pert

ies

wer

e al

low

ed to

rea

ct w

ith

the

sulf

hydr

yl g

roup

in t

he a

ctiv

e si

te o

f rho

dane

se. T

he E

llm

an r

eage

nt;

5,5'

-dit

hiob

is (

2-

nitr

oben

zoic

aci

d),

DT

NB

, re

acte

d ve

ry s

low

ly.

A m

arke

d in

crea

se in

the

rat

e of

rea

ctio

n of

rho

dane

se w

ith

DT

NB

was

obs

erve

d w

hen

cati

onic

dis

ulfi

des,

cys

tam

ine

and

cyst

ine,

w

ere

pres

ent.

No

chan

ge w

as s

een

wit

h no

n-ca

tion

ic d

isul

fide

s di

met

hyl

disu

lfid

e, 2

- hy

drox

y et

hyl d

isul

fide

, dit

hiog

lyco

lic

acid

oxi

dize

d gl

utat

hion

e, 2

,2'-

sali

cyli

c ac

id,

2,2'

- di

pyri

dyl

disu

lfid

e (2

-PD

S) a

nd 4

,4-D

ipyr

idyl

dis

ulfi

de (

4-PD

S).

A K

m o

f 2.

0 an

d 2.

5 m

mol

/L

was

ob

tain

ed

for

cyst

amin

e an

d cy

stin

e,

resp

ecti

vely

, w

hile

th

e K

m

for

thio

sulf

ate,

the

com

mon

sub

stra

te o

f th

e en

zym

e w

as 3

.0 m

mol

/L.

The

se r

esul

ts i

ndic

ate

exis

tenc

e of

ani

onic

gro

ups

in o

r in

the

vic

init

y of

the

acti

ve s

ite

whi

ch f

acil

itat

e bi

ndin

g of

the

cat

ioni

c di

sulf

ides

. Fu

rthe

rmor

e, t

hese

dat

a m

ight

sug

gest

tha

t th

ese

disu

lfid

e ar

e th

e tr

ue p

hysi

olog

ical

sub

stra

tes

of r

hoda

nese

.

Key

wor

ds:

Rho

dane

se, c

ysta

min

e, d

isul

fide

s.

Al

US

ES

OF

GE

L E

LE

CT

RO

PH

OR

ES

IS T

O M

ON

ITO

R D

ET

ER

GE

NT

EN

ZY

ME

S:

AP

PL

ICA

TIO

N T

O S

AF

ET

Y A

SS

ES

SM

EN

TS

.

Tho

mas

Asq

uith

and

Kat

heri

ne S

arlo

C

orpo

rate

Pro

fess

iona

l & R

egul

ator

y Se

rvic

es/H

uman

Saf

ety

Dep

t., T

he P

roct

er &

Gam

ble

Co.

, P.O

. B

ox 3

9870

7, C

inci

nnat

i, O

hio,

452

39-8

707.

Occ

upat

iona

l ex

posu

re g

uide

line

s ar

e on

e to

ol u

sed

to s

ucce

ssfu

lly

cont

rol a

ller

gy to

de

terg

ent e

nzym

es in

man

ufac

turi

ng f

acil

itie

s. T

hese

exp

osur

e gu

idel

ines

are

bas

ed o

n sa

fety

ass

essm

ents

whi

ch m

easu

re th

e al

lerg

enic

pot

enti

al o

f enz

ymes

. B

ecau

se p

rote

in

com

posi

tion

is o

ne f

acto

r tha

t det

erm

ines

all

erge

nic

pote

ntia

l, it

is im

port

ant t

hat

com

posi

tion

rem

ains

ess

enti

ally

unc

hang

ed a

fter

exp

osur

e gu

idel

ines

are

det

erm

ined

. G

el

elee

trop

hore

sis (

SD

S-P

AG

E a

nd I

EF)

pro

vide

s a

fast

and

cos

t eff

ecti

ve m

eans

to m

onit

or th

e co

mpo

siti

on o

f enz

ymes

. Si

gnif

ican

t cha

nges

in c

ompo

siti

on m

ay tr

igge

r ad

diti

onal

saf

ety

test

ing.

Gui

deli

nes

for

the

min

imum

acc

epta

ble

vari

abil

ity

are

no c

hang

es in

ele

ctro

phor

etic

m

obil

ity

or in

crea

se in

rel

ativ

e am

ount

gre

ater

than

10%

for

any

com

pone

nt c

ompr

isin

g 10

%

or g

reat

er o

f the

tota

l pro

tein

. T

hey

have

bee

n ap

plie

d to

mon

itor

sta

bili

ty o

f sk

in p

rick

test

re

agen

ts, t

o de

tect

con

tam

inan

ts, t

o as

sess

cha

nges

in p

rodu

ctio

n te

chni

ques

and

to q

uali

fy

new

sou

rces

of a

ppro

ved

enzy

mes

. Po

ssib

le a

ppli

cati

on o

f the

se g

uide

line

s to

oth

er

man

ufac

turi

ng e

nvir

onm

ents

(i.e

., fo

od p

roce

ssin

g an

d pr

otei

n ph

arm

aceu

tica

ls)

wil

l als

o be

di

scus

sed.

C-T

ER

MIN

AL

SE

QU

EN

CE

AN

ALY

SIS

OF

PO

LYP

EP

TID

ES

CO

NTA

ININ

G C

- TE

RM

INA

L P

RO

LIN

E.

Jero

me

M.

Bai

ley,

Oan

h Tu

, G

ilber

t Is

sai,

Alic

e H

a, a

nd J

ohn

E. S

hive

ly

Bec

kman

Res

earc

h In

stitu

te o

f the

City

of H

ope,

Div

isio

n of

Imm

unol

ogy,

145

0 E.

D

uart

e R

oad,

Dua

rte,

CA

91

010.

The

inab

ility

to

deri

vatiz

e C

-ter

min

al p

rolin

e ha

s be

en a

maj

or im

pedi

men

t to

the

de

velo

pmen

t of

a r

outin

e ch

emic

al m

etho

d fo

r C

-ter

min

al s

eque

nce

anal

ysis

. Pr

evio

us w

ork

in o

ur la

bora

tory

des

crib

ed c

hem

istr

y w

hich

ded

vatiz

ed t

he C

-ter

min

al

amin

o ac

id to

a t

hioh

ydan

toin

and

per

mitt

ed t

he s

eque

nce

anal

ysis

of a

ll of

the

com

mon

am

ino

acid

s w

ith t

he e

xcep

tion

of p

rolin

e. T

his

new

che

mis

try

com

bine

d th

e ac

tivat

ion

and

dedv

atiz

atio

n st

eps,

obv

iatin

g th

e ne

ed fo

r in

term

edia

te o

xazo

linon

e fo

rmat

ion.

Th

is c

hem

istr

y ha

s no

w p

erm

itted

the

dedv

atiz

aUon

and

hyd

roly

sis

of C

- te

rmin

al p

rolin

e as

a t

hioh

ydan

toin

am

ino

acid

. W

e ha

ve a

utom

ated

this

che

mis

try

with

pep

tidas

cov

alen

tly a

ttac

hed

to c

arbo

xylic

aci

d m

odifi

ed p

olye

thyl

ene

and

prot

eins

non

-cov

alen

tly a

pplie

d to

Zite

x (p

orou

s Te

flon)

. It

is n

ow p

ossi

ble,

for

the

first

tim

e, t

o ro

utin

ely

sequ

ence

thro

ugh

all t

wen

ty o

f the

com

mon

am

ino

acid

s.

Prog

ram

s an

d m

etho

ds fo

r au

tom

ated

seq

uenc

ing

are

curr

ently

bei

ng o

ptim

ized

and

will

be

pres

ente

d.

This

wor

k w

as s

uppo

rted

by

NIH

Gra

nt G

M 4

6022

.

B1

EXPLOITING AUTOMATED PROTEIN SEQUENCE ANALYSIS: IN SITU CLEAVAGE AND RE-

SEQUENCING

Alexander W. Bell, Nicole C.

Baur, John J.M. Bergeron~, Wei-Jia Ou z, David ~.

Thomas 2, Katherine Cianflone 3, Allain B

aldo 3, Maxwell T. Hincke 4, Richard

.

Momparler 5, JesSe Lalibert6 5, David M.P. Thomson% and M. Sutherland

Protein Analytic Facility,

Sheldon Biotechnelogy Centre;

IDepartment of

Anatomy; 3Division of Cardiology, Royal Victoria Hospital; & 6Department of

Immunology,

Montreal

General

Hospital;

McGill

University,

Montreal.

ZBiotechnology Research

Institute,

NRC Canada;

&

5Centre

de Recherche

Pediatrique,

H6pital

Ste-Justine,

Universitd

de

Montrdal,

Montreal.

4Department of Anatomy, University of Ottawa Faculty of Health Science,

Ottawa.

A technique i

nvolving re-sequence analysis of a sample after exposure to

conditions which a

ffect cleavage at the C-terminal side of methionyl r

esidues

is r

outinely employed a) to d

irectly assess t

he quantity eriginally s

ubjected

to sequence analysis; b) to estimate the number of cleavable methionyl

residues; c) to provide a

dditional s

equence information and/or d) to c

larify

ambiguous sequencing results. The technique involves removal of the sample

from the gas-phase sequencer followed by I) exposure to cyanogen bromide

(CNBr) in the p

resence of 70% formic a

cid a

nd 2) re-sequence analysis of the

cleavage mixture. The technique takes advantage of CNBr cleavage s

pecificity,

differential wash-out characteristics of peptldes, sequencer reliability a

nd

reproducibility, ease of manipulation and reduced labour costs related to

both sample regeneration and/or ensuing work-up. The only requirement for

success, that is the generation of new sequence information, is limited to

the s

ample c

ontaining a c

leavable methionyl residue 8-10 r

esidues from t

he C-

terminus of the protein, although the null result is informative. Several

unknown proteins, N-terminally blocked or displaying ragged N-termini have

been identified u

sing this technique.

> >

A2

B

2

Page 10

STUDY

OF

A REARRANG~MENT

AT

THE

C-TERMINUS

OF

PEPTIDES

DURING

THE

COLLISION ACTIVATED DISSOCIATION EXPERIMENTS.

Vladimir

BCeada I ,

Javier

Gonzalez I ,

Gabriel

Padron I ,

Hilda

Garay I ,

Osvaldo Reyes I,

Toshifumi Takao 2,

Yasutsugu Shimonishi 2 .

i)

Center

for

Genetic

Engineering

& Biotechnology,

P.O.Box

6162,

La

Habana,

CUBA

2) Institute for Protein Research,

Osaka University,

Suita

Campus, Osaka 565, JAPAN.

An

internal

rearrangement

involving

the

loss

of

the

c-terminal

amino

acid residue of peptides in Low Energy Collision Activated Dissociation

(CAD

) experiments,

has

been

reported

i.

Specific

sequences

at

the

C-

terminus

of

peptidee

often

tend

to

be

misassigned

when

this

rearrangement

appears

as a

very intense signal

in the CAD spectrum.

We

designed

and

synthesized

a set

of

peptides

in

order

to

study

the

influence of the nature of amino acids at the rearrangement

site and the

position

of

a basic

residue

within

the

peptide

sequence

in

the

occurrence

of

this

rearrangement.

Our

results

confirm

that

this

phenomenon

is strongly

influenced

by the basicity

of

the

amino

acids

within the sequence,

thus

it could be useful

for

differentiating

two

isobaric amino acids such as Lysine and Glutamine in peptide sequencing.

Here,

we also

report

the use of

the enzymatic

18C-labeling

method

to

identify

the

C-terminal

rearrangement

of

peptides

and

avoid

misassignments of the sequence 2

l-Gaekell.

S.J et al., J.~un. Soc Mass Spectrom,

i, 249

(1990).

2-Takao. T

et al., A.Chemistry 65, 2394

(1993).

CHARACTERIZATION OF A

RECOMBINANT CYTOSOLIC DOMAIN OF CD45 PHOSPHATASE

Patricia G. Brake, Anne Pacitti, Terry Higgins, Panos Stevis~ John

Hallnowski, Sue McElhlney I,

James Huan~, Christine Vestal 3

Sterling winthrop Inc., Collegeville, PA, ZLexln Pharmaceutical Corporation,

Horsham, PA, 3PerSeptive Biosystems, Houston, TX

CD45 is a

large transmembrane glycoprotein required for antigen driven T-

and B-cell activation. The intracellular portion of CD45 contains two

protein domains one of which is an active protein tyrosine phosphatase.

The

phosphatase activity of the intraoellular portion of CD45 is believed to

initiate the cascade of events leadlng to B- and T-cell proliferation by

activating one or more src family klnases. The 82kDa cytosolic domain of

this protein was expressed at high levels in yeast and purified for use in

the initiation of biophysical studies.

But, despite an apparent purity of

this preparation on SDS-PAGE of >95%, the protein did not crystallize.

Microcharacterization of a

purified preparation of this protein by amino

acid sequence analysis and laser desorption mass spectrometry showed

heterogeneity at the N-terminus of the protein caused by cleavage at dibasic

residues.

This cleavage correlated with decreased specific activity of the

enzyme.

Subsequently, the DNA was modified by deletion of sequence coding

for residues N-terminal to the identified cleavage site and the resulting

derivative protein was expressed and purified.

Amino acid sequence analysis

showed that the identified N-terminal heterogeneity was eliminated by this

modification.

Crystallization trials of this preparation are in progress.

In addition,

SDS-PAGE analysis suggests a

small amount of internal protein

cleavage, potentially from a

similar dibasic amino acid sequence which is

present in the protein,

DNA sequence coding for this protein has been

mutated to eliminate this potential cleavage; analysis of this construction

is in progress.

t~q

B3

B5

ANAL

YSIS

OF

THE

MIC

ROHE

TERO

GEN

ITY

OF

RECO

MBI

NANT

SCH

ISTO

SOM

A MAN

SONI

ANTI

GEN

rS

mp2

8 RE

VEAL

S N-

FORM

YL-A

LANI

NE A

S A

NEW

PO

ST-T

RANS

LATI

ONA

L M

ODI

FICA

TIO

N IN

SA

CCHA

ROM

YCES

CERE

VISI

AE

Rain

er B

isch

off, D

omin

ique

Roec

klin

, Ber

nade

tte B

ouch

on, K

laus

Kla

rsko

v ~ an

d Al

ain V

an D

orss

elae

r 2

TRAN

SGEN

E S.

A., P

rote

in A

naly

tical

Gro

up, 1

1 Ru

e de

Mol

shei

m, F

-670

82 S

trasb

ourg

Ced

ex, F

RANC

E,

Vakg

roep

Bio

chem

ie Fy

siol

ogie

en M

icro

biol

ogie

, Led

egan

ckst

raat

35, 9

000 G

ent, B

ELG

IUM

, 2 Un

iver

sit(~

Loui

s Pa

steu

r, La

bora

t. de

Spe

ctro

met

rie de

Mas

se B

ioor

gani

que,

URA

31,

CNR

S, 5

Rue

Bla

ise P

asca

l, 670

08

Stra

sbou

rg C

edex

, FRA

NCE.

rSm

p28

was

exp

ress

ed in

trace

llula

rly in

a r

ecom

bina

nt s

train

of

Sacc

haro

myc

es ce

revi

siae

and

purif

ied b

y affi

nity

chr

omat

ogra

phy o

n gl

utat

hion

e-Se

phar

ose.

The

isol

ated

pro

tein

was

hom

ogen

ous

by S

DS-P

AGE

unde

r red

ucin

g co

nditi

ons

but s

how

ed h

eter

ogen

eity

upo

n is

oele

ctric

focu

sing

and

an

ion-

exch

ange

HPL

C. T

he t

wo

maj

or fo

rms

(rSm

p28/

1 an

d rS

mp2

8/2)

wer

e is

olat

ed b

y an

ion-

ex

chan

ge H

PLC

and

subi

ecte

d to

pro

tein

mic

rose

q-en

cing

reve

alin

g tha

t rSm

p28/

2 w

as N

.term

inat

ly

bloc

ked,

Com

para

tive

trypt

ic m

aps o

f the

two

form

s wer

e id

entic

al ex

cept

for o

ne a

dditi

onal

frag

men

t in

the

dige

st o

f rSm

p28/

2 w

hich

was

sho

wn

to b

e N-

term

inal

ly b

lock

ed a

nd to

cor

resp

ond

to th

e N-

te

rmin

al tr

yptic

fra

gmen

t of

rSm

p28

as d

eter

min

ed b

y am

ino

acid

ana

lysi

s, M

ass

mea

sure

men

t re

veal

ed a

diff

eren

ce o

f app

r, 26

Da

betw

een

the

expe

cted

mas

s of

the

N.te

rmin

al fr

agm

ent a

nd th

e m

easu

red

mas

s in

dica

ting

that

the

N-te

rmin

us w

as fo

rmyt

ated

(~m

= 2

8 O

a), P

artia

t deb

tock

ing

ol th

e pe

ptid

e fra

gmen

t w

ith t

riflu

oroa

cetic

aci

d al

low

ed t

o ob

tain

the

exp

ecte

d N-

term

inal

seq

uenc

e co

nfirm

ing

that

the

N-te

rmin

al a

lani

ne w

as b

lock

ed w

ith a

n ac

id la

bile

form

yl g

roup

dur

ing

prot

ein

bios

ynth

esis

in S

. cer

evis

iae,

N-fo

rmyl

-ala

nine

repr

esen

ts a

new

pos

t-tra

nsla

tiona

l mod

ifica

tion

in S

. ce

revi

siae

pote

ntia

lly im

plic

atin

g so

far n

ot d

escr

ibed

enz

ymat

ic a

ctiv

ities

.

B4

PR

EP

AR

ATI

ON

AN

D C

HA

RA

CTE

RIZ

ATI

ON

OF

THE

SIT

E-D

IRE

CTE

D E

211Q

MU

TA

NT

OF

YE

AS

T

EN

OLA

SE

1

John

M.

Bre

wer

1, V

ani

S,

San

gada

la 1

, Rob

ert

t,. R

obso

n =,

Cla

ibor

ne C

,V.

Glo

ver 1

, Mic

hael

J.

Hol

land

~,

and

Luke

sz L

ebio

da 4

~Dep

t. o

f B

ioch

emis

try,

Uni

v. G

eorg

ia,

Ath

ens,

GA

30

602;

aD

ept,

of M

icro

biol

ogy,

Uni

v. R

eadi

ng,

Eng

land

U

K;

3Dep

t. of

Bio

logi

cal

Che

mis

try,

Uni

v, C

alifo

rnia

Med

ical

Sch

ool,

Dav

is,

CA

95

616;

4D

ept.

of

Che

mis

try,

Uni

v. S

outh

Car

olin

a, C

olum

bia,

SC

29

208

Yea

st e

nola

se 1

, a

dim

eric

enz

yme,

bin

ds o

ne M

g++

/sub

unit

enab

ling

subs

trat

e bi

ndin

g, w

hich

in

turn

ena

bles

a s

econ

d M

~3~ *

lsub

unit

bin

ding

and

cat

alys

is I

lh

The

'ch

arge

shu

ttle

' m

echa

nism

of

enol

ase

cata

lysi

s, b

ased

on

X-r

ay c

ryst

allo

grap

hic

stud

ies,

inv

olve

s tr

ansf

er o

f th

e pr

oton

on

carb

on-2

o

f sa

bstr

ete

to a

H2O

, the

n to

the

car

boxy

late

of

Glu

-168

, ov

er t

o th

e ca

rbox

ylat

e o

f G

lu-2

11 a

nd

subs

eque

ntly

to

the

solv

ent

(2).

T

o te

st t

he i

nvol

vem

ent

of G

lu-2

11 i

n th

is m

echa

nism

a s

ite-d

irec

ted

mut

ant,

E21

1Q,

wa

s pr

epar

ed a

nd c

hara

cter

ized

as

desc

ribe

d fo

r E

168Q

(3)

. T

he i

dent

ity o

f E

211Q

w

as c

onfir

med

by

SD

S-g

el e

lect

roph

ores

is a

nd a

min

o ac

id s

eque

ncin

g.

E21

1Q s

how

ed 0

.O1%

of

the

na~L

ve e

nzym

e ac

tivity

in t

he s

tand

ard

assa

y,

Diff

eren

tial s

cann

ing

calo

rim

etry

(D

SC

) sh

owed

tha

t E

211Q

and

nat

ive

enol

ases

res

pond

sim

ilarl

y to

Mg

++ a

nd s

ubst

rate

. A

ll th

e E

211Q

enz

yme

is f

olde

d co

rrec

tly.

Spe

ctro

phot

omet

ric

titra

tions

wit

h a

sub

stra

ta a

nalo

gue,

D-t

artr

onat

e se

mia

ldeh

yde-

2-

phos

phat

e (4

) co

nfir

med

tha

t th

e bi

ndin

g af

fini

ty w

as n

ot a

ffec

ted

in t

he m

utan

t.

Rea

ctio

n o

f th

e an

alog

ue w

ith

E21

IQ

sug

gest

s pa

rt o

f th

e re

actio

n is

abo

ut t

wo

ord

ers

of

mag

nitu

de s

low

er

than

th

at w

ith

the

nat

ive

enzy

me,

T

hese

obs

erva

tions

are

con

sist

ent

wit

h t

he p

redi

cted

par

ticip

atio

n o

f G

lu-2

11 i

n th

e 'c

harg

e sh

uttle

' m

echa

nism

. 1.

B

rew

er,

J.M

. C

rit.

Rev

. B

ioch

emis

try

11

209

(18

81).

2.

Le

biod

a, L

. an

d S

tec,

B.

Bio

chem

istr

y 30

281

7 (1

991)

. 3.

B

rew

er,

J.M

., R

obso

n, R

.L.,

Glo

ver,

C.V

.C.,

Hol

land

, M

.J.

and

Lebi

oda,

L.

P

rote

ins:

S

truc

ture

, F

unct

ion

and

Gen

etic

s 1

7 4

26 (

1993

).

4.

Spr

ing,

T.G

. an

d W

old,

F,

Bio

chem

istr

y 1

0 4

655

(197

1).

86

Page 11

Dir

ect

Mas

s Sp

ectr

omet

ric

Ana

lyse

s fo

r P

rote

in C

hem

istr

y St

udie

s

Sco

tt D

. B

ucke

l, T

racy

Ste

vens

on a

nd J

osep

h A

. L

oo,

Par

ke-D

avis

Pha

rmac

euti

cal

Res

earc

h,

Div

isio

n of

War

ner

Lam

bert

Com

pany

, A

nn A

rbor

, M

I 48

105

In o

ur s

tudi

es o

f th

e st

ruct

ure

of v

ario

us p

rote

ins,

we

have

use

d a

com

bin

atio

n o

f pr

otei

n se

quen

ce a

naly

sis,

mat

rix-

assi

sted

las

er d

esor

ptio

n/io

niza

tion

(M

AL

DI)

ti

me-

of-f

ligh

t m

ass

spec

trom

etry

and

ele

ctro

spra

y io

niza

tion

(E

SI)

mas

s sp

ectr

omet

ry.

MA

LD

I-M

S i

s le

ss s

ensi

tive

to

the

pres

ence

of

extr

aneo

us s

alts

tha

n E

SI.

H

owev

er,

the

abil

ity

to d

irec

tly

mas

s an

alyz

e pr

otei

n m

ater

ials

via

ES

I w

itho

ut a

ddit

iona

l sa

mpl

e cl

eanu

p pr

oced

ures

res

ults

fro

m t

he d

iscr

imin

atin

g na

ture

of

a fo

cal-

plan

e m

ay

dete

ctor

. T

he a

rray

det

ecto

r ca

n be

"tu

ned"

for

lo

w l

evel

, hi

gher

ch

arge

d sp

ecie

s in

a c

ompl

ex m

ixtu

re a

nd]o

r in

the

pre

senc

e of

low

mol

ecul

ar m

ater

ial.

M

AL

DI-

MS

ca

n be

u

sed

to

m

on

ito

r th

e d

egre

e o

f tr

un

cati

on

an

d

the

seq

uen

ce

by

carb

oxyp

epti

dase

tre

atm

ent

of p

epti

des

and

smal

l pr

otei

ns w

itho

ut r

emo

val

of

extr

aneo

us b

uffe

r m

ater

ials

. T

he

cap

abil

ity

of

anal

yzi

ng

pro

tein

s se

para

ted

by

elec

tro

ph

ore

sis

wit

h

mas

s sp

ectr

omet

ry i

s a

pow

erfu

l to

ol.

We

have

bee

n ab

le t

o de

term

ine

the

mo

lecu

lar

wei

ghts

of

spot

s fr

om t

wo-

dim

ensi

onal

gel

s by

ES

I th

at h

ad b

een

blot

ted

onto

PV

DF

mem

bran

es,

stai

ned

wit

h C

oo

mas

sie

blue

, an

d ex

trac

ted

wit

h he

xafl

uoro

isop

ropa

nol

(HF

IP),

an

d a

lso

det

erm

ined

the

se

quen

ce o

f pe

ptid

es v

ia c

hem

ical

dig

esti

on o

f th

e m

ater

ial

not

used

for

mas

s de

term

inat

ion.

W

e ha

ve t

aken

sam

ples

dir

ectl

y fr

om a

n af

fini

ty c

olum

n el

uate

und

er h

igh

salt

con

diti

ons,

bou

nd t

he

prot

ein

to t

he m

emb

ran

e in

a P

roS

pin|

ca

rtri

dge,

ext

ract

ed t

he m

emb

ran

e w

ith

HF

IP,

and

dete

rmin

ed t

he m

ass

of

prot

ein

by M

AL

DI-

MS

and

ES

I-M

S.

The

nu

mb

er o

f di

sulf

ide

bond

s pr

esen

t in

sm

all

tig

hd

y-b

rid

ged

pep

tide

s ca

n be

det

erm

ined

by

the

dif

fere

nce

in m

ass

of t

he

oxid

ized

mat

eria

l an

d th

e m

ater

ial

redu

ced

by t

ris-

(2-c

arb0

xyet

hyl)

phos

phin

e w

itho

ut a

ddit

iona

l de

riva

tiza

fion

of

the

resu

ltin

g fr

ee c

yste

ines

..

B7

8~IA

ME

MB

RA

NE

-AN

D M

ICR

OFI

LA

ME

NT

-ASS

OC

IAT

ED

gag

-LIK

E P

RO

TE

IN F

RO

M A

SC1T

ES

OR

CE

LL

MIC

RO

VIL

LI,

BIN

DS

SR

C S

H3

DO

MA

IN T

HR

OU

GH

A P

OL

Y-P

RO

LIN

E M

OT

IF

C.A

. C

arot

hers

Car

raw

ay, l

J. H

uang

, l Y

. Li,

t S.-

H.

Juan

g, 1

A.

Gal

hi, 1

B.J

. May

er, 3

andK

. L

. Car

raw

ay. 2

D

epts

. of

Bio

chem

. &

Mol

ec.

Bio

l) a

nd.C

eU B

iol.

& A

nat.

,2 U

niv.

of

Mia

mi S

ch.

of M

edic

ine,

Mia

mi,

F

L 3

3136

and

The

Roc

kefe

ller

Inst

itute

," N

ew Y

ork,

NY

100

21

Asc

ites

subl

ines

of t

he 1

3762

rat

mam

mm

'y ad

enoc

arci

nom

avro

vide

a us

eful

mod

el s

yste

m fo

r inv

esti

gati

ng

mem

bran

e-m

iero

fila

men

t (M

F) i

nter

acti

ons t

. T

he M

AT

-Cf

subl

ine

diff

ers

from

til

e M

AT

-BI

subf

fam

in

havi

ng h

ighl

y st

able

, bra

n~he

r m

icro

vill

i (M

V) 2

and

im

mob

ile

cell

sur

face

rec

epto

rs. 3

A 5

8 kD

a pr

otei

n (p

58),

exp

ress

ed in

the

MA

T-C

1 bu

t not

tlie

MA

T-B

1 ce

lls 4

, has

bee

n im

plic

ated

in th

e st

abil

izat

ion o

f the

~

AT

-C1

cell

sur

face

, v5

8 is

pre

sent

in

MV

in

a tr

ansm

embr

ene

com

vl~x

(TM

C) 5

wit

h ac

tin

and

a hi

gh

M,

com

plex

of

at l

east

"fiv

e gi

ycop

rote

ins

(55,

65,

80,

110

and

120

kO

a). 6

The

TM

C s

erve

s as

the

cor

e fo

r a

larg

e si

gnal

tran

sduc

tion

pa~

cle

cont

aini

ng p1

85m

/Erb

B27

and

sev

eral

kno

wn

sign

al tr

ansd

ucti

on

com

pone

nts,

[nc

hidi

ng p

60~.

=. P

urif

ied

p58

bind

s -ol

iosv

holiv

ids

and

bloc

ks a

ctin

pol

ymer

]zai

lon,

act

ing

as

a fi

lam

ent c

appi

ng p

rote

in.4

The

se m

embr

ane-

and

MI*

-bin

ding

pro

pert

ies

are

cons

iste

nt w

ith

the

prop

osed

ro

le o

f p58

in s

tabi

lizi

ng th

e hi

ghly

mal

igna

nt M

AT

-C1

cell

suTr

face

by s

tabi

lizin

g, th

e in

tera

ctio

ns b

etw

een

the

mie

rovi

llar

MF

s an

d th

e m

embr

ane.

T

he c

ompl

ete

eDN

A s

eque

nce

of p

5~ h

as b

een

obta

ined

S, e

nd

the

dedu

ced

amin

o ad

td s

eque

m~

sho

wed

a s

urpt

lsin

g ~

_tt_

~i" ty

te m

anim

alia

n re

trov

lral

sag

__

l~3~_

, i

nclu

ding

re

gion

s cor

resp

ondi

ng tu

p15

, pl2

mtd

the

N-t

erm

inal

80%

of p

30 b

ut n

o p

l0 s

eque

nce,

p58

als

o co

ntai

ned

a se

quen

ce 0

PPPY

PVPT

APP

) ~t

4thi

n a

long

er p

roli

ne-r

leh

sequ

ence

sim

ilar

to t

hose

fo~n

d in

Sro

and

AIM

SH

3 do

mai

n-bi

ndin

g pr

otei

ns 3

BPI

and

3B

P2.

p58

boun

d to

Sro

SH

3 do

mai

n on

blo

ts w

ith

biot

i93~

late

d SH

3 do

mai

n an

d on

SH

3 do

mai

n-ag

aros

e of

a m

ic~v

illa

r ext

ract

. In

viw

o-tr

ansl

ated

p58

eo-

imm

unop

reci

#ita

ted

wit

h pl

atel

et c

-Src

in

the

abse

nce

but n

ot i

n th

e pr

esen

ce o

f sy

nOet

ic p

epil

de.

The

se r

esul

ts s

ugge

st th

at

p58

is a

n im

port

ant e

lem

ent i

n or

gani

zing

a M

F-as

soci

ated

si~

ml t

rans

duct

ion

part

icle

at t

he c

ytop

lasm

ic

surf

ace

of th

e pl

asm

a m

embr

ane

hi th

e as

clte

s cel

ls.

We

prop

ose

and

are

test

ing

ihe

hypo

thes

is th

at b

indi

ng

of p

58 t

o Sr

c m

ay a

lso

mod

ulat

e Sr

c ac

tivi

ty a

nd s

igns

[ trd

nsdu

ctio

n in

the

tum

or 6

ells

.

1.

C. C

arra

way

and

K.

Car

raw

ay (1

989)

Bio

chim

. Bio

phys

. Act

a B

iom

embr

anes

Rev

iew

s 988

,147

-171

. 2.

K

. C

arra

way

et

al.

(198

0)

Nat

ure

285,

508

-510

. 3.

K

. C

arra

way

et

al.

(197

9)

J. C

ell B

iol.

83,5

29-5

43.

4.

Y.

Liu

et

aI.

(198

9)

J. B

iol.

Che

m.

264,

120

8-1,

214.

5.

C

. C

arra

way

et

al.

(198

3)

Proc

. N

ail.

Aca

d. S

ci.

80,4

30-4

34).

6.

C

. C

arra

way

et

al.

(199

1)

J. B

iol.

Che

m.

266,

162

38-1

6246

. 7.

C

. C

arra

way

et

al.

(199

3)

J. B

iol.

Che

m.

268,

558

2-55

87.

8.

S.-H

. Ju

ang

at a

l.

(199

4) J

. B

iol.

Che

m.,

in p

ress

.

C2

STR

UC

TUR

E C

HAR

ACTE

RIZ

ATIO

N O

F M

EMBR

ANE B

OU

ND

AN

D S

UR

FAC

E AD

SOR

BED

PR

OTE

IN

Mar

tin C

affre

y, J

in W

ang,

Car

mic

hael

J.

A.

Wal

lace

1,

lan

Cla

rk-L

ewis

2 D

ept.

of C

hem

istry

, Th

e O

hio

Sta

te U

nive

rsity

, C

olum

bus,

OH

432

10.

U.S

.A.,

1Dep

t. of

B

ioch

emis

try,

Dal

hous

ie U

nive

rsity

, H

alifa

x, N

ova

Sco

tia,

Can

ada

B3H

4H

7, 2

Bio

med

ical

R

esea

mh

Cen

ter,

Uni

vers

ity o

f B

ritis

h C

olum

bia,

Van

couv

er,

B.C

., C

anad

a V

6T 1

W5.

The

x-ra

y st

andi

ng w

ave

(XS

W)

met

hod

(1-6

) ha

s be

en u

sed

to s

tudy

the

stru

ctur

e an

d to

polo

gy o

f th

e pr

otei

n, c

ytoc

hrom

e c,

bou

nd to

a n

egat

ivel

y ch

arge

d m

odel

lipi

d m

embr

ane

and

adso

rbed

at

a m

etal

sur

face

. A

t the

met

al s

urfa

ce,

cyto

chro

me

c fo

rms

an h

exag

onal

ly

clos

e-pa

cked

mon

olay

er.

A s

imila

r pa

ckin

g ar

rang

emen

t is

obs

erve

d at

the

sur

face

of

a se

lf-as

sem

bled

lip

id m

onol

ayer

on

silv

er.

In t

he c

ase

of a

Lan

gmui

r-B

lodg

ett

(LB

) fil

m,

prot

ein

mul

tilay

ers

are

form

ed.

The

data

sug

gest

tha

t cy

toch

rom

e c

mai

ntai

ns i

ts n

ativ

e gl

obul

ar s

truct

ure

upon

sur

face

bin

ding

an

d su

bseq

uent

sto

rage

for

an

exte

nded

per

iod.

Fu

rther

, th

e da

ta a

re c

onsi

sten

t w

ith a

pro

tein

doc

king

mec

hani

sm w

here

in th

e he

me

plan

e is

orie

nted

per

pend

icul

ar t

o an

d w

ith i

ts e

xpos

ed e

dge

faci

ng t

he s

urfa

ce.

This

stu

dy

dem

onst

rate

s th

e ut

ility

of

XS

Ws

as a

new

and

pow

erfu

l st

ruct

ural

too

l fo

r in

vest

igat

ing

mem

bran

e- a

nd s

urfa

ce-a

ssoc

iate

d pr

otei

ns.

1.

M.J

. B

edzy

k et

al..

Sci

ence

241,

17

88 (

1990

) 2.

M

.J.

Bed

zyk

et a

l., S

cien

ce 2

48,

52 (

1990

) 3.

M

. C

affre

y et

al.,

Far

aday

Dis

cuss

. 94,

In

pres

s (1

993)

4.

J.

Wan

g et

al..

Nat

ure

354,

377

(19

91)

5.

J. W

ang

et a

l.. S

cien

ce 2

58,

775

(199

2)

6.

Jin

Wan

g et

al..

J.

Mol

. B

iol.

In p

ress

.

C1

DIR

EC

T D

ET

ER

MIN

AT

ION

O

F IM

MO

BIL

IZE

D

PR

OT

EIN

CO

NC

EN

TR

AT

ION

.

Pat

rick

L.

Col

eman

& D

anie

l S

arpo

ng.

Bio

scie

nces

Lab

orat

ory.

3M

Co.

St.

Pau

l, M

N

One

of

the

mos

t fu

ndam

enta

l ne

eds

in b

ioch

emic

al r

esea

rch

is k

now

ledg

e of

the

co

ncen

trat

ion

of p

rote

in.

Thi

s is

gen

eral

ly n

ot t

oo d

iffic

ult

whe

n th

e pr

otei

n is

in

solu

tion,

whe

n an

y on

e of

sev

eral

diff

eren

t op

tical

or

chem

ical

met

hods

can

giv

e re

liabl

e re

sults

. In

con

tras

t, w

ork

with

im

mob

ilize

d pr

otei

ns i

s of

ten

ham

pere

d by

in

abili

ty to

acc

urat

ely

dete

rmin

ethe

mas

s of

pro

tein

pre

sent

. M

any

of th

e co

mm

on

supp

ort

mat

eria

ls p

recl

ude

the

use

of o

ptic

al m

etho

ds.

We

desc

ribe

in

this

pos

ter

a di

rect

che

mic

al m

etho

d fo

r th

e de

term

inat

ion

of th

e co

ncen

trat

ion

(den

sity

) of

pro

tein

cou

pled

to

seve

ral t

ypes

of

poro

us m

edia

freq

uent

ly

used

as

supp

orts

for

im

mob

iliza

tions

in a

ffin

ity s

epar

atio

ns a

nd e

nzym

ic c

atal

ysis

. T

he

met

hod

empl

oys

the

read

ily a

vaila

ble

BC

A (

bici

ncho

nini

c ac

id)

whi

ch i

nter

acts

with

th

e C

u+ f

orm

ed b

y re

duct

ion

of C

u2+

in t

he p

rese

nce

of p

rote

in a

mid

e bo

nds

(Ana

l. B

ioch

em.

150,

76,

198

5).

The

rea

ctio

n ta

kes

plac

e at

roo

m t

empe

ratu

re a

nd c

an b

e re

ad in

as

little

as

30 m

in.,

thou

gh m

ore

typi

cally

in 2

h.

The

ass

ay is

sen

sitiv

e to

as

little

as

50 I

lg o

f im

mob

ilize

d pr

otei

n an

d is

gen

eral

ly in

depe

nden

t of

the

am

ount

of

supp

ort

pres

ent.

G

reat

er s

ensi

tivity

is f

easi

ble

but

has

not

yet

prov

en n

eces

sary

. T

he

assa

y ha

s pr

oved

inv

alua

ble

in o

ptim

izin

g th

e ac

tivity

of

imm

obili

zed

prot

ein

ligan

ds

for

affin

ity s

epar

atio

ns,

sinc

e hi

gh c

oupl

ing

effic

ienc

es m

ake

use

of in

dire

ct m

etho

ds

(det

erm

inat

ion

of t

he a

mou

nt o

f pr

otei

n w

hich

has

not

cou

pled

) hi

ghly

ina

ccur

ate.

W

e de

velo

ped

the

met

hod

in c

onju

nctio

n w

ith E

mph

aze T

M B

iosu

ppor

t M

ediu

m,

but

have

de

term

ined

tha

t it

is w

idel

y ap

plic

able

to

mos

t of

the

com

mer

cial

ly a

vaila

ble

affin

ity

supp

orts

. C

3

Page 12

Con

form

atio

nal

Fle

xibi

lity

of

th

e G

ly-G

ly

dipe

ptid

e w

ithi

n pr

otei

n st

ruct

ures

, ti

t

Dav

id W

. Dee

rliel

d 111

Am

anda

Hol

land

-Min

kley

1, Jo

hn D

. Hem

pel 2

and

Hug

h B

. Nic

hola

s Jr.

1

1 .) P

ittsb

urgh

Supe

rcom

putin

g Cen

ter,

4400

Fif

th A

venu

e. P

ittsb

urgh

, PA

152

13

2.) D

epar

tmen

t of M

olec

ular

Gen

etic

s/B

ioch

emis

try, U

nive

rsity

of P

ittsb

urgh

, Pitt

sbur

gh, P

A 1

5261

We e

xam

ined

the

conf

orm

atio

n of d

ipep

tides

segm

ents

from

ove

r 100

pro

tein

s con

tain

ed w

ithin

the

Prot

ein

Dat

a Ban

k as d

efin

ed by

Qia

n an

d Se

jnow

ski (

J. M

ol. B

iol.

1988

, 202

, 865

-884

). W

e in

itial

ly cr

eate

d a

Prot

ein S

truct

ure d

ata b

ase (

PSdb

) ent

ry (i

nclu

ding

mol

ecul

ar su

rfac

e acc

essi

bilit

y, se

cond

ary s

truc

ture

in

form

atio

n and

oth

er de

scrip

tors

) for

each

of t

he p

rote

ins w

ith ea

ch re

sidu

e's c

onfo

rmat

ion c

lass

ified

ac

cord

ing t

o Sc

hera

ga's d

efin

ition

s of p

eptid

e con

form

atio

nal a

ngle

s (M

acro

j~ol

ecul

es 19

83, 1

6, 1

043-

10

49).

We t

hen s

earc

h thr

ough

the P

Sdb e

ntri

es de

term

inin

g the

rela

tive f

requ

ency

for e

ach

of th

e va

riou

s co

nfor

mat

ions

for b

oth m

ono-

and d

ipep

tides

. We

obse

rved

the s

ame g

ener

al pe

ptid

e con

form

atio

na]

tend

enci

es re

porte

d by

Kam

imur

a and

Tak

ahas

hi (C

AB

IO$1

994,

10, 1

63-1

69).

We d

ivid

ed th

e R

araa

chan

dran

plot

for e

ach r

esid

ue in

to h

igh

ener

gy (g

? > 0

) and

low

ener

gy (~

< 0

) are

as w

hich

, for

the

dipe

ptid

e, yi

elds

four

dist

inct

ener

gy qu

adra

nts (

L-L

, H-L

, L-H

, H-H

). T

he "t

ypic

al" d

ipep

tide s

egm

ent (

for

non-

glyc

ine d

ipep

tides

) was

foun

d to

be: L

-L=9

1%, H

-L=4

%, L

-H=4

% an

d H

-H=I

%. F

or G

ly-G

ly, th

ese

num

bers

wer

e: L

-L=2

4%, H

-L=2

7%, L

-H=I

9%, H

-H=3

0%. T

he fr

eque

ncy f

or a

sing

le G

ly re

sidu

e was

H

=53%

and L

=47%

. Thu

s, fo

r the

Gly

-Gly

dipe

pfid

e seg

men

t in p

rote

ins,

the

stat

istic

ally

expe

cted

and

obse

rved

valu

es w

ere f

ound

to b

e sim

iliar

with

in ex

pect

ed er

ror.

Thi

s ind

icat

es th

at th

ere

is n

ot a

syne

rgis

tic

influ

ence

on m

ainc

hain

conf

orm

atio

ns of

Gly

-Gly

segm

ents

; but

rath

er, i

s sh

nply

the p

rodu

ct o

f the

co

nfor

mat

iona

l flex

ibili

ty of

each

resi

due.

D1

A

HIG

HL

Y,

CO

NS

ER

VE

D

N-T

ER

MIN

AL

S

EQ

UE

NC

E

FO

R

TE

LE

OS

T

VIT

EL

LO

GE

NIN

S

Nan

cy D

. D

ensl

ow,

Ler

oy C

. F

olm

ar t,

Cra

ig V

. S

ulli

van 2

, D

ept.

Bio

ehem

. an

d M

ol.

Bio

l.,

Uni

vers

ity

of F

lori

da,

Gai

nesv

ille

, F

L,

tU.S

. E

nvir

onm

enta

l Pro

tect

ion

Age

ncy,

G

ulf

Bre

eze,

FL

, an

d 2D

ept.

Zoo

logy

, N

orth

Car

olin

a St

ate

Uni

vers

ity,

Ral

eigh

, N

C.

N-t

erm

inal

am

ino

acid

seq

uenc

es w

ere

obta

ined

for

vit

ello

geni

n (V

tg)

from

sev

eral

ph

ylog

enet

ieal

ly d

iver

se fi

sh in

clud

ing:

str

iped

bas

s (M

oron

e sax

atili

s), p

infi

sh (L

agod

on

rhom

boid

es),

brow

n bu

llhe

ad (

Amei

urus

neb

ulos

us),

med

aka

(Ory

zias

lat

ipes

), an

d ye

llow

per

ch (

perc

afla

vesc

ens)

. Th

ese

sequ

ence

s ar

e co

mpa

re~

wit

h se

quen

ces

deri

ved

from

eD

NA

of

m

umm

icho

g (F

undu

lus

hete

rocl

itus)

and

st

urge

on

(Aci

pens

er

tran

smon

tanu

s) a

nd w

ith

publ

ishe

d N

-ter

min

al a

min

o ac

id s

eque

nces

fro

m t

he l

ampr

ey

(Ict

hyom

yzon

uni

cusp

is),

claw

ed f

rog

(Xen

opus

lae

vis)

and

dom

esti

c ch

icke

n (G

allu

s do

mes

tica)

. A

seg

men

t bet

wee

n am

ino

acid

s 7

and

20 i

s hi

ghly

sii

nila

r am

ong

the

fish

sp

ecie

s.

Usi

ng

the

stri

ped

bass

se

quen

ce

as

the

tem

plat

e fo

r co

mpa

riso

ns,

the

mum

mic

hog

show

ed 1

00%

ide

ntit

y in

thi

s se

gmen

t,

pinf

ish,

87%

; br

own

bull

head

s,

93%

, w

hite

stu

rgeo

n, 6

0% a

nd s

ilve

r la

mpr

ey 4

7%.

The

yel

low

per

ch a

nd m

edak

a sh

owed

10

0%

iden

tity

wit

h th

e st

ripe

d ba

ss b

etw

een

posi

tion

s 5

and

10.

We

have

m

odel

ed a

pep

tide

to

the

cons

erve

d re

gion

s in

thi

s se

quen

ce a

nd h

ave

used

it t

o m

ake

a po

lycl

onal

ant

ibod

y in

rab

bits

. T

he a

ntib

ody

has

a w

ide

cros

s-re

acti

vity

to v

itel

loge

nin

from

dif

fere

nt s

peci

es a

nd m

ay b

e a

usef

ul d

iagn

osti

c to

ol t

o ex

amin

e vi

tell

ogen

esis

in

fish

.

D2

KA

PID

~FIN

GE

RP

RIN

TIN

G O

F P

RO

TE

INS

WIT

H I

MM

OB

IL~

D

PR

OT

EA

SE

CO

LU

MN

S

Jam

es D

. D

ixon

, Jo

nath

an P

. M

ark,

Chr

isto

pher

P. E

lico

ne,

Sim

in D

. M

alek

nia,

Bri

an F

. M

cGui

nnes

s, F

red

E.

Reg

nier

and

Nou

bar

B.

Afe

yan

Per

Sep

tive

Bio

syst

ems,

Inc.

38

Sid

ney

Stre

et,

Cam

brid

ge, M

A 0

2139

We

desc

ribe

an

appr

oach

uti

lizi

ng ir

mno

bilD

ed p

rote

ase'

s fo

r ra

pidl

y ge

nera

ting

repr

oduc

ible

pr

oteo

lyti

c di

gest

s of

pro

tein

s. T

hese

pro

teol

ytic

dig

ests

are

then

ana

lyze

d us

ing

high

per

form

ance

li

quid

chr

omat

ogra

phy

(HP

LC

) or

mas

s sp

ectr

omet

ric

(MS

) te

chni

ques

to f

inge

rpri

nt th

e pr

otei

ns.

The

pro

cedu

re h

as b

een

auto

mat

ed u

sing

a m

ulti

-col

umn

swit

chin

g de

vice

to s

elec

tive

ly p

lace

in-

line

a v

arie

ty o

fim

mob

iliT

ed p

rote

ase

colu

mns

. Im

mob

iliz

ed p

rote

ase

colu

mns

con

tain

ing

tryp

sin,

ct

-chy

mot

ryps

in, c

ucum

isin

, pap

ain,

pep

sin,

and

end

opro

tein

ase

Lys

-C h

ave

been

eva

luat

ed i

n th

is

conf

igur

atio

n. T

he p

rote

olyt

ic d

iges

tion

pro

duct

s ar

e di

rect

ly c

aptu

red

on a

C-1

8 re

vers

e-ph

ase

colu

mn

for

a su

bseq

uent

HP

LC

pep

tide

map

ping

ste

p.

The

imm

obil

ized

pre

tens

e co

lum

ns a

re

stab

le to

cha

otro

pic

solu

tion

s su

ch a

s 3.

0 M

gua

nidi

ne h

ydro

chlo

ride

or

4.0

M u

rea.

At

a te

mpe

ratu

re o

f 60

oc,

com

plet

e di

gest

s o

f pr

otei

ns a

re g

ener

ated

in

unde

r on

e m

inut

e w

ith

the

imm

obil

ized

pre

tens

e co

!um

ns.

Fur

ther

mor

e, p

assi

ve s

ampl

e lo

sses

due

to a

dsor

ptio

n to

su

rfac

es d

urin

g m

anua

l sam

ple

man

ipul

atio

ns a

re r

educ

ed b

y us

ing

the

tand

em c

olum

n ca

ptur

e ap

proa

ch.

Ove

r on

e th

ousa

nd s

ampl

e in

ject

ions

hav

e be

en l

ogge

d on

a s

ingl

e co

lum

n ov

er a

pe

riod

of

seve

ral w

eeks

.

Rap

id,

repr

oduc

ible

pro

teol

ytic

dig

ests

of

prot

eins

are

obt

aine

d us

ing

thes

e im

mob

iliz

ed p

rote

ase

colu

mns

. T

he d

iges

ts a

re s

uita

ble

for

use

in c

ompa

rati

ve p

epti

de m

appi

ng a

ppli

cati

ons

or f

or M

S

anal

yses

to g

ener

ate

mas

s m

aps

for

data

base

sea

rchi

ng.

D3

PR

OTE

IN: F

AR

NE

SY

L TR

AN

SFE

RA

SE

. E

VID

EN

CE

FO

R A

N E

LEC

TRO

PH

ILIC

S

UB

STI

TUTI

ON

ME

CH

AN

ISM

.

Julia

M.

Dol

ence

and

C.

Dal

e P

oulte

r. D

epar

tmen

t o~

Che

mis

try, U

nive

rsity

of U

tah.

Pro

tein

pre

nyl t

rans

fera

ses

cata

lyze

the

post

tran

slat

iona

l m

odifi

catio

n of

a w

ide

varie

ty o

f pro

tein

s, in

clud

ing

fung

al m

atin

g fa

ctor

s, n

ucle

ar la

min

s, a

nd R

as.

Pro

tein

: fa

rnes

yl tr

ansf

eras

e (P

FTas

e) c

atal

yzes

the

trans

fer

of a

farn

esyl

gro

up to

a c

yste

ine

resi

due

form

ing

a th

ioet

her b

ond.

A

ser

ies

of fa

rnes

yl d

ipho

spha

te (F

PP

) an

alog

s co

ntai

ning

fluo

rom

ethy

l, tri

fluor

omet

hyl,

and

hydr

ogen

at t

he 3

' pos

ition

wer

e sy

nthe

size

d an

d us

ed a

s al

tern

ate

subs

trate

s an

d in

hibJ

tors

. M

axim

um v

eloc

ities

and

M

icha

elis

con

stan

ts w

ere

mea

sure

d, a

nd th

e re

sults

sup

port

an e

lect

roph

ilic

alky

latio

n m

echa

nism

, si

mila

r to

oth

er p

reny

l enz

ymes

invo

lvin

g al

kyla

tions

of c

arbo

n-ca

rbon

do

uble

bon

ds.

Res

ults

from

thi

s st

udy

may

be

exte

nded

to u

nder

stan

ding

the

m

echa

nism

of o

ther

pre

nyl t

rans

fera

se e

nzym

es.

D4

Page 13

DIS

UL

PH

ID. E

M

AP

PIN

G

OF

PR

OL

AM

INS

Ts

ez

i E

g~

rov

, A

lex

an

de

r M

us

~ly

am

ov

, Y

ve

s

Po

pin

ea

u I

,

Je

ns

A

nd

ers

en

,

Pe

ter

Ro

ep

sto

rff

An

aly

tic

al

Pro

tein

C

he

mis

try

R

es

ea

rch

G

rou

p,

En

ge

l~a

rdt

Ins

t.

of

Mo

lec

ula

r B

iolo

gy

R

us

sia

n

Ac

ad

o

f S

ci.

, R

us

sia

, 2

-1N

RA

, L

ab

. o

f B

ioc

he

mis

try

a

nd

T

ec

hn

olo

gy

o

f P

rote

ins

, F

ran

ce

, P

rote

in

Re

se

arc

h

Gro

up

, D

ep

t.

of

Mo

lec

ula

r B

iolo

gy

, O

de

ns

e

Un

iv.,

D

en

ma

rk

Pro

lam

ins

, th

e

ma

jor

ce

rea

l s

ee

d

sto

rag

e

pro

tein

s,

co

mp

ris

e

a

fam

ily

of

h

ete

rog

en

eo

us

p

rote

ins

w

ith

co

mm

on

p

rop

ert

ies

, a

mo

un

d

wh

ich

s

olu

bil

ity

in

a

q.

alc

oh

ol

an

d

hig

h

glu

tam

in

an

d

pro

lin

c

on

ten

ts.

Th

e

pu

rpo

se

o

f th

is

stu

dy

Is

th

e

ide

nti

fic

ati

on

o

f in

tra

- a

nd

in

ter-

c

ha

in

dis

ulp

hid

e

bo

nd

s

of

wh

ea

t p

rola

min

s

mo

no

me

ric

g

lla

din

s

, a

s

we

ll

as

of

oa

t o

ne

s

(av

en

ins

).

Th

e

co

va

len

t c

hro

ma

tog

rap

hy

,

HP

LC

, g

el

ele

ctr

op

ho

res

is,

mlc

ros

eq

ue

n-

cin

g

an

d

ma

ss

sp

ec

tro

me

try

w

ere

u

se

d

in

this

s

tud

y

for

pro

tein

p

uri

fic

ati

on

a

nd

d

isu

lph

ide

m

ap

pin

g

of

pro

lam

ins

. A

s

a

res

ult

, s

ev

era

l p

ure

p

rola

min

c

om

po

ne

nts

h

av

e

be

en

is

ola

ted

a

nd

c

ha

rac

teri

ze

d

(1).

T

he

e

xa

ct

nu

mb

er

of

cyst

ein

e re

sid

ue

s

In

pro

lam

lns

w

as

de

term

ine

d

by

ES

MS

b

efo

re

an

d

aft

er

red

uc

tio

n

an

d

alk

yla

tio

n.

All

S

-S-b

on

ds

o

f a

ve

nin

-3

(2)

an

d

~-4

B

gli

ad

in

we

re

ide

nti

fie

d.

I.

T.

Eg

oro

v

et

al~

, J.

Ce

rea

l.

Sc

i.

(ac

ce

pte

d).

2.

T.

E

go

rov

e

t a

l.,

Eu

r.

J.

Bio

ch

em

, (a

cc

ep

ted

).

MA

PP

ING

AN

D C

HA

RA

CT

ER

IZA

TIO

N O

F P

RO

TE

INS

AS

SO

CIA

TE

D W

ITH

M

OR

PH

OL

OG

ICA

L T

RA

NS

FO

RM

AT

ION

IN

TH

E S

HE

CE

LL

AS

SA

Y.

D. L

. G

augg

el, T

. N

. Asq

uith

, R. J

. Isf

ort,

N.

S. M

ille

r an

d D

. B.

Cod

y C

orpo

rate

Pro

fess

iona

l & R

egul

ator

y Se

rvic

es/H

uman

Saf

ety

Dep

t., T

he P

roct

er &

G

ambl

e C

o., P

.O.

Box

398

707,

Cin

cinn

ati,

Ohi

o, 4

5239

-870

7.

The

Syr

ian

Ham

ster

Em

bryo

(SH

E)

Cel

l Ass

ay is

a g

ood

scre

en fo

r pot

enti

al

carc

inog

enic

act

ivit

y of

che

mic

als.

Und

er re

duce

d pH

cel

l cul

ture

con

diti

ons,

it d

etec

ts

both

gen

otox

ic a

nd n

onge

noto

xic

carc

inog

ens

wit

h an

ove

rall

con

cord

ance

of

86%

whe

n co

mpa

red

to c

hem

ical

s te

sted

in a

nim

al m

odel

s. C

hang

es in

cel

lula

r mor

phol

ogy

are

used

to a

sses

s ca

rcin

ogen

ic a

ctiv

ity.

Pro

tein

s fr

om n

orm

al a

nd tr

ansf

orm

ed c

ells

wer

e m

appe

d by

2D

PA

GE

and

vis

uali

zed

by C

oom

assi

e B

lue

R25

0 st

aini

ng o

r 35

S/an

tora

diog

raph

y. P

rote

ins

of in

tere

st w

ere

iden

tifi

ed b

y E

dman

seq

uenc

ing

or b

y co

mpa

riso

n ag

ains

t pub

lish

ed 2

D m

aps.

Pro

tein

dis

ulfi

de is

omer

ase

(PD

I) w

as i

dent

ifie

d as

a p

oten

tial

mar

ker

for

tran

sfor

mat

ion.

Pro

tein

s w

hich

cop

reci

pita

ted

wit

h PD

I w

ere

also

iden

tifi

ed a

nd c

hara

cter

ized

. T

hese

resu

lts

wil

l be

disc

usse

d w

ithi

n th

e co

ntex

t of

SHE

cel

l tra

nsfo

rmat

ibn.

> >

E1

CQNFORNATIONAL COa~PJ~RXSON IN THE SN~KB TOXXN FAMILY

Roberto J. Falkensteln, Mirtha J. Biscoglio de Jim~nez Bonino and Clara Pefla

Instltuto de Qulmica y Fisicoqu~mlca Biol6gicas

(UBA-CONICET),

Facultad de

Farmacia y Bioqu~mlca, Jun~n 956, 1113 Buenos Aires, Argentina

The aim of this work is to perform a theoretical study of the conformational

homology between several members of the polypeptldlc snake toxin family. In

spite of displaying a common three-dlmensional folding pattern - a compact

core rich in disulfide bridges, three loops and a triple stranded ~-sheet -

show various pharmacological activities.

We applied the method of Kubota et al.

[Biochim. Biophys. Acta, 701, 242-252,

(1982) ].

Consensus sequences were inferred on the basis of function, genus and

sequence homology. The method establishes the relationship between an amino

acid sequence and its native conformation in terms of correlation coefficients

<c(i,j)> and identifies homologous sequences which have homologous native

conformations.

When long ~-neurotoxins,

binding to nicotinic acetylcholine receptor,

were

compared, a high variability in the N-terminal loop was detected. On the other

hand, a high homology degree between the consensus sequences from genera NaJa

and Ophiophagus,

and Dendroaspls was observed even when a C(i,j)=0.8 was

selected. The same conclusion applies to comparisons between genera Astrotia

and Pelamls, and Bungarus. However,

some conformational differences

around

position 30 appear when the consensus

sequence from genus Lauticauda

is

included in the matrix.

A high homology degree

[C(i,j)=0.8]

was observed also in the comparison

between sequences from most members of citotoxln subgroups.

An unexpected high conformational similarity was found between k-neurotoxins

- binding to the neuronal nicotinic receptor - and long u-neurotoxins.

In

contrast,

long and short u-neurotoxins

sharing the same target, display a

lower similarity degree.

Summarizing, Kubota'smethod allowed us to obtain important conclusions about

conformational homology in the snake toxin family, even though many of its

members have not yet been crystallized.

F]

GI aOLUTION STRUCTURE OF A CYCLIC ~-NSHANALOCUE-INITIALI~4RSTODIES

Michael F. Giblin I,

Tuck C. Wong 2 and~Thomas P. Quinn I,

Departments

of iBiochemfstry and 2Chemistry, University of Missouri, Columbla,MO 65211

x-Melanocyte Stimulating Hormone (~-MSH) is a 13 amino acid peptlde hormone

that stimulates melanin biosynthesis in vertebrates.

Cyclic analogues of

~-MSH have been designed which exhibit increased bloactlvity in vitro~

One

of these analogues is a disulfide cycllzed decapeptide which includes a D-Phe

replacement at position 4.

It has been hypothesized that a reverse turn

could be important for hormone activity due to the superpotency of such

cyclic analogues.

In the present study,th~ solution structure of this substituted analogue

is being investigated using NMR techniques.

NeE measurements in 80%H20/20%D~O

indicate a relatively constrained region between residues 3-5 of the peptide~

with considerably more conformational freedom around the disulfide bridge and

in the three residue tail.

Temperature dependent chemical shift studies of

the ~mide protons indicate no involvement in intramolecular hydrogen bonds,

and =J~NH measurements coupled with the pattern of sequential and non-

sequential NOE's do not conform to the classical picture of the~-turn.

The solvent 2,2,2-Trifluoroethanol-d 3 (TFE) has been postulated to act

as a membrane mimic, and is known to favor ~-hellx formation in linear

peptides.

NeE data obtained in 70% TFE/30% H20 is currently being compared

with the earlier data to determine whether this solvent mixture has an

ordering effect on the peptide.

G2

Page 14

IDE

NT

IFIC

AT

ION

O

F

MO

DIF

IED

PT

H-A

MIN

O

AC

IDS

IN P

RO

TE

IN

SEQ

UE

NC

E

AN

AL

YSI

S G

rego

ry A

. G

rant

and

Mar

k W

. Cra

nksh

aw

Ass

ocia

tion

of

Bio

mol

ecul

ar

Res

ourc

e F

acil

itie

s an

d W

ashi

ngto

n U

nive

rsit

y Sc

hool

of

M

edic

ine,

St.

Lou

is,

MO

631

10.

The

ide

ntif

icat

ion

of

amin

o ac

id r

esid

ues

in m

oder

n pr

otei

n se

quen

ce

anal

ysis

em

ploy

ing

auto

mat

ed E

dman

deg

rada

tion

is

depe

nden

t on

the

elu

tion

pos

itio

n of

the

PT

H-

amin

o ac

ids

on h

igh

pres

sure

liq

uid

chro

mat

ogra

phy

syst

ems.

T

his

me

tho

d

reli

es o

n

a co

mpa

riso

n of

the

mig

rati

on o

f th

e un

know

n P

TH

-am

ino

acid

wit

h th

at o

f re

fere

nce

stan

dard

s.

Thi

s is

rel

ativ

ely

stra

ight

forw

ard

for

the

gene

tica

lly

code

d am

ino

acid

s,

but

beco

mes

pr

oble

mat

ic w

hen

mod

ifie

d or

unu

sual

am

ino

acid

res

idue

s ar

e pr

esen

t in

the

sam

ple

bein

g se

quen

ced.

Sin

ce t

he m

etho

d do

es n

ot p

rovi

de a

dir

ect

iden

tifi

cati

on o

f th

e P

TH

-am

ino

acid

, ad

diti

onal

ana

lysi

s by

che

mic

al o

r ph

ysic

al m

eans

is

nece

ssar

y. H

owev

er,

it is

oft

en h

elpf

ul t

o ha

ve s

ome

know

ledg

e of

whe

re k

now

n m

odif

ied

PT

H-a

min

o ac

ids

elut

e in

the

se s

yste

ms,

Thi

s pr

ovid

es a

sta

rtin

g po

int

for

the

inve

stig

ator

and

pro

vide

s an

add

itio

nal

leve

l of

kno

wle

dge

upon

whi

ch t

o pr

ocee

d.

A c

ompi

lati

on o

f un

usua

l PT

H-a

min

o ac

ids

has

been

und

erta

ken

by t

he A

ssoc

iati

on o

f B

iom

olec

ular

Res

ourc

e Fa

cilit

ies

(AB

RF

) in

an

atte

mpt

to

cons

olid

ate

info

rmat

ion

of t

his

type

fo

r ea

sy r

efer

ence

. It

sho

uld

be n

oted

tha

t an

exh

aust

ive

revi

ew o

f th

e lit

erat

ure

has

not

been

at

tem

pted

and

it h

as n

ot b

een

poss

ible

to

inde

pend

entl

y ve

rify

the

info

rmat

ion

pres

ente

d he

re.

Rat

her,

mem

bers

of

the

AB

RF

wer

e as

ked

to s

ubm

it a

ny in

form

atio

n th

ey h

ad in

thi

s re

gard

for

incl

usio

n in

thi

s bo

okle

t. W

hen

liter

atur

e re

fere

nces

hav

e be

en p

rovi

ded

they

are

inc

lude

d~

The

refo

re,

this

inf

orm

atio

n is

int

ende

d to

be

used

onl

y as

a g

uide

. A

ddit

iona

l su

ppor

ting

an

alys

es s

houl

d be

em

ploy

ed t

o ve

rify

the

iden

tity

of

any

unko

wn

resi

due.

Thi

s co

mpi

lati

on i

s in

tend

ed f

or u

se b

y th

e en

tire

sci

enti

fic

com

mun

ity

and

is a

vail

able

to

any

one

who

is

inte

rest

ed.

G3

CH

AR

AC

TER

IZA

TIO

N

OF

RE

CO

MB

INA

NT

PO

RIN

BY

MA

SS

SP

EC

TRO

ME

TRY

F. G

uine

t 1 , Y

. Pe

tilto

t 2, J

.M.

Cha

psal

3,

J. D

ubay

le I

, F.

Gre

co 1

, O.

Bar

ge 3,

E.

Fore

st 2,

C

. Va

lent

in I

Mas

s m

easu

rem

ent

of a

re

com

bina

nt

pori

n of

M

r =

4547

0 w

as

perf

orm

ed

on

an

elec

tros

pray

mas

s sp

ectr

omet

er (

Perk

in E

lmer

Sci

ex A

PIII)

. Th

is m

embr

ane

prot

ein

was

po

orly

sol

uble

in

solv

ents

and

buf

fers

(cl

assi

caly

use

d fo

r m

ass

spec

trom

etry

), an

d w

e pe

rfor

med

the

ana

lysi

s in

pur

e fo

rmic

aci

d. P

erfo

rman

t sp

ectr

a w

ere

obta

ined

, ho

wev

er

we

obse

rved

a r

apid

evo

lutio

n of

the

spe

ctra

und

er s

tora

ge i

n fo

rmic

aci

d. T

he i

ncre

ase

of m

ass

obse

rved

cou

ld b

e du

e to

for

myl

atio

ns.

Oth

er c

ondi

tions

of

solu

biliz

atio

n ha

ve

been

set

whi

ch g

aran

tee

any

poly

pept

ide

chan

ge.

Usi

ng t

his

tech

niqu

e w

e an

alys

ed

seve

ral

batc

hes

of th

is p

rote

in,

and

we

saw

mas

s m

odifi

catio

ns f

or c

erta

in p

repa

ratio

ns.

Ana

lysi

s (b

y se

quen

cing

an

d m

ass

dete

rmin

atio

n)

of

the

pept

ide

map

of

th

ose

poly

pept

ides

(e

nzym

atic

fin

qer

prin

ting)

al

low

ed

us t

o ge

t a

bett

er u

nder

stan

ding

of

th

ose

mod

ifica

tions

mai

nly

due

to C

-ter

min

al d

egra

datio

n.

1 R

esea

rch

and

Dev

elop

men

t D

epar

tmen

t/Pr

otei

n Q

ualit

y C

ontr

ol

Pas

teur

Med

eux

S~ru

ms

de V

acci

ns,

Fran

ce

2 La

bora

toir

e de

Spe

ctro

met

rie

de M

asse

des

Pro

tein

es

Inst

itut d

e B

iolo

gie

Stru

ctur

ale,

Gre

nobl

e, F

ranc

e 3

Res

earc

h an

d D

evel

opm

ent

Depa

rtmen

t~Ba

cter

iolo

gy

Pas

teur

Mer

ieux

Ser

ums

de V

acci

ns,

Fran

ce

G5

t~

CRYSTALLIZATION OF A THERMALSTABLETRYPSIN I~IBITORFROMP~SEOLUS

LUNATUS

Scott Griffith, Steve Schroeder, Thomas Quinn

Department of Biochemistry, University of Missouri, Columbia, MO 65211

In contrast to all other known Kunitz-type trypsin inhibitors, lima beans

contain a Kunitz-type trypsin inhibitor which can be subjected to high

temperatures for a significant time without destroying its ability to

actigely inhibit trypsin (i). Due to this attribute, Lima Bean Trypsin

Inhibitor (LBTI) has been made the subject of a structural investigation

as a m

odel of protein thermal stability.

Crude LBTI extract was obtained

and purified by an initial heat treatment followed by affinity chromatography.

The thermal stability of LBTI was examined by a trypsin activity assay.

Pure

LBTI and Soybean Trypsin Inhibitor (STI) were heated from 85~

to 92~

over

15 m

inutes.

The LBTI retained its activity while STI lost its ability to

actively inhibit trypsin.

Circular Diehroism thermal melt data suggests

similar LBTI secondary structure at 25 ~ and 80~

Two crystallization

conditions identified by sparse-matrix sampling were optimized, yielding

cubic crystals with dimensions of 0.5 mm.

Initial diffraction studies

have shown diffraction to a resolution of 2.7 Angstroms.

Future investigation

will reveal the tertiary structure of this protein and provide insight on

the physical nature of LBTI thermal stability.

Supported by Hughes

Undergraduate Research Internship.

Reference:

Tauber, H., Kershaw, B.B., and Wright R.D. (1949) J. Biol. Chem. 17___99:1155

G4

Insights into the Structure and Active-Site Architecture of IPP:DMAPP

Isomerase

Frederick M Hahn, Jonathan A. Baker and C. Dale Poulter,

University of

Utah

Genetic and biochemical methods have allowed for the further

characterization

of the enzymic structural elements that facilitate

the essential interconversion

of isopentenyl diphosphate and

dimethylallyl

diphosphate,

Isolation of the S. pombe isomerase gene

was performed by a plasmid shuffle mediated complementation strategy.

Amino acid sequence alignment from three isomerase sources allowed for

the identification of possible active sight regions in the proteins

primary structure and identified 52 n-terminal amino acids of the S.

cerevisiae protein as catalytically non-essential.

The lack of

catalytic importance for these n-terminal amino acids was confirmed by

complementation analysis with a truncated S. cerevisaie isomerase

gene.

A high yield overexpression

and purification protocol has been

developed for recombinant wild type and mutant S. pombe isomerases

that has greatly facilitated the crystallization

of the enzyme for x-

ray crystallographic

analysis.

Two amino acids that are beleived to

be important in the protonation/deprotonation

steps of the enzymatic

reaction have been mutated and characterized.

These studies support

the putative roles of Cys87 and Glu152 as important catalytic

components of the S. pombe isomerase active site.

HI

rj~

> > P'

I