- Email: [email protected]
Protein engineering of α-crystallin
Thursday, Sep 24, 1992 Palazzo Dei Congressi/B
X ICER Abstracts
554
7 PROTEIN Merck.
ENGINEERING K.B.. de &
de Jona. Department Nijmegen,
Ii.
W.W. of Biochemistry, The Netherlands.
the
exact
weight for
arrangement
get insight into aggregate formation, terminal sequences
of the
of
800,000. quaternary
of subunits
interactions we expressed of aA-crystallin, site-directed mutants. Aggregate truncated and mutated chains has of gel permeation chromatography.
suggest
hydrophobic
terminal
that
the
aggregate
aggregates.
dimers
Some initiating studies on the directed (LA mutants have been be presented.
FOLDING EXPRESSED
AND STABILITY IN ESCHERICHIA
by
stability done. The
we chose
C-
of the results
OF RECOMBINANT COLI
for constitutive
cdi.
CRYSTALLINS 31,
yll-crystaltin compemaW the low intrinsic atabitity domain at acidic pH. The role of the connecting
1) 2) 3) 4)
of the domaina will be discussed. (3) and c-cryatallh
and the aaaociation Further studies (4) of duck lens
of the C-terminal peptide for the behaviour of the whole $I on recombinant rat DBIIexpressed in E.co/i will be
Wiatow. G.J. et al., J. Mol. Biol. m 175-202 (1983) Rudolph, R. et al., Proc. Natl. Acad. Sci. USA Bz, 4625-2629 Aarta, H.J. el al., Eur.J. B&hem. m 31-36 (1989) Hendriks, W. et al., Proc. Natl. Acad. Sci. USA &, 7114-7116
(1990) (1988)
553
6 FUNCTIONALLY IMPORTANT POINT MUTATIONS GAPClA CRYSTALLINS OF %"MALS Chirgadze, Yu.N. Institute of Protein Research, Academy Russia, 142292 Pushchino, MOSCOW Region,
IN
THE
of
Sciences RUSSIA
Genetics, Baylor College of Medicine,
AVIAN
6
Houston, TX
expression
was employed.
In this system
the 62 crystallins
a bacterial
expression
system based
on the PET vector
system.
Both
Both
proteins
could
be expressed
at greater
than 30%
of the soluble
protein in extracts and the 82 protein was enzymatically active as expected. Analysis of the kinetic properties of the 62 protein revealed a K,,, of 0.35 for argininosuccinate and a V, of 3.47 units/mg. The availability of an active and an inactive protein with nearly identical amino acid sequence can reveal a great deal about amino acid residues critical to the catalytic function of the molecule. We have used this system and site directed mutation to evaluate the role of various amino acid residues on the active site of the enzyme. Our initial studies have focussed on the role of histidine in the catalytic process.
sitewill
aatra6e
mteracUon molecule cryatatlin presented.
OF
61 and 62 cDNAs were inserted into the PET-3d vector and expressed in E.
no
but
PROPERTIES
complemented the arg4 mutation in S. cerevisiae whereas the 61 crystallins failed to support growth of this mutant strain in arginine deficient medium. Although these data demonstrated that the 62 cry&&in was an enzymatically active protein we were unable to obtain a significant level of production of the protein in yeast. To allow for the expression of large amounts of the protein
the
hydrophylic
tetramers,
ENZYMATIC
We have utilized both yeast and bacterial expression systems to evaluate. the enzymatic properties of the 61 and 62 crystallins from both the chicken and duck. A yeast expression system using the alcoholdehydrogenase promoter
by means
observations
caused
THE
Institute for Molecular 11030, USA.
from
studied Our
The
or
Ni jmegen,
cr-crystallin
is
domain.
and
aggregates with an Several models structure, but is still unclear. To responsible for in E.coli, N-and Caa well as some formation of the
been
formation
N-terminal domain forms
of
University
The 20 kDa aA and aB subunits mammalian eye lenses form large average molecular have been proposed
EXPLORING CRYSTALLINS
OF U-CRYSTALLIN
EYE LENS of
Twenty eye lens gamma-crystallin sequences belonqinq to four farcilies from mouse, rat, calf and human have been analyzed. Conservative amino acid residues in the sequence of mammals were shown to be responsible for the existence of a unique two-domain molecular structure (~01. Biol., USSR, 21, 310, 1987). Among variable residues those which are related to hydrophobic-hydrophilic point mutations were assiqned to the significant variants. They could essentially determine intermolecular surface interactions. We considered the known crystal packing of calf eye lens gamma-crystallins of B-, C- and E-types, that is the fractions II, IIIb and 1va, respectively. It has been found that in crystal medium the intermolecular contact reqions include significant variants in positions 23 and 27 for the N-domain, and 101, 103 and 115 for the C-domain of the molecule. Hydrophobic-hydrophilic patterns of these residues show remarkable specificity for different family types of eye lens gamma-crystallins, i.e. for 8A. dB, FC, .fD, dE and SF. Such point mutations in different qene products of gamma-crystallins suqqest different types of surface interactions and, consequently, different intermolecular associations which occur in native lens medium.
S.165
X ICER Abstracts
554
7 PROTEIN Merck.
ENGINEERING K.B.. de &
de Jona. Department Nijmegen,
Ii.
W.W. of Biochemistry, The Netherlands.
the
exact
weight for
arrangement
get insight into aggregate formation, terminal sequences
of the
of
800,000. quaternary
of subunits
interactions we expressed of aA-crystallin, site-directed mutants. Aggregate truncated and mutated chains has of gel permeation chromatography.
suggest
hydrophobic
terminal
that
the
aggregate
aggregates.
dimers
Some initiating studies on the directed (LA mutants have been be presented.
FOLDING EXPRESSED
AND STABILITY IN ESCHERICHIA
by
stability done. The
we chose
C-
of the results
OF RECOMBINANT COLI
for constitutive
cdi.
CRYSTALLINS 31,
yll-crystaltin compemaW the low intrinsic atabitity domain at acidic pH. The role of the connecting
1) 2) 3) 4)
of the domaina will be discussed. (3) and c-cryatallh
and the aaaociation Further studies (4) of duck lens
of the C-terminal peptide for the behaviour of the whole $I on recombinant rat DBIIexpressed in E.co/i will be
Wiatow. G.J. et al., J. Mol. Biol. m 175-202 (1983) Rudolph, R. et al., Proc. Natl. Acad. Sci. USA Bz, 4625-2629 Aarta, H.J. el al., Eur.J. B&hem. m 31-36 (1989) Hendriks, W. et al., Proc. Natl. Acad. Sci. USA &, 7114-7116
(1990) (1988)
553
6 FUNCTIONALLY IMPORTANT POINT MUTATIONS GAPClA CRYSTALLINS OF %"MALS Chirgadze, Yu.N. Institute of Protein Research, Academy Russia, 142292 Pushchino, MOSCOW Region,
IN
THE
of
Sciences RUSSIA
Genetics, Baylor College of Medicine,
AVIAN
6
Houston, TX
expression
was employed.
In this system
the 62 crystallins
a bacterial
expression
system based
on the PET vector
system.
Both
Both
proteins
could
be expressed
at greater
than 30%
of the soluble
protein in extracts and the 82 protein was enzymatically active as expected. Analysis of the kinetic properties of the 62 protein revealed a K,,, of 0.35 for argininosuccinate and a V, of 3.47 units/mg. The availability of an active and an inactive protein with nearly identical amino acid sequence can reveal a great deal about amino acid residues critical to the catalytic function of the molecule. We have used this system and site directed mutation to evaluate the role of various amino acid residues on the active site of the enzyme. Our initial studies have focussed on the role of histidine in the catalytic process.
sitewill
aatra6e
mteracUon molecule cryatatlin presented.
OF
61 and 62 cDNAs were inserted into the PET-3d vector and expressed in E.
no
but
PROPERTIES
complemented the arg4 mutation in S. cerevisiae whereas the 61 crystallins failed to support growth of this mutant strain in arginine deficient medium. Although these data demonstrated that the 62 cry&&in was an enzymatically active protein we were unable to obtain a significant level of production of the protein in yeast. To allow for the expression of large amounts of the protein
the
hydrophylic
tetramers,
ENZYMATIC
We have utilized both yeast and bacterial expression systems to evaluate. the enzymatic properties of the 61 and 62 crystallins from both the chicken and duck. A yeast expression system using the alcoholdehydrogenase promoter
by means
observations
caused
THE
Institute for Molecular 11030, USA.
from
studied Our
The
or
Ni jmegen,
cr-crystallin
is
domain.
and
aggregates with an Several models structure, but is still unclear. To responsible for in E.coli, N-and Caa well as some formation of the
been
formation
N-terminal domain forms
of
University
The 20 kDa aA and aB subunits mammalian eye lenses form large average molecular have been proposed
EXPLORING CRYSTALLINS
OF U-CRYSTALLIN
EYE LENS of
Twenty eye lens gamma-crystallin sequences belonqinq to four farcilies from mouse, rat, calf and human have been analyzed. Conservative amino acid residues in the sequence of mammals were shown to be responsible for the existence of a unique two-domain molecular structure (~01. Biol., USSR, 21, 310, 1987). Among variable residues those which are related to hydrophobic-hydrophilic point mutations were assiqned to the significant variants. They could essentially determine intermolecular surface interactions. We considered the known crystal packing of calf eye lens gamma-crystallins of B-, C- and E-types, that is the fractions II, IIIb and 1va, respectively. It has been found that in crystal medium the intermolecular contact reqions include significant variants in positions 23 and 27 for the N-domain, and 101, 103 and 115 for the C-domain of the molecule. Hydrophobic-hydrophilic patterns of these residues show remarkable specificity for different family types of eye lens gamma-crystallins, i.e. for 8A. dB, FC, .fD, dE and SF. Such point mutations in different qene products of gamma-crystallins suqqest different types of surface interactions and, consequently, different intermolecular associations which occur in native lens medium.
S.165