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WSN: forthcoming papers in Protein Science (vol.3, #10)
(from URL: gopher://orion.oac.uci.edu/protein/)
AU - Blow DM
AU - Chayen NE
AU - Lloyd LF
AU - Saridakis E
TI - Control of nucleation of protein crystals
AD - L.F. Lloyd, Blackett Laboratory, Imperial College, London
SW7 2BZ, UK; or e-mail: d.blow@ic.ac.uk.
AB - Control of nucleation may be needed to obtain a reliable
supply of large protein crystals, when standard
techniques give many small or twinned crystals.
Heterogeneous nucleation may be controlled by the use
of fine filters, with the elimination of airborne
contaminants by working under paraffin oil. The area of
contact with the supporting vessel also has an
important effect. A heterogeneous nucleant for lysozyme
(identified earlier) has been shown to be effective for
carboxypeptidase G_2. Control of homogeneous nucleation
(previously demonstrated by dilutions of a nucleating
sample after various times of incubation) may also be
achieved by incubating a sample at 1 temperature, where
nucleation can occur, and changing the temperature to
conditions where there is growth but no nucleation.
SO - Protein Science 1994;3:1638-1643
AU - Rossmann MG
TI - Viral cell recognition and entry
AD - Michael G. Rossmann, Department of Biological Sciences,
Purdue University, West Lafayette, Indiana 47907-1392; e
-mail: b4p@mace.cc.purdue.edu.
AB - Rhinovirus infection is initiated by the recognition of a
specific cell-surface receptor. The major group of
rhinovirus serotypes attach to intercellular adhesion
molecule-1 (ICAM-1). The attachment process initiates a
series of conformational changes resulting in the loss
of genomic RNA from the virion. X-ray crystallography
and sequence comparisons suggested that a deep crevice
or canyon is the site on the virus recognized by the
cellular receptor molecule. This has now been verified
by electron microscopy of human rhinovirus 14 (HRV14)
and HRV16 complexed with a soluble component of ICAM-1.
A hydrophobic pocket underneath the canyon is the site
of binding of various hydrophobic drug compounds that
can inhibit attachment and uncoating. This pocket is
also associated with an unidentified, possibly cellular
in origin, ``pocket factor.'' The pocket factor binding
site overlaps the binding site of the receptor. It is
suggested that competition between the pocket factor
and receptor regulates the conformational changes
required for the initiation of the entry of the genomic
RNA into the cell.
SO - Protein Science 1994;3:1712-1725
AU - Chao H
AU - Sonnichsen FD
AU - Deluca CI
AU - Sykes BD
AU - Davies PL
TI - Structure-function relationship in the globular type III
antifreeze protein: Identification of a cluster of
surface residues required for binding to ice
AD - Peter L. Davies, Department of Biochemistry, Queen's
University, Kingston, Ontario K7L 3N6, Canada.
AB - Antifreeze proteins (AFPs) depress the freezing point of
aqueous solutions by binding to and inhibiting the
growth of ice. Whereas the ice-binding surface of some
fish AFPs is suggested by their linear, repetitive,
hydrogen bonding motifs, the 66-amino-acid-long Type
III AFP has a compact, globular fold without any
obvious periodicity. In the structure, 9 beta-strands
are paired to form 2 triple-stranded antiparallel
sheets and 1 double-stranded antiparallel sheet, with
the 2 triple sheets arranged as an orthogonal beta-
sandwich (Sonnichsen FD, Sykes BD, Chao H, Davies PL,
1993, Science 259:1154-1157). Based on its structure
and an alignment of Type III AFP isoform sequences, a
cluster of conserved, polar, surface-accessible amino
acids (N14, T18, Q44, and N46) was noted on and around
the triple-stranded sheet near the C-terminus. At 3 of
these sites, mutations that switched amide and hydroxyl
groups caused a large decrease in antifreeze activity,
but amide to carboxylic acid changes produced AFPs that
were fully active at pH 3 and pH 6. This is consistent
with the observation that Type III AFP is optimally
active from pH 2 to pH 11. At a concentration of 1 mg/mL,
Q44T, N14S, and T18N had 50%, 25%, and 10% of the
activity of wild-type antifreeze, respectively. The
effects of the mutations were cumulative, such that the
double mutant N14S/Q44T had 10% of the wild-type
activity and the triple mutant N14S/T18N/Q44T had no
activity. All mutants with reduced activity were shown
to be correctly folded by NMR spectroscopy. Moreover, a
complete characterization of the triple mutant by 2-
dimensional NMR spectroscopy indicated that the
individual and combined mutations did not significantly
alter the structure of these proteins. These results
suggest that the C-terminal beta-sheet of Type III AFP
is primarily responsible for antifreeze activity, and
they identify N14, T18, and Q44 as key residues for the
AFP-ice interaction.
SO - Protein Science 1994;3:1760-1769
AU - Pawlak M
AU - Meseth U
AU - Dhanapal B
AU - Mutter M
AU - Vogel H
TI - Template-assembled melittin: Structural and functional
characterization of a designed, synthetic channel-
forming protein
AD - Horst Vogel, Institute of Physical Chemistry IV, Swiss
Federal Institute of Technology Lausanne (EPFL),
Ecublens, CH-1015 Lausanne, Switzerland; e-mail: vogel@
icphp1.epfl.ch.
AB - Template-assembled proteins (TASPs) comprising 4 peptide
blocks, each of either the natural melittin sequence
(melittin-TASP) or of a truncated melittin sequence
(amino acids 6-26, melittin_6-26-TASP), C-terminally
linked to a (linear or cyclic) 10-amino acid template
were synthesized and characterized, structurally by CD,
by fluorescence spectroscopy, and by monolayer
experiments, and functionally, by electrical
conductance measurements on planar bilayers and release
experiments on dye-loaded vesicles. Melittin-TASP and
the truncated analogue preferentially adopt alpha-
helical structures in methanol (56% and 52%,
respectively) as in lipid membranes. Unlike in methanol,
the melittin-TASP self-aggregates in water. On an air-
water interface, the differently sized molecules can be
self-assembled and compressed to a compact structure
with a molecular area of around 600 Angstrom^2,
compatible with a 4-helix bundle preferentially
oriented perpendicular to the interface. The proteins
reveal a strong affinity for lipid membranes. A
partition coefficient of 1.5 x 10^9 M^-1 was evaluated
from changes of the Trp fluorescence spectra of the
TASP in water and in the lipid bilayer. In planar lipid
bilayers, TASP molecules are able to form defined ion
channels, exhibiting a small single-channel conductance
of 7 pS (in 1 M NaCl). With increasing protein
concentration in the lipid bilayer, additional, larger
conductance states of up to 1 nS were observed. These
states are likely to be formed by aggregated TASP
structures as inferred from a strongly voltage-
dependent channel activity on membranes of large area.
In this respect, melittin-TASP reveals channel features
of the native peptide, but with a considerably lower
variation in the size of the channel states. Compared
to the free peptide, template-assembled melittin has a
much higher membrane activity: it is about 100 times
more effective in channel formation and 20 times more
effective in releasing dye molecules from lipid vesicles.
This demonstrates that the lytic properties are not
solely related to channel formation.
SO - Protein Science 1994;3:1788-1805
AU - Damberger FF
AU - Pelton JG
AU - Harrison CJ
AU - Nelson HCM
AU - Wemmer DE
TI - Solution structure of the DNA-binding domain of the heat
shock transcription factor determined by
multidimensional heteronuclear magnetic resonance
spectroscopy
AD - David E. Wemmer, Department of Chemistry, University of
California, Berkeley, California 94720; e-mail: dewemmer
@lbl.gov.
AB - The solution structure of the 92-residue DNA-binding
domain of the heat shock transcription factor from
Kluyveromyces lactis has been determined using
multidimensional NMR methods. Three-dimensional (3D)
triple resonance, 1H-13C-13C-1H total correlation
spectroscopy, and 15N-separated total correlation
spectroscopy-heteronuclear multiple quantum correlation
experiments were used along with various 2D spectra to
make nearly complete assignments for the backbone and
side-chain 1H, 15N, and 13C resonances. Five-hundred
eighty-three NOE constraints identified in 3D 13C- and
15N-separated NOE spectroscopy (NOESY)-heteronuclear
multiple quantum correlation spectra and a 4-
dimensional 13C/^13C-edited NOESY spectrum, along with
35 phi, 9 chi_1, and 30 hydrogen bond constraints, were
used to calculate 30 structures by a hybrid distance
geometry/simulated annealing protocol, of which 24 were
used for structural comparison. The calculations
revealed that a 3-helix bundle packs against a small 4-
stranded antiparallel beta-sheet. The backbone RMS
deviation (RMSD) for the family of structures was 1.03 +
- 0.19 Angstrom with respect to the average structure.
The topology is analogous to that of the C-terminal
domain of the catabolite gene activator protein and
appears to be in the helix-turn-helix family of DNA-
binding proteins. The overall fold determined by the
NMR data is consistent with recent crystallographic
work on this domain (Harrison CJ, Bohm AA, Nelson HCM,
1994, Science 263:224) as evidenced by RMSD between
backbone atoms in the NMR and X-ray structures of 1.77 +
- 0.20 Angstrom. Several differences were identified
some of which may be due to protein-protein
interactions in the crystal.
SO - Protein Science 1994;3:1806-1821
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