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WSN: forthcoming papers in Protein Science (vol.3, #8)
(from URL: gopher://orion.oac.uci.edu/protein/)
AU - Williams MA
AU - Goodfellow JM
AU - Thornton JM
TI - Buried waters and internal cavities in monomeric proteins
AD - Janet M. Thornton, Biomolecular Structure and Modelling
Unit, Department of Biochemistry and Molecular Biology,
University College London, Gower Street, London WC1E 6BT,
UK; e-mail: thornton@uk.ac.ucl.bioc.bsm.
AB - We have analyzed the buried water molecules and internal
cavities in a set of 75 high-resolution, nonhomologous,
monomeric protein structures. The number of hydrogen
bonds formed between each water molecule and the
protein varies from 0 to 4, with 3 being most common.
Nearly half of the water molecules are found in pairs
or larger clusters. Approximately 90% are shown to be
associated with large cavities within the protein, as
determined by a novel program, PRO_ACT. The total
volume of a protein's large cavities is proportional to
its molecular weight and is not dependent on structural
class. The largest cavities in proteins are generally
elongated rather than globular. There are many more
empty cavities than hydrated cavities. The likelihood
of a cavity being occupied by a water molecule
increases with cavity size and the number of available
hydrogen bond partners, with each additional partner
typically stabilizing the occupied state by 0.6 kcal/mol.
SO - Protein Science 1994;3:1224-1235
AU - Bromberg S
AU - Licata VJ
AU - Mallikarachchi D
AU - Allewell NM
TI - Ligation alters the pathway of urea-induced denaturation
of the catalytic trimer of Escherichia coli aspartate
transcarbamylase
AD - N.M. Allewell, Department of Biochemistry, University of
Minnesota, 1479 Gortner Avenue, St. Paul, Minnesota
55108; e-mail: norma@molbio.cbs.umn.edu.
AB - We have examined the pathway and energetics of urea-
induced dissociation and unfolding of the catalytic
trimer (c_3) of aspartate transcarbamylase from
Escherichia coli at low temperature in the absence and
presence of carbamyl phosphate (CP; a substrate), N-
(phosphonacetyl)-L-Asp (PALA; a bisubstrate analog),
and 2 anionic inhibitors, Cl^- and ATP, by analytical
gel chromatography supplemented by activity assays and
ultraviolet difference spectroscopy. In the absence of
active-site ligands and in the presence of ATP, c_3
dissociates below 2 M urea into swollen c chains that
then gradually unfold from 2 to 6 M urea with little
apparent cooperativity. Linear extrapolation to 0 M
urea of free energies determined in 3 independent types
of experiments yields estimates for [Delta]G_
dissociation at 7.5 degrees C of about 7-10 kcal m^-1
per interface.[Delta]G_unfolding of dissociated chains
when modeled as a 2-state process is estimated to be
very small, on the order of [approximately equal to]2
kcal m^-1. The data are also consistent with the
possibility that the unfolding of the dissociated
monomer is a 1-state swelling process. In the presence
of the ligands CP and PALA, and in the presence of Cl^-,
c_3 dissociates at much higher urea concentrations, and
trimer dissociation and unfolding occur simultaneously
and apparently cooperatively, at urea concentrations
that increase with the affinity of the ligand.
SO - Protein Science 1994;3:1236-1244
AU - Tominaga N
AU - Jameson DM
AU - Uyeda K
TI - Reversible unfolding of fructose 6-phosphate, 2-kinase:
fructose 2,6-bisphosphatase
AD - Kosaku Uyeda, Department of Veterans Affairs Medical
Center, 4500 S. Lancaster Road, Dallas, Texas 75216.
AB - Reversible unfolding of rat testis fructose 6-phosphate,2-
kinase:fructose 2,6-bisphosphatase in guanidine
hydrochloride was monitored by following enzyme
activities as well as by fluorescence methodologies
(intensity, emission maximum, polarization, and
quenching), using both intrinsic (tryptophan) and
extrinsic (5((2-(iodoacetyl)amino) ethyl)naphthalene-1-
sulfonic acid) probes. The unfolding reaction is
described minimally as a 4-state transition from folded
dimer to partially unfolded dimer to monomer unfolded
monomer. The partially unfolded dimer had a high
phosphatase/kinase ratio due to preferential unfolding
of the kinase domain. The renaturation reaction
proceeded by very rapid conversion (less than 1 s) of
unfolded monomer to dimer, devoid of any enzyme activity,
followed by slow (over 60 min) formation of the active
enzyme. The recovery rates of the kinase and the
phosphatase were similar. Thus, the refolding appeared
to be a reversal of the unfolding pathway involving
different forms of the transient dimeric intermediates.
Fluorescence quenching studies using iodide and
acrylamide showed that the tryptophans, including Trp-
15 in the N-terminal peptide, were only slightly
accessible to iodide but were much more accessible to
acrylamide. Fructose 6-phosphate, but not ATP or
fructose 2,6-bisphosphate, diminished the iodide
quenching, but all these ligands inhibited the
acrylamide quenching by 25%. These results suggested
that the N-terminal peptide (containing a tryptophan)
was not exposed on the protein surface and may play an
important role in shielding other tryptophans from
solvent.
SO - Protein Science 1994;3:1245-1252
AU - Cohen DS
AU - Pielak GJ
TI - Stability of yeast iso-1-ferricytochrome c as a function
of pH and temperature
AD - Gary J. Pielak, Department of Chemistry, Campus Box 3290,
University of North Carolina, Chapel Hill, North
Carolina 27599; e-mail: gjpielak@uncvx1.oit.unc.edu.
AB - Absorbance-detected thermal denaturation studies of the
C102T variant of Saccharomyces cerevisiae iso-1-
ferricytochrome c were performed between pH 3 and 5.
Thermal denaturation in this pH range is reversible,
shows no concentration dependence, and is consistent
with a 2-state model. Values for free energy ([Delta]G_D),
enthalpy ([Delta]H_D), and entropy ([Delta]S_D) of
denaturation were determined as functions of pH and
temperature. The value of [Delta]G_D at 300 K, pH 4.6,
is 5.1 +- 0.3 kcal mol^-1. The change in molar heat
capacity upon denaturation ([Delta]C_p), determined by
the temperature dependence of [Delta]H_D as a function
of pH (1.37 +- 0.06 kcal mol^-1 K^-1), agrees with the
value determined by differential scanning calorimetry.
pH-dependent changes in the Soret region indicate that
a group or groups in the heme environment of the
denatured protein, probably 1 or both heme propionates,
ionize with a pK near 4. The C102T variant exhibits
both enthalpy and entropy convergence with a [Delta]H_D
of 1.30 kcal mol^-1 residue^-1 at 373.6 K and a [Delta]S
_D of 4.24 cal mol^-1 K^-1 residue^-1 at 385.2 K. These
values agree with those for other single-domain,
globular proteins.
SO - Protein Science 1994;3:1253-1260
AU - Guenot J
AU - Fletterick RJ
AU - Kollman PA
TI - A negative electrostatic determinant mediates the
association between the Escherichia coli trp repressor
and its operator DNA
AD - Jeanmarie Guenot at her present address: Physical
Chemistry Department, Hoffmann-La Roche, Inc., 340
Kingsland Avenue, Nutley, New Jersey 07110; e-mail:
guenot@rnch01.dnet.roche.com.
AB - The electrostatic potential surfaces were characterized
for trp repressor models that bind to DNA with sequence
specificity, without specificity, and not at all.
Comparisons among the surfaces were used to isolate
protein surface features likely to be important in DNA
binding. Models that differ in protein conformation and
tryptophan-analogue binding consistently showed
positive potential associated with the protein surfaces
that interact with the DNA major groove. However,
negative potential is associated with the trp repressor
surface that contacts the DNA minor groove. This
negative potential is significantly neutralized in the
protein conformation that is bound to DNA. Positive
potential is also associated with the tryptophan binding
-site surface, a consequence of the tryptophan- or
tryptophan analogue-induced allosteric change. This
protein region is complementary to the strongest
negative potential associated with the DNA phosphate
backbone and is also present in the isolated protein
structure from the protein-DNA complex. The effects of
charge-change mutation, pH dependence, and salt
dependence on the electrostatic potential surfaces were
also examined with regard to their effects on protein-
DNA binding constants. A consistent model is formed
that defines a role for long-range electrostatics early
in the protein-DNA association process and complements
previous structural, molecular association, and
mutagenesis studies.
SO - Protein Science 1994;3:1276-1285
AU - Liu T
AU - Derose EF
AU - Mullen GP
TI - Determination of the structure of the DNA binding domain
of gammadelta resolvase in solution
AD - Gregory P. Mullen at his present address: Department of
Biochemistry, University of Connecticut Health Center,
263 Farmington Avenue, Farmington, Connecticut 06032.
AB - The DNA binding domain (DBD) of gammadelta resolvase
(residues 141-183) is responsible for the interaction
of this site-specific DNA recombinase with consensus
site DNA within the gammadelta transposable element in
Escherichia coli. Based on chemical-shift comparisons,
the proteolytically isolated DBD displays side-chain
interactions within a hydrophobic core that are highly
similar to those of this domain when part of the intact
enzyme (Liu T, Liu DJ, DeRose EF, Mullen GP, 1993, J
Biol Chem 268:16309-16315). The structure of the DBD in
solution has been determined using restraints obtained
from 2-dimensional proton NMR data and is represented
by 17 conformers. Experimental restraints included 458
distances based on analysis of nuclear Overhauser
effect connectivities, 17 phi and chi_1 torsion angles
based on analysis of couplings, and 17 backbone
hydrogen bonds determined from NH exchange data. With
respect to the computed average structure, these
conformers display an RMS deviation of 0.67 Angstrom
for the heavy backbone atoms and 1.49 Angstrom for all
heavy atoms within residues 149-180. The DBD consists
of 3 alpha-helices comprising residues D149-Q157, S162-
T167, and R172-N183. Helix-2 and helix-3 form a
backbone fold, which is similar to the canonical helix-
turn-helix motif. The conformation of the NH_2-terminal
residues, G141-R148, appears flexible in solution. A
hydrophobic core is formed by side chains donated by
essentially all hydrophobic residues within the helices
and turns. Helix-1 and helix-3 cross with a right-
handed folding topology. The structure is consistent
with a mechanism of DNA binding in which contacts are
made by the hydrophilic face of helix-3 in the major
groove and the amino-terminal arm in the minor groove.
This structure represents an important step toward
analysis of the mechanism of DNA interaction by gamma
delta resolvase and provides initial structure-function
comparisons among the divergent DBDs of related
resolvases and invertases.
SO - Protein Science 1994;3:1286-1295
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Iosif Vaisman