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WSN: Protein Science (vol.3, #12)
(from URL: gopher://orion.oac.uci.edu/protein/)
AU - Neet KE, Timm DE
TI - Conformational stability of dimeric proteins:
Quantitative studies by equilibrium denaturation
AD - Kenneth E. Neet, Department of Biological Chemistry, UHS/
Chicago Medical School, 3333 Green Bay Road, North
Chicago, Illinois 60064; e-mail: neetk@mis.fuhscms.edu.
AB - The conformational stability of dimeric globular proteins
can be measured by equilibrium denaturation studies in
solvents such as guanidine hydrochloride or urea. Many
dimeric proteins denature with a 2-state equilibrium
transition, whereas others have stable intermediates in
the process. For those proteins showing a single
transition of native dimer to denatured monomer, the
conformational stabilities,[Delta]G_u(H_2O), range from
10 to 27 kcal/mol, which is significantly greater than
the conformational stability found for monomeric
proteins. The relative contribution of quaternary
interactions to the overall stability of the dimer can
be estimated by comparing [Delta]G_u(H_2O) from
equilibrium denaturation studies to the free energy
associated with simple dissociation in the absence of
denaturant. In many cases the large stabilization
energy of dimers is primarily due to the intersubunit
interactions and thus gives a rationale for the
formation of oligomers. The magnitude of the
conformational stability is related to the size of the
polypeptide in the subunit and depends upon the type of
structure in the subunit interface. The practical use,
interpretation, and utility of estimation of
conformational stability of dimers by equilibrium
denaturation methods are discussed.
SO - Protein Science 1994;3:2167-2174
AU - Xie D, Fox R, Freire E
TI - Thermodynamic characterization of an equilibrium folding
intermediate of staphylococcal nuclease
AD - Ernesto Freire, Department of Biology, The Johns Hopkins
University, 34th and Charles Streets, Baltimore,
Maryland 21218; e-mail: bcc@biocal2.bio.jhu.edu.
AB - High-sensitivity differential scanning calorimetry and CD
spectroscopy have been used to probe the structural
stability and measure the folding/unfolding
thermodynamics of a Pro^117 to Gly variant of
staphylococcal nuclease. It is shown that at neutral pH
the thermal denaturation of this protein is well
accounted for by a 2-state mechanism and that the
thermally denatured state is a fully hydrated unfolded
polypeptide. At pH 3.5, thermal denaturation results in
a compact denatured state in which most, if not all, of
the helical structure is missing and the beta subdomain
apparently remains largely intact. At pH 3.0, no
thermal transition is observed and the molecule exists
in the compact denatured state within the 0-100 degrees
C temperature interval. At high salt concentration and
pH 3.5, the thermal unfolding transition exhibits 2
cooperative peaks in the heat capacity function, the
first one corresponding to the transition from the
native to the intermediate state and the second one to
the transition from the intermediate to the unfolded
state. As is the case with other proteins, the enthalpy
of the intermediate is higher than that of the unfolded
state at low temperatures, indicating that, under those
conditions, its stabilization must be of an entropic
origin. The folding intermediate has been modeled by
structural thermodynamic calculations. Structure-based
thermodynamic calculations also predict that the most
probable intermediate is one in which the beta
subdomain is essentially intact and the rest of the
molecule unfolded, in agreement with the experimental
data. The structural features of the equilibrium
intermediate are similar to those of a kinetic
intermediate previously characterized by hydrogen
exchange and NMR spectroscopy.
SO - Protein Science 1994;3:2175-2184
AU - Hubbard SJ, Argos P
TI - Cavities and packing at protein interfaces
AD - Simon J. Hubbard, European Molecular Biology Laboratory,
Postfach 10.2209, Meyerhofstrasse 1, 69012 Heidelberg,
Germany; e-mail: hubbard@embl-heidelberg.de.
AB - An analysis of internal packing defects or ``cavities''
(both empty and water-containing) within protein
structures has been undertaken and includes 3 cavity
classes: within domains, between domains, and between
protein subunits. We confirm several basic features
common to all cavity types but also find a number of
new characteristics, including those that distinguish
the classes. The total cavity volume remains only a
small fraction of the total protein volume and yet
increases with protein size. Water-filled ``cavities
'' possess a more polar surface and are typically larger.
Their constituent waters are necessary to satisfy the
local hydrogen bonding potential. Cavity-surrounding
atoms are observed to be, on average, less flexible
than their environments. Intersubunit and interdomain
cavities are on average larger than the intradomain
cavities, occupy a larger fraction of their resident
surfaces, and are more frequently water-filled. We
observe increased cavity volume at domain-domain
interfaces involved with shear type domain motions. The
significance of interfacial cavities upon subunit and
domain shape complementarity and the protein docking
problem, as well as in their structural and functional
role in oligomeric proteins, will be discussed. The
results concerning cavity size, polarity, solvation,
general abundance, and residue type constituency should
provide useful guidelines for protein modeling and
design.
SO - Protein Science 1994;3:2194-2206
AU - Marqusee S, Sauer RT
TI - Contributions of a hydrogen bond/salt bridge network to
the stability of secondary and tertiary structure in
lambda repressor
AD - Susan Marqusee at her present address: Division of
Biochemistry and Molecular Biology, MCB: 229 Stanley
Hall, University of California, Berkeley, California
94720; e-mail: susan_marqusee@maillink.berkeley.edu.
AB - In the N-terminal domain of lambda repressor, the Asp 14
side chain forms an intrahelical, hydrogen bond/salt
bridge with the Arg 17 side chain and a tertiary
hydrogen bond with the Ser 77 side chain. By measuring
the stabilities to urea denaturation of the wild-type N-
terminal domain and variants containing single, double,
and triple alanine substitutions at positions 14, 17,
and 77, the side-chain interaction energies, the
coupling energy between interactions, and the intrinsic
effects of each wild-type side chain on protein
stability have been estimated. These studies indicate
that the Asp 14-Arg 17 and Asp 14-Ser 77 interactions
are stabilizing by roughly 0.8 and 1.5 kcal/mol,
respectively, but that Asp 14, by itself, is
destabilizing by roughly 0.9 kcal/mol. We also show
that a peptide model of alpha-helix 1, which contains
Asp 14 and Arg 17, forms a reasonably stable, monomeric
helix in solution and responds to alanine mutations at
positions 14 and 17 in the fashion expected from the
intact protein studies. These studies suggest that it
is possible to view the stability effects of mutations
in intact proteins in a hierarchical fashion, with the
stability of units of secondary structure being
distinguishable from the stability of tertiary structure.
SO - Protein Science 1994;3:2217-2225
AU - Schulman BA, Kim PS
TI - Hydrogen exchange in BPTI variants that do not share a
common disulfide bond
AD - Peter S. Kim, Howard Hughes Medical Institute, Whitehead
Institute for Biomedical Research, Department of Biology,
Massachusetts Institute of Technology, Nine Cambridge
Center, Cambridge, Massachusetts 02142.
AB - Bovine pancreatic trypsin inhibitor (BPTI) is stabilized
by 3 disulfide bonds, between cysteines 30-51, 5-55,
and 14-38. To better understand the influence of
disulfide bonds on local protein structure and dynamics,
we have measured amide proton exchange rates in 2
folded variants of BPTI,[5-55] Ala and [30-51; 14-38]
V5A55, which share no common disulfide bonds. These
proteins resemble disulfide-bonded intermediates that
accumulate in the BPTI folding pathway. Essentially the
same amide hydrogens are protected from exchange in
both of the BPTI variants studied here as in native BPTI,
demonstrating that the variants adopt fully folded,
native-like structures in solution. However, the most
highly protected amide protons in each variant differ,
and are contained within the sequences of previously
studied peptide models of related BPTI foldiing
intermediates containing either the 5-55 or the 30-51
disulfide bond.
SO - Protein Science 1994;3:2226-2232
AU - Lim K, Ho JX, Keeling K, Gilliland GL, Ji X, Ruker F, Carter DC
TI - Three-dimensional structure of Schistosoma japonicum
glutathione S-transferase fused with a six-amino acid
conserved neutralizing epitope of gp41 from HIV
AD - Daniel C. Carter, ES 76 Biophysics Branch, George C.
Marshall Space Flight Center, NASA, Huntsville, Alabama
35812; e-mail: carter@lsb.msfc.nasa.gov.
AB - The 3-dimensional crystal structure of glutathione S-
transferase (GST) of Schistosoma japonicum (Sj) fused
with a conserved neutralizing epitope on gp41
(glycoprotein, 41 kDa) of human immunodeficiency virus
type 1 (HIV-1)(Muster T et al., 1993, J Virol 67:6642-
6647) was determined at 2.5 Angstrom resolution. The
structure of the 3-3 isozyme rat GST of the mu gene
class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992,
Biochemistry 31:10169-10184) was used as a molecular
replacement model. The structure consists of a 4-
stranded beta-sheet and 3 alpha-helices in domain 1 and
5 alpha-helices in domain 2. The space group of the Sj
GST crystal is P4_32_12, with unit cell dimensions of a
= b = 94.7 Angstrom, and c = 58.1 Angstrom. The crystal
has 1 GST monomer per asymmetric unit, and 2 monomers
that form an active dimer are related by
crystallographic 2-fold symmetry. In the binding site,
the ordered structure of reduced glutathione is observed.
The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the
C-terminus of Sj GST forms a loop stabilized by symmetry
-related GSTs. The Sj GST structure is compared with
previously determined GST structures of mammalian gene
classes mu,alpha, and pi. Conserved amino acid residues
among the 4 GSTs that are important for hydrophobic and
hydrophilic interactions for dimer association and
glutathione binding are discussed.
SO - Protein Science 1994;3:2233-2244
AU - Vijayalakshmi J, Padmanabhan KP, Mann KG, Tulinsky A
TI - The isomorphous structures of prethrombin2, hirugen-, and
PPACK-thrombin: Changes accompanying activation and
exosite binding to thrombin
AD - A. Tulinsky, Department of Chemistry, Michigan State
University, East Lansing, Michigan 48824-1322; e-mail:
tulinsky@cemvax.cem.msu.edu.
AB - The X-ray crystal structure of prethrombin2 (pre2), the
immediate inactive precursor of alpha-thrombin, has
been determined at 2.0 Angstrom resolution complexed
with hirugen. The structure has been refined to a final
R-value of 0.169 using 14,211 observed reflections in
the resolution range 8.0-2.0 Angstrom. A total of 202
water molecules have also been located in the structure.
Comparison with the hirugen-thrombin complex showed that,
apart from the flexible beginning and terminal regions
of the molecule, there are 4 polypeptide segments in
pre2 differing in conformation from the active enzyme
(Pro 186-Asp 194, Gly 216-Gly 223, Gly 142-Pro 152, and
the Arg 15-Ile 16 cleavage region). The formation of
the Ile 16-Asp 194 ion pair and the specificity pocket
are characteristic of serine protease activation with
the conformation of the catalytic triad being conserved.
With the determination of isomorphous structures of
hirugen-thrombin and D-Phe-Pro-Arg chloromethyl ketone
(PPACK)-thrombin, the changes that occur in the active
site that affect the kinetics of chromogenic substrate
hydrolysis on binding to the fibrinogen recognition
exosite have been determined. The backbone of the Ala
190-Gly 197 segment in the active site has an average
RMS difference of 0.55 Angstrom between the 2
structures (about 3.7sigma compared to the bulk
structure). This segment has 2 type II beta-bends, the
first bend showing the largest shift due to hirugen
binding. Another important feature was the 2 different
conformations of the side chain of Glu 192. The side
chain extends to solvent in hirugen-thrombin, which is
compatible with the binding of substrates having an
acidic residue in the P3 position (protein-C, thrombin
platelet receptor). In PPACK-thrombin, the side chain
of Asp 189 and the segment Arg 221A-Gly 223 move to
provide space for the inhibitor, whereas in hirugen-
thrombin, the Ala 190-Gly 197 movement expands the
active site region. Although 8 water molecules are
expelled from the active site with PPACK binding, the
inhibitor complex is resolvated with 5 other water
molecules.
SO - Protein Science 1994;3:2254-2271
AU - Schendel SL, Cramer WA
TI - On the nature of the unfolded intermediate in the in
vitro transition of the colicin E1 channel domain from
the aqueous to the membrane phase
AD - William A. Cramer, Department of Biological Sciences,
Purdue University, West Lafayette, Indiana 47907; e-mail:
wac@bilbo.bio.purdue.edu.
AB - The transition of the colicin E1 channel polypeptide from
a water-soluble to membrane-bound state occurs in vitro
at acid pH values that are associated with an unfolded
channel structure whose properties qualitatively
resemble those of a ``molten globule,'' or ``compact
unfolded,'' intermediate state. The role of such a
state for activity was tested by comparing the pH
dependence of channel-induced solute efflux and the
amplitude of the near-UV CD spectrum. The requirement
of a partly unfolded state for activity was shown by
the coincidence of the onset of channel activity
measured for 4 different lipid compositions with the
decrease in near-UV CD amplitude as a function of pH.
Tertiary constraints on the 3 tryptophans of the
colicin channel, assayed by the amplitude of the near-
UV CD spectrum, are retained over the pH range 3-4
where channel activity could be measured and, as well,
at pH 2. In addition, the tryptophan fluorescence
emission spectrum is virtually unchanged over the pH
range 2-6. The temperature independence of the near-UV
spectrum at pH 3-6 up to 70 degrees C implies that the
colicin E1 channel polypeptide is more stable than that
of colicin A. A transition between 53 and 58 degrees C
in the amplitude of the near-UV CD is consistent with
preservation of part of the hydrophobic core in a
destabilized state at pH 2. Thus, the unfolded state
associated with colicin activity at acidic pH has the
properties of a ``compact unfolded'' state, having some,
but not all of the properties of a ``molten globule.''
The small effect on local membrane acidity of a
physiological acidic membrane lipid content, the
retention of significant near-UV CD amplitude down to
pH 2, and the small extent of immersion of the 40-
Angstrom globular colicin channel polypeptide in the 10-
Angstrom lower pH layer at the membrane surface make it
unlikely that a local lower pH at the membrane surface
significantly facilitates formation of an unfolded
intermediate.
SO - Protein Science 1994;3:2272-2279
AU - Zegers I, Maes D, Thi MHD, Wyns L, Poortmans F, Palmer R
TI - The structures of RNase A complexed with 3'-CMP and d(CpA):
Active site conformation and conserved water molecules
AD - Ingrid Zegers, Instituut Moleculaire Biologie, Dienst
Ultrastructuur, Paardenstraat 65, B-1640 St. Genesius
Rode, Belgium; e-mail: igzegers@vub.ac.be.
AB - The interactions of RNase A with cytidine 3'-
monophosphate (3'-CMP) and deoxycytidyl-3',5'-
deoxyadenosine (d(CpA)) were analyzed by X-ray
crystallography. The 3'-CMP complex and the native
structure were determined from trigonal crystals, and
the d(CpA) complex from monoclinic crystals. The
differences between the overall structures are
concentrated in loop regions and are relatively small.
The protein-inhibitor contacts are interpreted in terms
of the catalytic mechanism. The general base His 12
interacts with the 2' oxygen, as does the electrostatic
catalyst Lys 41. The general acid His 119 has 2
conformations (A and B) in the native structure and is
found in, respectively, the A and the B conformation in
the d(CpA) and the 3'-CMP complex. From the present
structures and from a comparison with RNase T1, we
propose that His 119 is active in the A conformation.
The structure of the d(CpA) complex permits a detailed
analysis of the downstream binding site, which includes
His 119 and Asn 71. The comparison of the present RNase
A structures with an inhibitor complex of RNase T1
shows that there are important similarities in the
active sites of these 2 enzymes, despite the absence of
any sequence homology. The water molecules were
analyzed in order to identify conserved water sites.
Seventeen water sites were found to be conserved in
RNase A structures derived from 5 different space groups.
It is proposed that 7 of those water molecules play a
role in the binding of the N-terminal helix to the rest
of the protein and in the stabilization of the active
site.
SO - Protein Science 1994;3:2322-2339
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