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WSN: forthcoming papers in Protein Science (vol.3, #9)
(from URL: gopher://orion.oac.uci.edu/protein/)
AU - Hagihara Y
AU - Oobatake M
AU - Goto Y
TI - Thermal unfolding of tetrameric melittin: Comparison with
the molten globule state of cytochrome c
AD - Yuji Goto, Department of Biology, Faculty of Science,
Osaka University, Toyonaka, Osaka 560, Japan; e-mail:
c62490a@center.osaka-u.ac.jp.
AB - Whereas melittin at micromolar concentrations is unfolded
under conditions of low salt at neutral pH, it
transforms to a tetrameric alpha-helical structure
under several conditions, such as high peptide
concentration, high anion concentration, or alkaline pH.
The anion- and pH-dependent stabilization of the
tetrameric structure is similar to that of the molten
globule state of several acid-denatured proteins,
suggesting that tetrameric melittin might be a state
similar to the molten globule state. To test this
possibility, we studied the thermal unfolding of
tetrameric melittin using far-UV CD and differential
scanning calorimetry. The latter technique revealed a
broad but distinct heat absorption peak. The heat
absorption curves were consistent with the unfolding
transition observed by CD and were explainable by a 2-
state transition mechanism between the tetrameric alpha-
helical state and the monomeric unfolded state. From
the peptide or salt-concentration dependence of
unfolding, the heat capacity change upon unfolding was
estimated to be 5 kJ (mol of tetramer) -1 K^-1 at 30
degrees C and decreased with increasing temperature.
The observed change in heat capacity was much smaller
than that predicted from the crystallographic structure
(9.2 kJ (mol of tetramer) -1 K^-1), suggesting that the
hydrophobic residues of tetrameric melittin in solution
are exposed in comparison with the crystallographic
structure. However, the results also indicate that the
structure is more ordered than that of a typical molten
globule state. We consider that the conformation is
intermediate between the molten globule state and the
native state of globular proteins.
SO - Protein Science 1994;3:1418-1429
AU - Norin M
AU - Haeffner F
AU - Achour A
TI - Computer modeling of substrate binding to lipases from
Rhizomucor miehei, Humicola lanuginosa, and Candida
rugosa
AD - Karl Hult, Department of Biochemistry and Biotechnology,
Royal Institute of Technology, S-100 44 Stockholm,
Sweden; e-mail: kalle@biochem.kth.se.
AB - The substrate-binding sites of the triacyl glyceride
lipases from Rhizomucor miehei, Humicola lanuginosa,
and Candida rugosa were studied by means of computer
modeling methods. The space around the active site was
mapped by different probes. These calculations
suggested 2 separate regions within the binding site.
One region showed high affinity for aliphatic groups,
whereas the other region was hydrophilic. The aliphatic
site should be a binding cavity for fatty acid chains.
Water molecules are required for the hydrolysis of the
acyl enzyme, but are probably not readily accessible in
the hydrophobic interface, in which lipases are acting.
Therefore, the hydrophilic site should be important for
the hydrolytic activity of the enzyme.
Lipases from R. miehei and H. lanuginosa are excellent
catalysts for enantioselective resolutions of many
secondary alcohols. We used molecular mechanics and
dynamics calculations of enzyme-substrate transition-
state complexes, which provided information about
molecular interactions important for the
enantioselectivities of these reactions.
SO - Protein Science 1994;3:1493-1503
AU - Ottiger M
AU - Szyperski T
AU - Luginbuhl P
AU - Ortenzi C
AU - Luporini P
AU - Bradshaw RA
AU - Wuthrich K
TI - The NMR solution structure of the pheromone Er-2 from the
ciliated protozoan Euplotes raikovi
AD - K. Wuthrich, Institut fur Molekularbiologie und Biophysik,
Eidgenossische Technische Hochschule, 8093 Zurich,
Switzerland.
AB - The NMR structure of the pheromone Er-2 from the ciliated
protozoan Euplotes raikovi has been determined in
aqueous solution. The structure of this 40-residue
protein was calculated with the distance geometry
program DIANA from 621 distance constraints and 89
dihedral angle constraints; the program OPAL was
employed for the energy minimization. For a group of 20
conformers used to characterize the solution structure,
the average pairwise RMS deviation from the mean
structure calculated for the backbone heavy atoms N, C^
alpha, and C' of residues 3-37 was 0.31 Angstrom. The
molecular architecture is dominated by an up-down-up
bundle of 3 short helices of residues 5-11, 14-20, and
23-33, which is similar to the structures of the
homologous pheromones Er-1 and Er-10. Novel structural
features include a well-defined N-cap on the first helix,
a 1-residue deletion in the second helix resulting in
the formation of a 3_10-helix rather than an alpha-
helix as found in Er-1 and Er-10, and the simultaneous
presence of 2 different conformations for the C-
terminal tetrapeptide segment, i.e., a major
conformation with the Leu 39-Pro 40 peptide bond in the
trans form and a minor conformation with this peptide
bond in the cis form.
SO - Protein Science 1994;3:1515-1526
AU - Spassov VZ
AU - Karshikoff AD
AU - Ladenstein R
TI - Optimization of the electrostatic interactions in
proteins of different functional and folding type
AD - Andrej D. Karshikoff, Centre for Structural Biochemistry,
Karolinska Institute, NOVUM, S-14157 Huddinge, Stockholm,
Sweden; e-mail: aka@csb.ki.se.
AB - The 3-dimensional optimization of the electrostatic
interactions between the charged amino acid residues
was studied by Monte Carlo simulations on an extended
representative set of 141 protein structures with known
atomic coordinates. The proteins were classified by
different functional and structural criteria, and the
optimization of the electrostatic interactions was
analyzed. The optimization parameters were obtained by
comparison of the contribution of charge-charge
interactions to the free energy of the native protein
structures and for a large number of randomly
distributed charge constellations obtained by the Monte
Carlo technique. On the basis of the results obtained,
one can conclude that the charge-charge interactions
are better optimized in the enzymes than in the
proteins without enzymatic functions. Proteins that
belong to the mixed alphabeta folding type are
electrostatically better optimized than pure alpha-
helical or beta-strand structures. Proteins that are
stabilized by disulfide bonds show a lower degree of
electrostatic optimization. The electrostatic
interactions in a native protein are effectively
optimized by rejection of the conformers that lead to
repulsive charge-charge interactions. Particularly, the
rejection of the repulsive contacts seems to be a major
goal in the protein folding process. The dependence of
the optimization parameters on the choice of the
potential function was tested. The majority of the
potential functions gave practically identical results.
SO - Protein Science 1994;3:1556-1569
AU - Lebowitz J
AU - Kar S
AU - Braswell E
AU - Mcpherson S
AU - Richard DL
TI - Human immunodeficiency virus-1 reverse transcriptase
heterodimer stability
AD - Jacob Lebowitz, Department of Microbiology, 520 CHSB,
University of Alabama at Birmingham, Birmingham,
Alabama 35294.
AB - Structural and biochemical evidence strongly supports a
heterodimeric (p66p51) active form for human
immunodeficiency virus-1 reverse transcriptase (RT).
Heterodimer stability was examined by sedimentation
analysis as a function of temperature and ionic strength.
Using NONLIN regression software, monomer-dimer-trimer
and monomer-dimer-tetramer association models gave the
best fit to the analytical ultracentrifuge
sedimentation equilibrium data. The heterodimer is the
predominant form of RT at 5 degrees C, with a
dimerization K_a value of 5.2 x 10^5 M^-1 for both
models. K_a values of 2.1 x 10^5 and 3.8 x 10^5 M^-1
were obtained for the respective association models at
20 degrees C. RT in 50 and 100 mM Tris, pH 7.0,
completely dissociates at 37 degrees C and behaves as
an ideal monomeric species. The dissociation of RT as a
function of increasing temperature was also observed by
measuring the decrease in sedimentation velocity (s_w,20).
If the stabilization of the heterodimer was due
primarily to hydrophobic interactions we would
anticipate an increase in the association from 21
degrees C to 37 degrees C. The opposite temperature
dependence for the association of RT suggests that
electrostatic and hydrogen bond interactions play an
important role in stabilizing heterodimers. To examine
the effect of ionic strength on p66p51 association we
determined the changes in s_w,20 as a function of NaCl
concentration. There is a sharp decrease in s_w,20
between 0.10 and 0.5 M NaCl, leading to apparent
complete dissociation. The above results support a
major role for electrostatic interactions in the
stabilization of the RT heterodimer.
SO - Protein Science 1994;3:1374-1382
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Iosif Vaisman