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WSN: Protein Science (vol.3, #11)
(from URL: gopher://orion.oac.uci.edu/protein/)
AU - Loo RRO
AU - Dales N
AU - Andrews PC
TI - Surfactant effects on protein structure examined by
electrospray ionization mass spectrometry
AD - P.C. Andrews, Department of Biological Chemistry,
University of Michigan, 2552 MSRB II, Ann Arbor,
Michigan 48109; e-mail: andrews@brcf.med.umich.edu.
AB - Electrospray ionization mass spectrometry (ESI-MS) has
proven to be a useful tool for examining noncovalent
complexes between proteins and a variety of ligands. It
has also been used to distinguish between denatured and
refolded forms of proteins. Surfactants are frequently
employed to enhance solubilization or to modify the
tertiary or quaternary structure of proteins, but are
usually considered incompatible with mass spectrometry.
A broad range of ionic, nonionic, and zwitterionic
surfactants was examined to characterize their effects
on ESI-MS and on protein structure under ESI-MS
conditions. Solution conditions studied include 4%
acetic acid/50% acetonitrile/46% H_2O and 100% aqueous.
Of the surfactants examined, the nonionic saccharides,
such as n-dodecyl-beta-D-gluco-pyranoside, at 0.1% to
0.01% (w/v) concentrations, performed best, with
limited interference from chemical background and
adduct formation. Under the experimental conditions used,
ESI-MS performance in the presence of surfactants was
found to be unrelated to critical micelle concentration.
It is demonstrated that surfactants can affect both the
tertiary and quaternary structures of proteins under
conditions used for ESI-MS. However, several of the
surfactants caused significant shifts in the charge-
state distributions, which appeared to be independent
of conformational effects. These observations suggest
that surfactants, used in conjunction with ESI-MS, can
be useful for protein structure studies, if care is
used in the interpretation of the results.
SO - Protein Science 1994;3:1975-1983
AU - Monera OD
AU - Kay CM
AU - Hodges RS
TI - Protein denaturation with guanidine hydrochloride or urea
provides a different estimate of stability depending on
the contributions of electrostatic interactions
AD - Robert S. Hodges, Department of Biochemistry, University
of Alberta, Edmonton, Alberta T6G 2H7, Canada.
AB - The objective of this study was to address the question
of whether or not urea and guanidine hydrochloride
(GdnHCl) give the same estimates of the stability of a
particular protein. We previously suspected that the
estimates of protein stability from GdnHCl and urea
denaturation data might differ depending on the
electrostatic interactions stabilizing the proteins.
Therefore, 4 coiled-coil analogs were designed, where
the number of intrachain and interchain electrostatic
attractions (A) were systematically changed to
repulsions (R): 20A, 15A5R, 10A10R, and 20R. The GdnHCl
denaturation data showed that the 4 coiled-coil analogs,
which had electrostatic interactions ranging from 20
attractions to 20 repulsions, had very similar [GdnHCl]
1/2 values (average of [similar or equal to]3.5 M) and,
as well, their [Delta][Delta]G_u values were very close
to 0 (0.2 kcal/mol). In contrast, urea denaturation
showed that the [urea] 1/2 values proportionately
decreased with the stepwise change from 20
electrostatic attractions to 20 repulsions (20A, 7.4 M;
15A5R, 5.4 M; 10A10R, 3.2 M; and 20R, 1.4 M), and the
[Delta][Delta]G_u values correspondingly increased with
the increasing differences in electrostatic
interactions (20A - 15A5R, 1.5 kcal/mol; 20A - 10A10R,
3.7 kcal/mol; and 20A - 20R, 5.8 kcal/mol). These
results indicate that the ionic nature of GdnHCl masks
electrostatic interactions in these model proteins, a
phenomenon that was absent when the uncharged urea was
used. Thus, GdnHCl and urea denaturations may give
vastly different estimates of protein stability,
depending on how important electrostatic interactions
are to the protein.
SO - Protein Science 1994;3:1984-1991
AU - Covell DG
AU - Smythers GW
AU - Gronenborn AM
AU - Clore GM
TI - Analysis of hydrophobicity in the alpha and beta
chemokine families and its relevance to dimerization
AD - David G. Covell, Biomedical Supercomputing Laboratory, PRI
/Dyncorp, Frederick Cancer Research and Development
Center, National Cancer Institute, Frederick, Maryland
21702; or G. Marius Clore or Angela M. Gronenborn,
Laboratory of Chemical Physics, Building 5, National
Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, Maryland 20892-
0520.
AB - The chemokine family of chemotactic cytokines plays a key
role in orchestrating the immune response. The family
has been divided into 2 subfamilies,alpha and beta,
based on the spacing of the first 2 cysteine residues,
function, and chromosomal location. Members within each
subfamily have 25-70% sequence identity, whereas the
amino acid identity between members of the 2
subfamilies ranges from 20 to 40%. A quantitative
analysis of the hydrophobic properties of 11 alpha and
9 beta chemokine sequences, based on the coordinates of
the prototypic alpha and beta chemokines, interleukin-8
(IL-8), and human macrophage inflammatory protein-1beta
(hMIP-1beta), respectively, is presented. The monomers
of the alpha and beta chemokines have their strongest
core hydrophobic cluster at equivalent positions,
consistent with their similar tertiary structures. In
contrast, the pattern of monomer surface hydrophobicity
between the alpha and beta chemokines differs in a
manner that is fully consistent with the observed
differences in quaternary structure. The most
hydrophobic surface clusters on the monomer subunits
are located in very different regions of the alpha and
beta chemokines and comprise in each case the amino
acids that are buried at the interface of their
respective dimers. The theoretical analysis of
hydrophobicity strongly supports the hypothesis that
the distinct dimers observed for IL-8 and hMIP-1beta
are preserved for all the alpha and beta chemokines,
respectively. This provides a rational explanation for
the lack of receptor crossbinding and reactivity
between the alpha and beta chemokine subfamilies.
SO - Protein Science 1994;3:2064-2072
AU - Kumar A
AU - Sekharudu C
AU - Ramakrishnan B
AU - Dupureur CM
AU - Zhu H
AU - Tsai MD
AU - Sundaralingam M
TI - Structure and function of the catalytic site mutant Asp
99 Asn of phospholipase A_2: Absence of the conserved
structural water
AD - Muttaiya Sundaralingam or Ming-Daw Tsai, Department of
Chemistry, Biotechnology Center, The Ohio State
University, 120 West 18th Avenue, Columbus, Ohio 43210-
1002; e-mail: sunda%biot@mps.ohio-state.edu.
AB - To probe the role of the Asp-99[center dot][center dot]
[center dot]His-48 pair in phospholipase A_2(PLA2)
catalysis, the X-ray structure and kinetic
characterization of the mutant Asp-99 to Asn-99 (D99N)
of bovine pancreatic PLA2 was undertaken. Crystals of
D99N belong to the trigonal space group P3_121 and were
isomorphous to the wild type (WT)(Noel JP et al., 1991,
Biochemistry 30:11801-11811). The 1.9-Angstrom X-ray
structure of the mutant showed that the carbonyl group
of Asn-99 side chain is hydrogen bonded to His-48 in
the same way as that of Asp-99 in the WT, thus
retaining the tautomeric form of His-48 and the
function of the enzyme. The NH_2 group of Asn-99 points
away from His-48. In contrast, in the D102N mutant of
the protease enzyme trypsin, the NH_2 group of Asn-102
is hydrogen bonded to His-57 resulting in the inactive
tautomeric form and hence the loss of enzymatic activity.
Although the geometry of the catalytic triad in the
PLA2 mutant remains the same as in the WT, we were
surprised that the conserved structural water, linking
the catalytic site with the ammonium group of Ala-1 of
the interfacial site, was ejected by the proximity of
the NH_2 group of Asn-99. The NH_2 group now forms a
direct hydrogen bond with the carbonyl group of Ala-1.
SO - Protein Science 1994;3:2082-2088
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