From the National Creative Research Initiative Center, Korea Research Institute of Bioscience and Biotechnology, P. O. Box 115, Yusong, Taejon 305-600, Korea
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ABSTRACT |
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Metastability of the native form of proteins has
been recognized as a mechanism of biological regulation. The
energy-loaded structure of the fusion protein of influenza virus and
the strained native structure of serpins (serine protease inhibitors)
are typical examples. To understand the structural basis and functional
role of the native metastability of inhibitory serpins, we
characterized stabilizing mutations of
Facile conversion of the metastable native structure of proteins
into an alternative more stable form, accompanying the execution of
their functions, has been recognized as a mechanism of biological regulation. The energy-loaded structure of the fusion protein of
influenza virus (1), the strained native structure of plasma serpins
(serine protease inhibitors) (2), and possibly the surface glycoprotein
of human immunodeficiency virus
(HIV)1 (3) are typical
examples. The native strain of serpins is considered to be crucial to
their physiological functions, such as plasma protease inhibition (2,
4), hormone delivery (5), Alzheimer filament assembly (6, 7), and
extracellular matrix remodeling (8). The inhibitory serpins, which
include The serpin structure is composed of three To understand the structural basis of the loaded energy in the native
structure, we have been characterizing stabilizing amino acid
substitutions of Recombinant Chemicals--
Ultrapure urea was purchased from ICN
Biochemicals. Porcine pancreatic elastase and
N-succinyl-(Ala)3-p-nitroanilide were purchased from Sigma. All other chemicals were reagent grade.
Mutagenesis and Screening of Thermostable Mutants--
Random
mutagenesis at the target region was performed as described previously
using degenerative oligonucleotides (24), and thermostable mutations of
Urea-induced Equilibrium Unfolding Transition--
Equilibrium
unfolding as a function of urea was monitored by fluorescence
spectroscopy ( Determination of the Stoichiometry of Inhibition--
The
stoichiometry of inhibition was determined by titration reactions as
described (28). The active concentration of porcine pancreatic elastase
was determined by measuring the initial rates of hydrolysis of 1 mM
N-succinyl-(Ala)3-p-nitroanilide.
Various amounts of purified recombinant wild-type or mutant
Complex Formation with a Protease--
Complex formation of
mutant Molecular Properties of Mutant We found various stabilizing single amino acid substitutions at
over 10 sites in the target region (Fig.
1). The conformational stability of
representative mutant 1-antitrypsin in a region presumably involved in
complex formation with a target protease. We found various unfavorable
interactions such as overpacking of side chains, polar-nonpolar
interactions, and cavities as the structural basis of the native
metastability. For several stabilizing mutations, there was a
concomitant decrease in the inhibitory activity. Remarkably, some
substitutions at Lys-335 increased the stability over 6 kcal mol
1 with simultaneous loss of activity over 30% toward
porcine pancreatic elastase. Considering the location and energetic
cost of Lys-335, we propose that this lysine plays a pivotal role in
conformational switch during complex formation. Our current results are
quite contradictory to those of previously reported hydrophobic core mutations, which increased the stability up to 9 kcal
mol
1 without any significant loss of activity. It appears
that the local strain of inhibitory serpins is critical for the
inhibitory activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1-antitrypsin (
1AT), antithrombin
III,
1-antichymotrypsin, and plasminogen activator
inhibitor-1, serve as a good model system to study the native
metastability; several crystal structures of both the strained native
(9-13) and the relaxed cleaved forms (14-16) are available. In
addition, the inhibitory activity that presumably is related to the
native metastability is easy to assay.
-sheets and several
-helices (Fig. 1). Upon binding a target protease, the reactive center loop of inhibitory serpins is thought to be inserted into the
major
-sheet, A sheet, to form a very stable complex between the
inhibitor and the protease (17, 18). Various biochemical (19, 20) and
structural (21-23) studies suggest that the loop insertion is
necessary for the formation of a stable complex but not sufficient to
confer inhibitory activity. Instead, the rate of loop insertion is
thought to be critical for inhibitory function. The inhibition process
of serpins can be described as a suicide substrate mechanism (17), in
which serpins, upon binding with proteases, partition between cleaved
serpins and stable serpin-enzyme complexes in a ratio represented by
the stoichiometry of inhibition (SI, number of moles of inhibitors
required to completely inhibit 1 mol of a target protease). The SI
values of most inhibitory serpins are close to one for cognate target
proteases. Retardation of the loop insertion, however, would alter the
partitioning between the inhibitory and substrate pathways in such a
way that the SI value would increase since the SI value is defined by 1 + ksubstrate/kinhibition (17, 18, 20). It is conceivable that the energy loaded in the strained
native structure of serpins is utilized for the facile loop insertion.
1AT, a prototype inhibitory serpin. We previously reported that decrease in the size of side chains at the
hydrophobic core of
1AT (Fig. 1, blue)
confers increased stability (24). That was quite unusual, as a decrease
in size inside the hydrophobic core usually yields a cavity that causes loss of stability (25). We proposed that side chain locking in the
native
1AT prohibits rearrangement of the side chains for maximal packing (24). Such structural defects are likely to be the
basis of the native strain in the inhibitory serpins. If the energy
loaded in the strained native structure of serpins is utilized for the
inhibitory function, mutations that decrease the loaded energy should
also decrease the activity. In the present study, we tested this
concept of the native strain. We searched for mutations that increased
the stability and simultaneously affected the inhibitory activity. We
characterized stabilizing amino acid substitutions of
1AT in a region that is presumably involved in the
conformational change for the insertion of the reactive center loop
during the inhibitory complex formation: strands 3 and 5 of A sheet
(s3A and s5A), helix F (hF), and the connecting loop (Fig. 1,
purple). Characterization of the mutant proteins and
structural examination of the mutation sites revealed various unusual
interactions as the structural basis of the native metastability.
Functional analyses provide direct evidence that the energy loaded in
the native inhibitory serpins is utilized for the biological activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1AT Proteins--
Plasmids for
1AT expression in Escherichia coli, and the
refolding and purification of recombinant
1AT protein
were described previously (26). Concentrations of
1AT
were determined in 6 M guanidine hydrochloride using a
value of A1 cm1% = 4.3 at 280 nm, calculated from the tyrosine and tryptophan content of the
1AT protein (27) and based upon
Mr = 44,250.
1AT were screened after heat treatment of cell lysates
at 60 °C for 1 h as described previously (26). Substitutions at
specific sites were generated by oligonucleotide-directed mutageneses.
ex = 280 nm and
em = 360 nm, excitation and emission slit widths = 5 nm for both), details
of which were described previously (24, 26). The buffer was 10 mM phosphate, 50 mM NaCl, 1 mM
EDTA, and 1 mM
-mercaptoethanol, pH 6.5, and the protein
concentration was 10 µg/ml. The native protein was incubated in the
buffer containing various concentrations of urea at 25 °C.
Experimental data of the fluorescence measurement were fitted to a
two-state unfolding model.
1AT proteins were incubated in 50 µl of assay buffer
(30 mM phosphate, 160 mM NaCl, 0.1% PEG 6000, and 0.1% Triton X-100, pH 7.4) with 100 nM porcine
pancreatic elastase at designated molar ratios of
1AT to
protease. After incubation with protease for 10 min at 37 °C, the
reaction mixture was diluted 10-fold with the assay buffer and the
residual enzyme activity was determined using 1 mM
N-succinyl-(Ala)3-p-nitroanilide as a
substrate (29).
1AT with porcine pancreatic elastase was examined
by monitoring the SDS-resistant
1AT-proteinase complex
(28). The active concentration of porcine pancreatic elastase was
determined as described above. Purified
1AT was incubated in assay buffer with the protease at designated molar ratios
of
1AT to protease. Samples were incubated at 37 °C
for 10 min and were analyzed by 10% SDS-polyacrylamide gel
electrophoresis. The protein bands were visualized by Coomassie
Brilliant Blue.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1AT
1AT was measured by equilibrium unfolding in the presence of urea in which changes in intrinsic tryptophan fluorescence intensity were monitored. The changes in free
energy of stabilization (
G) of the mutant proteins are summarized in Table I. For many of the
sites, additional substitutions were made by introducing alanine, among
which stabilizing mutations were further characterized (Table I). One
remarkable result is that K335A and K335G mutations showed a profound
effect on stability (Fig. 2A),
shifting the midpoint of the unfolding transition from 1.8 M to 3.9 M and 4.0 M urea,
respectively, which resulted in the increase of stability over 6 kcal
mol
1. This is the biggest increase in stability of
1AT by a single amino acid substitution studied so far.
The effect of the mutations in
1AT on the inhibitory
activity was examined by determining the SI values against a target
protease. The SI values of K335A and K335G
1AT, which
showed dramatic increase in stability (Fig. 2A), were
increased substantially (Fig. 2B). The SI value of K168A, which showed little increase in stability (Fig. 2B), was
also increased (Fig. 2B). The changes in SI value were due
to the alteration of the partitioning between the inhibitory and
substrate pathways. When the mutational effects on the formation of
protease-inhibitor complex were analyzed on SDS-polycrylamide gel, the
ratios of cleaved proteins over complexes were increased by K335A and
K335G mutations compared with wild-type
1AT (Fig.
3), indicating that there was a shift in
partitioning toward substrate pathway in mutant proteins. The effects
of other stabilizing mutations on inhibitory activity were determined
and the results are summarized in Table I.
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Fig. 1.
A schematic diagram of the native structure
of 1AT (12). The region
subjected to the present studies is colored purple. The
designation of secondary structures are described in the text. The
identified mutation sites are marked by beads, and the amino
acid residues are shown by one-letter codes. The colors of
the beads are the same as in Fig. 4. The hydrophobic core
region which was targeted in the previous study (24) is represented in
blue. The figure was prepared using MOLSCRIPT program.
Characteristics of mutant 1AT in the loop-insertion region
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Fig. 2.
Molecular properties of K335A, K335G, and
K168A mutant 1AT. A,
urea-induced equilibrium unfolding transition. Unfolding transitions
were measured by the increase in fluorescence emission intensity at 360 nm (
ex = 280 nm). Samples were equilibrated in each urea
solution containing 10 mM potassium phosphate, 50 mM NaCl, 1 mM EDTA, 1 mM
-mercaptoethanol (final pH, 6.5) for 8 h at 25 °C. The
protein concentration was 10 µg/ml.
, wild-type;
, K168A;
,
K335A;
, K335G. B, stoichiometry of inhibition of the
mutant
1AT against porcine pancreatic protease. A
constant concentration of the protease was mixed with varying amounts
of
1AT. After incubation at 37 °C for 10 min in the
assay buffer (30 mM phosphate, 160 mM NaCl,
0.1% PEG 6000, 0.1% Triton X-100, pH 7.4), the residual protease
activity was measured with 1 mM
N-succinyl-Ala-Ala-Ala-p-nitroanilide as a
substrate.
, wild-type;
, K168A;
, K335A;
, K335G.
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Fig. 3.
Mutational effect on the formation of
1AT-protease complex. The
wild-type and Lys-335 mutant
1AT proteins were incubated
with porcine pancreatic elastase at designated molar ratios
(I/E), shown on the top of the figure, of
1AT to protease. Samples were incubated at 37 °C for
10 min in 30 mM phosphate, 160 mM NaCl, 0.1%
PEG 6000, and 0.1% Triton X-100, pH 7.4. The formation of
SDS-resistant
1AT-protease complex was analyzed on 10%
SDS-polyacrylamide gel electrophoresis. 5 µg of
1AT
was loaded on each lane except lane Ez. Lanes:
1AT,
1AT protein incubated
without porcine pancreatic elastase; wt, wild-type
1AT protein incubated with porcine pancreatic elastase;
A, K335A mutant
1AT protein incubated with
porcine pancreatic elastase; G, K335G mutant
1AT protein incubated with porcine pancreatic elastase;
Ez, porcine pancreatic elastase (1 µg is loaded).
Cp, inhibitor-protease complex; N, native
1AT; Cl, cleaved
1AT;
E, porcine pancreatic elastase.
Correlation between Loaded Energy and Inhibitory Activity
If the loaded energy in the native serpins is utilized for
inhibitory complex formation, there should be a correlation between stability increase and loss of inhibitory activity. The activity change
was plotted as the function of stability increase for each mutant
protein (Fig. 4), and the mutations were
grouped into three classes according to their properties (Table I). For
group I mutations (top group in Table I), there seems to be a
correlation between the increase in conformational stability and the
loss in inhibitory activity (Fig. 4, circles; Fig. 1,
yellow beads). Remarkably, the substitutions at
Lys-335, K335A and K335G, increased the stability over 6 kcal
mol1 with concomitant losses of activity over 30% toward
porcine pancreatic elastase. These results suggest that the loaded
energy is related to the inhibitory function at Lys-335, and possibly
at other sites. For the group II substitutions (middle group in Table
I), K168A, K168I, or I169V barely increased the conformational
stability but affected inhibitory activity substantially (Fig. 4,
triangles; Fig. 1, orange beads). It
seems, however, that for these mutations the loss of activity is not
due to an increase in conformational stability of the native form
per se but to a decrease in the stability of the
serpin-protease complex. Such mutations can be obtained in our initial
screening for thermostable variants, because the screening is based on
enhanced kinetic stability against aggregation (26). It has been
suggested that the mechanism of heat-induced aggregation of
1AT is loop-sheet polymerization in which the reactive
center loop of one molecule is inserted into A sheet of another (30).
For the group III mutations (bottom group in Table I), K163T, G164V, or
A183V, the activity of
1AT was less affected than
expected from the increase in stability (Fig. 4, green
squares; Fig. 1, green beads). Unlike
group I mutations, which decreased the size of side chains, G164V and
A183V increased the size of side chains. The hydrophobic core mutation
reported previously, Multi-7, did not show any significant activity
change even with a stability increase up to 9 kcal mol
1
(28). It is also included in the plot for comparison, and the relationship between stability increase and activity change is also
indicated (Fig. 4, blue square; dashed
line).
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Structural Basis of Metastability
In the crystal structure of the native 1AT (12),
the stabilizing mutation sites are composed of many unusual
interactions, which appear to be the basis of the metastability. These
interactions are likely to be mobilized during the complex formation
with a target protease. From the mutational analysis, the following
modes were revealed as structural basis of the native metastability of
1AT.
Overpacking of Side Chains--
Lys-335 interacts with hydrophobic
residues, Ile-169 and Leu-172 (Fig.
5A). The N atom of Lys-335
side chain may be paired with the backbone carbonyl oxygen of Ile-188
(2.87 Å) and O
atom of Thr-165 (3.72 Å), but the aliphatic part of
Lys-335 is squeezed in hydrophobic interactions provided by Ile-169
(3.45 Å) and Leu-172 (3.24 Å). It appears that K335G or K335A
mutation relieved such strain by decreasing the size of the side chain.
Remarkably, many stabilizing substitutions such as T165S, I169V, L172V,
and L172A are the ones occurring at the residues interacting with
Lys-335 (Fig. 5A). Furthermore, the substitution pattern
showed size reduction as a common theme. Leu-172 is unusually close to
Lys-335 (3.24 Å). L172V as well as L172A, which increased the
stability even more (Table I), might have eliminated such an
overpacking of the side chains. Stabilizing mutations such as F189I and
F189V also showed the size reduction pattern. Size reduction to alanine at 189, however, resulted in the loss of stability. We previously reported that decrease in the size of side chains at the hydrophobic core of
1AT (Fig. 1, blue) confers increased
stability (24). Structural determination of
1AT variants
carrying stabilizing substitutions at the hydrophobic core revealed
better packing at the mutation sites (12, 13, 31). The substitution
pattern observed in the present study (Table I) revealed that size
reduction in the present target region (Fig. 1, purple) also
increases the stability of the molecule. It appears that overpacking of
the side chains is one mechanism of the native strain of
1AT.
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Polar-Nonpolar Interactions--
The present studies revealed
various polar-nonpolar interactions as an additional mode of
destabilization in the target region. The side chain of Lys-335 is not
only squeezed but also surrounded by hydrophobic residues. It is
unusual to find charged residues in the hydrophobic environment.
Studies on other mutant proteins revealed that the energetic cost of
burying a charge in a hydrophobic environment is estimated be 3-9 kcal
mol1 (32). Mutations at Lys-335 of
1AT
increased the stabilizing energy by 3-7 kcal mol
1 (Table
I). Phe-189 is on a surface pocket (Fig. 5A), interacting unfavorably with the backbone carbonyl oxygen of Gly-164 (2.89 Å).
Thr-165 appears to stabilize the end of helix F and the following turn
by providing hydrogen bonding to the backbone carbonyl oxygen of
Val-161 and backbone nitrogen of Ile-169. The C
2 atom of Thr-165, however, has close interactions with the polar atoms of the backbone, while the hydroxyl group of Thr-165 is close to the C
atom of Lys-335 (3.47 Å). T165S might have relieved such strain while maintaining the H-bond networks with backbone atoms of Val-161 and
Ile-169. As expected, substitution of Thr-165 by alanine decreased stability. Leu-172 also has an unfavorable interaction with the backbone carbonyl oxygen of Asn-186 on s3A (2.97 Å). Lys-331 is on a
surface pocket, interacting with Val-333 and Val-173. K331M increased
the stability (Table I) possibly by providing more favorable
interactions with the surrounding residues. It is likely that these
unfavorable polar-nonpolar interactions are another basis of the native
metastability of
1AT.
Cavities--
Structural examination of the target region also
revealed the existence of surface cavities. Two of the substitutions
identified in the present study, A183V and G164V, do not fit the common
theme of size reduction for stabilization of the native
1AT. In both cases, size increase rather than size
reduction caused the stability increase. In the native structure (12),
the region near Gly-164 and Ala-183 is not well packed, leaving an
empty patch on the surface (Fig. 6). The
surface cavity near Gly-164 is surrounded by the side chain atoms of
Phe-189 and Lys-335. Likewise, the side chain of Ala-183 does not show
much interaction with other side chains. Surrounding hydrophobic
residues such as Phe-147, Ile-157, Val-185, and Leu-172 are at least 5 Å apart. Cavities are very likely to be the source of energetic cost
in conformational stability (33). The stabilizing substitutions, G164V
and A183V, appear to provide better packing by filling the nearby
cavities.
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Favorable Interactions in the Complex Form
In the cleaved relaxed form, the identified mutation sites have
favorable interactions. The crystal structure of the serpin-protease complex is not available yet. However, it has been proposed that accompanying the structural transition to the relaxed complex form, the
reactive center loop is at least halfway (up to P9 position, the 9th
residue amino-terminal to the scissile peptide bond) inserted into A
sheet (34-36), as in the relaxed cleaved structure. If this is the
case, one can refer to the cleaved structure (14) to understand the
complex. The side chain of Lys-335, which is buried in the native state
(Fig. 5A), is exposed in the cleaved structure and makes a
salt bridge with Asp-171 (Fig. 5B). Likewise, Lys-168 is
paired with Glu-346 (P13 position) of the inserted reactive center loop
(Fig. 5B). The side chain of Ile-169, which is exposed on
the surface in the native form (Fig. 5A), interacts in the cleaved form with the side chain of Val-337 of s5A (Fig. 5B)
and C atom of Gly-349 (P10 position) of the inserted reactive center loop. Residues at other mutation sites also are engaged in favorable interactions in the cleaved structure. It is very likely that the
conversion from the native unfavorable interactions to more favorable
interactions in the complex is the driving force for the conformational
switch needed for the complex formation.
Mutations at Lys-168 and Ile-169, which decreased the inhibitory activity without increasing the stability (Fig. 4, triangles) suggest that the salt bridge between Lys-168 and Glu-346 shown in the cleaved structure (Fig. 5B) plays an important role in the complex stability. Likewise, Ile-169 appears to play a role in stabilizing the loop-inserted structure. It is not clear, however, if the salt bridge between Lys-335 and Asp-171 also plays a significant role in the complex formation, because D171N mutation did not affect the inhibitory activity significantly.2 It may be that Lys-335 mainly contributes to destabilization of the native form during the complex formation. It appears that each identified residue in the present target region contribute distinctively to the complex formation; some play a critical role in destabilizing the native form, some in the complex stability, and others in serving the dual role of destabilizing the native structure and stabilizing the complex. Many of these residues are conserved among inhibitory serpin sequences (2). The crystal structure of a serpin-protease complex will confirm the contribution of individual residues.
Importance of Local Strain in the Inhibitory Function
The mutational effect on the inhibitory activity observed in the
present study is in contrast to that obtained previously with the
stabilizing mutations at the hydrophobic core (Fig. 1, blue). The inhibitory activity of 1AT is very
sensitive to the stabilization in the target region (Table I; Fig. 4).
In contrast, the increase in the stabilization energy up to 9 kcal
mol
1 in the hydrophobic core by combining seven
stabilizing single amino acid substitutions (F51L, T59A, T68A, A70G,
M374I, S381A, and K387R; the mutant was named Multi-7) did not affect
the inhibitory activity of
1AT toward target elastases
(28). The results imply that local stability of the serpin is critical
to inhibitory activity. It may be that some parts of the serpin
molecule, especially the region where the reactive center loop is
inserted, have to be loosened during the complex formation, whereas
other parts like the hydrophobic core need not change as much. In this
regard, it is interesting that while side chain locking among nonpolar side chains is the major cause of metastability in the hydrophobic core
of
1AT (24), at the loop insertion site additional
schemes like unfavorable polar-nonpolar interactions are observed. The polar groups may govern structural specificity during the complex formation. Internal polar groups are often found to be destabilizing (37, 38), but have evolved to impart structural uniqueness (37, 39). It
appears that the local strain of inhibitory serpins is critical for
relating the loaded energy to functional regulation.
Implications for the Inhibitory Mechanism
Results in the present study strongly suggest that nature designed
the native form of inhibitory serpins to be poorly folded with the
purpose of carrying out a sophisticated regulation of protease
inhibition. Our study also revealed that various folding defects such
as side chain locking, buried polar groups in unfavorable hydrophobic
environments, and cavities are the structural basis of the
metastability of 1AT. These folding defects appear to be
designed to destabilize the interaction of helix F and the following
loop with A sheet (Fig. 1). Especially, Lys-335 is in a strategic
position for opening A sheet; the side chain of Lys-335 in the
uncleaved native form is tightly squeezed by the surrounding hydrophobic residues from helix F and the following loop that covers A
sheet (Fig. 5A). Considering the location (Fig. 1) and energetic cost (Fig. 2; Table I) of Lys-335, we propose that this
lysine plays a pivotal role during the complex formation, possibly by
lifting helix F and the following loop from A sheet, which is likely to
lead to the facile loop insertion during the complex formation. This
lysine is conserved among inhibitory members of serpins such as
antithrombin-III,
1-antichymotrypsin, and plasminogen
activator inhibitor-1, and hormone binding globulins of serpin family,
but not in non-inhibitory members like ovalbumin and angiotensinogen
(2). From our present data, however, it is not yet clear how much the
cavities in this region contribute to the conformational transition
during the complex formation, because the mutations supposedly filling
the cavities of
1AT, G164V and A183V did not affect the
inhibitory activity as much as expected from the stability increase
(Fig. 4, green squares).
It is worth noting that the recently determined crystal structure of gp120, the surface glycoprotein of HIV, complexed with soluble CD4 also revealed unusual interactions such as a large cavity and polar-aromatic interactions (40). Since the structure of free gp120 is not known, it is not yet clear how these unusual interactions contribute to conformational change of gp120 or gp41, the membrane fusion protein of HIV. Interestingly, the native form of hemagglutinin, the fusion protein of influenza virus, also has big cavities especially in the subunit contacts of the trimers (41). Although it was shown (42) that destabilization of the metastable native hemagglutinin yielded a fusion active state (43), structural basis underlying the conversion, including the role of the cavities, has not been understood.
Finally, a relationship between protein stability and function has not
been established unequivocally. According to still controversial
"stability-function hypothesis," residues contributed to function
may not be optimal for stability. This was clearly shown for the active
site residues of T4 lysozyme (44) and barnase (45). However, in case of
E. coli major cold shock protein, CspA, residues
constituting surface aromatic network has evolved for both function and
stability (46). In the case of 1AT, there is an inverse
correlation between protein stability and function in the protease
binding site. Still, suboptimal stability of
1AT appears
to be a prerequisite for functional execution rather than a consequence
of functional reconciliation. Interestingly, most of the substituting
residues in stabilizing mutations of
1AT are the ones
already existing in the sequence of ovalbumin (Table I), a
non-inhibitory member of the serpin family. Ovalbumin and inhibitory
serpins share a common ancestor (47). The molecule might have evolved
for better folding and stability in the ovalbumin line, but for
acquiring inhibitory function in the inhibitory serpin line.
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ACKNOWLEDGEMENTS |
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We thank S. E. Ryu, S.-H. Park, and C. Lee for helpful comments and S. E. Ryu for the graphic work.
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FOOTNOTES |
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* This work was supported by the Creative Research Initiatives Project of the Korean Ministry of Science and Technology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom reprint requests should be addressed. Tel.:
82-42-860-4140; Fax: 82-42-860-4593; E-mail:
mhyu{at}kribb4680.kribb.re.kr.
2 H. Im and M.-H. Yu, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
HIV, human
immunodeficiency virus;
1AT,
1-antitrypsin;
PEG, polyethylene glycol;
SI, stoichiometry of inhibition.
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