(Received for publication, November 13, 1995; and in revised form, January 18, 1996)
From the
Recombinant interstitial collagenase (rMMP-1) forms insoluble
inclusion bodies when over-expressed in Escherichia coli. We
surveyed conditions for renaturation of purified rMMP-1 in 6 M guandine hydrochloride (GdnHCl) and found that optimal folding
occurred when the denatured protein was diluted at 4 °C in 2 M guanidine HCl, 20% glycerol, 2.5 mM reduced and
oxidized glutathione, and 5 mM CaCl
, followed by
buffer exchange to remove denaturant and thiols. The circular dichroism
spectrum and catalytic constants of the refolded enzyme were similar to
those of native MMP-1. The propeptide, which comprises approximately
20% of the mass of proMMP-1, was not required for folding to a
functional enzyme. Size exclusion chromatography and spectroscopic
measurements at intermediate [GdnHCl] revealed two
intermediate folding states. The first, observed at 1 M GdnHCl, had a slightly larger Stokes' radius than the folded
protein. CD and fluorescence analysis showed that it contained ordered
tryptophan residues with a higher quantum yield than the fully folded
state. The second intermediate, which appeared between 2 and 4 M GdnHCl, exhibited properties consistent with the molten globule,
including secondary structure, lack of ordered tryptophan, exposed
hydrophobic binding sites, and a Stokes' radius between that of
the folded and unfolded states.
Interstitial collagenase (EC 3.4.24.7) is a member of the matrix
metalloproteinase family of enzymes which function in remodeling the
extracellular matrix. Elevated MMP ()activity is associated
with several physiological and pathological processes, including
embryogenesis, arthritis, corneal ulceration, cancer metastasis, and
periodontal disease(1, 2) . Procollagenase is secreted
from a variety of cell lines in culture, including fibroblasts (3) and umbilical vein endothelial cells(4) , as a
mixture of glycosylated 57-kDa and unglycosylated 52-kDa zymogens that
are activated by removal of an amino-terminal propeptide to give the
corresponding 48- and 42-kDa proteinases. MMP-1 (fibroblast
collagenase) and its neutrophil homolog (MMP-8) initiate the breakdown
of collagen fibrils by cleaving collagen types I, II, and III at a
single location in each
chain to generate three-quarter and
one-quarter length fragments, which are then degraded by other
proteinases. Sequence and homology analysis coupled with functional
comparison of full-length, truncated, and chimeric constructs indicate
that MMP-1 consists of three domains: an N-terminal propeptide of
approximately 80 residues, a 19-kDa catalytic domain of approximately
160 residues, and a C-terminal domain of 209 residues. The C-terminal
domain binds collagen and shares sequence similarity with the plasma
protein hemopexin and vitronectin, which also interacts with
extracellular matrix proteins(5) . The pexin domain is
essential for the expression of collagenolytic activity by MMP-1, since
the catalytic domain alone, while active against artificial substrates,
does not hydrolyze native collagen(6, 7, 8) .
The catalytic and pexin domains are linked by a proline-rich segment of
about 17 residues.
The three-dimensional structures of 19-kDa MMP-1
and the homologous domain of MMP-8 have been
determined(9, 10, 11, 12, 13) .
These studies reveal a catalytic unit consisting of a five-stranded
-sheet structure and three
-helical segments. Binding sites
for catalytic and structural zinc ions as well as three calcium ions in
MMP-1 and two in MMP-8 are present. More recently, the crystal
structure of full-length porcine MMP-1 was published(13) . The
collagen-binding pexin domain is composed of antiparallel
-sheets
that fold into a motif described as a
-propeller. The pexin domain
contains a single calcium binding site situated near the apex of the
propeller(13) . As expected, the catalytic domain of the pig
enzyme is similar to that of the human 19-kDa derivatives.
The precise role of the C-terminal pexin domain in conferring collagenolytic specificity to MMP-1 is not known. One means of exploring this question is through the study of mutant collagenases generated by recombinant DNA methodology. A prerequisite for such a study is to optimize the folding reaction of rMMP-1 and to compare the recombinant and wild-type proteins. This report summarizes a procedure developed to optimize the yield of renatured recombinant collagenase from inclusion bodies produced by over-expressing the protein from a plasmid in Escherichia coli. The unfolded and refolded states of the proteinase were characterized in terms of catalytic constants, Stokes' radius, fluorescence, and CD spectroscopic properties. To aid in elucidating the refolding pathway, we measured the dependence of various structural probes on denaturant concentration. The results suggest that unfolding in the presence of denaturant proceeds through two intermediates, one of which exhibits properties consistent with those of the ``molten globule'' state detected in the folding pathway of many proteins(14) .
The MMP substrate
Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH was
synthesized by Stack and Gray(17) . Guanidine HCl (ultrapure)
was from U. S. Biochemical Corp.; ANS and type I collagen from calf
skin were from Sigma; Brij 35 was obtained as a purified product from
Pierce Chemical Co.
Figure 1:
Effect of [GdnHCl] on
recovery of protein and catalytic activity in refolding. rMMP-1 in 6 M GdnHCl was diluted to a final concentration of 4.6
µM in different concentrations of GdnHCl (0.6 to 5.2 M). Turbidity () was estimated from the apparent
absorbance at 450 nm. Enzyme activity (
, measured by the
hydrolysis of a synthetic peptide substrate) and soluble protein
recovered (
) were determined after GdnHCl was removed by a
spin-column procedure as described under ``Experimental
Procedures.''
We next investigated the effect of the redox potential of the folding medium on the yield of active enzyme. MMP-1 contains a pair of cysteine residues in the COOH-terminal collagen binding domain that have been reported to form a disulfide bond essential for collagenolytic activity (8, 26) . To catalyze disulfide shuffling and promote intramolecular disulfide pairing rather than intermolecular pairing during refolding, rMMP-1 was incubated in the presence of different ratios of oxidized to reduced thiol reagent(27) . The data of Fig. 2demonstrate that the recovery of enzyme activity was highest when renaturation proceeded in the presence of 2.5 mM GSSG and 2.5 mM GSH. The glutathione redox buffer system was more effective than the buffer with DTT.
Figure 2: Effect of thiol reagents on the recovery of activity during refolding of rMMP-1. rMMP-1 was diluted to 4.6 µM in 2.3 M GdnHCl containing different ratios of oxidized and reduced thiol reagent. GSH=GSSG, 2.5 mM of each reagent; DTTox = DTTred, 2.5 mM each of oxidized and reduced DTT; GSSG, 5 mM GSSG; GSH, 5 mM GSH; DTTox, 5 mM oxidized DTT; 5 GSH:1GSSG, 5 mM GSH + 1 mM GSSG; 1GSH:5GSSG, 1 mM GSH + 5 mM GSSG. Buffers were purged with argon to eliminate oxygen. Enzyme activity was assayed with DnpS after GdnHCl and thiol reagent were removed by a spin column procedure.
The effects of glycerol, a protein stabilizing agent, and elevated temperature, which enhances hydrophobic interactions, were also assessed. Glycerol in the presence of GSH/GSSG increased the yield of active refolded protein and decreased the proportion of precipitated protein from 59 to 6.4%. The extent of precipitation was also found to be temperature-dependent. Protein precipitation increased from about 6% at 4 °C (as indicated above) to 32% at 25 °C and 47% at 35 °C.
MMP-1 binds
Ca which contributes to its stability (28, 29, 30, 31) . To determine if
Ca
plays a role in the folding reaction, rMMP-1 was
incubated in different concentrations of Ca
ranging
up to 10 mM. The different Ca
levels were
maintained at all folding steps until the enzyme activity assay. The
samples were then assayed in 11 mM Ca
. No
catalytically active protein was obtained when refolding took place in
the absence of added Ca
. The yield of active enzyme
increased with increasing Ca
to 5 mM and
remained constant to the highest concentration tested (9.5
mM).
Figure 3: Fluorescence emission spectra of folded and unfolded rMMP-1. Folded rMMP-1 was diluted to 0.2 µM in folding buffer without GdnHCl (-) or 6 M GdnHCl (- - -). After incubating the sample at 25 °C for 24 h, fluorescence emission spectra were recorded from 310 to 450 nm. Excitation wavelength was 280 nm.
Figure 4: Far UV CD of folded and unfolded rMMP-1 and pro-MMP-1. Panel A, folded rMMP-1 (1.9 µM) was incubated without GdnHCl (-) or with 6 M GdnHCl (- - - -) in folding buffer at 25 °C for 24 h. CD spectra were then recorded after centrifugation. Panel B, the CD spectrum of proMMP-1 (2.4 µM) was measured in folding buffer. In both panels, the dashed line represents the best fit of the experimental data using the program LINCOMB ( (33) and (34) ; see text and Table 1).
The CD spectra
were analyzed in terms of secondary structure using the fitting program
LINCOMB of Perczel et al.(33, 34) ; the
results are summarized in Table 1. Briefly, LINCOMB assumes that
the measured CD spectrum can be represented by a linear combination of
five reference CD spectra obtained from the CD spectra of 25 proteins
of known secondary structure. The reference or ``template''
spectra represent contributions to the far UV CD spectrum of a protein
by -helix,
-turn, and/or parallel
-sheet, aromatic and
disulfide, unordered and/or
-turn and antiparallel
-sheet. By
this method of analysis, both rMMP-1 and native proMMP-1 gave
comparable amounts of
-helix (approximately 30%) and
-structure (30-40%). However, the contribution of disulfide
and aromatic chromophores to the CD spectrum of the proenzyme appears
to be larger than in the recombinant enzyme missing the propeptide (28 versus 16%, respectively).
Figure 5:
SEC-HPLC elution profiles of rMMP-1 at
different concentrations of GdnHCl. Refolded rMMP-1 was diluted into
GdnHCl at the concentrations indicated and incubated for at least 1 h
prior to application to a Bio-Sil SEC 125-5 column equilibrated
with folding buffer containing the indicated GdnHCl concentrations. The
monitoring wavelength was 220 nm. The amount of protein injected at
each GdnHCl concentration was: 0 M, 20 µg; 1 and 2 M, 3.5 µg; 3 M, 8.5 µg; 4 and 6 M,
3.5 µg. Species with essentially identical elution times were
observed at 3 M GdnHCl when 3.5 µg of rMMP-1 was injected.
A peak representing buffer salts eluting at the void volume of the
column (10.1 min) is not shown. K values
and Stokes' radii are summarized in Table 2.
Figure 6:
Fluorescence emission spectra of ANS in
the presence and absence of rMMMP-1. Fluorescence emission spectra of
10 µ ANS in folding buffer + 1.8 M GdnHCl
with and without rMMP-1 (1 µM). Excitation wavelength was
394 nm. The peak at 450 nm is Raman scattering from the
solvent.
Figure 7: Near UV CD of folded rMMP-1 in different concentrations of GdnHCl. The CD spectrum of folded rMMP-1 (5.5 µM) was recorded from 310 to 250 nm. Solid GdnHCl was then added to the sample to give 0.8, 1.0, 2.0, 3.0, and 4.0 M denaturant. The increase in volume caused by the added GdnHCl was calculated using its partial specific volume of 0.75 µl/mg. The protein concentration of each sample was determined by dye-binding assay (18) after the spectrum was recorded.
Fig. 8summarizes
the effect of GdnHCl concentration on enzyme activity, secondary
structure, and tertiary structure as monitored by the tryptophanyl
residues. The parameters displayed are ellipticity at 222 and 290 nm,
fluorescence emission maximum, quantum yield, and enzyme activity.
Inspection of Fig. 8A shows that catalytic activity
disappeared by 1.5 M GdnHCl. In the range of
[GdnHCl] between 0 and 1 M, there was a slight
biphasic shift in emission maximum from 332 to about 340 nm, suggesting
an increase in solvent accessibility of one or more tryptophanyl
residues. Accompanying this shift was an increase in emission intensity
and in tryptophanyl asymmetry as assessed from the near UV CD data (Fig. 8B). The species displaying these characteristics
probably corresponds to I (elution time of 7.6 min) in
SEC-HPLC. Between
1.2 and 2 M GdnHCl, the emission
intensity declined and remained relatively constant at a level of about
65% of that of the refolded enzyme. Above 2 M GdnHCl the
emission maximum increased to about 352 nm and remained at this
wavelength as the CD shoulder at 290 nm disappeared, indicating
disruption of tertiary structure. Secondary structure, as indicated by
the relative constant ellipticity at 222 nm, was retained. This
probably corresponds to the SEC-HPLC state I
at
6.7
min. With a further increase in denaturant to 4 M GdnHCl,
secondary structure was lost, the emission maximum increased to 356 nm
(typical of fully exposed tryptophan), and emission intensity dropped
to about 15% of that of the refolded protein. This corresponds to the
unfolded state observed in the molecular sieve experiments at
6
min.
Figure 8:
Guanidine-induced denaturation of rMMP-1
detected by activity, fluorescence, and circular dichroism. Panel
A, folded rMMP-1 (0.2 µM) was incubated in different
concentrations of GdnHCl from 0 to 6 M at 25 °C for 24 h.
The fluorescence intensity at 332.5 nm (), wavelength of emission
maximum (+), and enzyme activity ([circf) were determined.
The relative change in fluorescence intensity was calculated from the
expression (F
- F
)/(F
- F
), where F
, F
, and F
are the
fluorescence intensity at 332.5 nm of highest, lowest, and any given
GdnHCl concentration, respectively. The relative change in enzyme
activity was estimated from (A
- A
)/(A
- A
), where A
, A
, and A
are the enzyme
activity of highest, lowest, and any given GdnHCl concentration,
respectively. For CD measurements (panel B), rMMP-1 was
incubated in different concentrations of GdnHCl at 25 °C for 24 h.
Secondary structure was approximated from the ellipticity at 220 nm
(
) at each GdnHCl concentration. The relative change was
calculated from the expression (
-
)/(
-
), where
,
, and
are the molar
ellipticities at 220 nm of highest, lowest, and any given GdnHCl
concentration, respectively. Changes in tertiary structure in the near
UV spectral region (
) were also analyzed. To increase the
accuracy of estimating the magnitude of the 290 nm shoulder of the CD
spectra of Fig. 7, the first derivative (d
/d
) of each spectrum was calculated and
the magnitude of the resulting peak-to-trough at 290 nm was used to
estimate the relative change in this folding parameter at each GdnHCl
concentration. The fluorescence emission maxima in the top panel are included for reference in the bottom
panel.
It is well known that heterologous proteins when overexpressed in bacteria often aggregate to form inclusion bodies(42) . While isolation of the inclusion bodies serves as a convenient enrichment step, it remains necessary to solubilize the recombinant protein, a process requiring strong denaturants. To generate the fully functional protein, one must then remove the denaturant to allow refolding. That folding often proceeds with poor yield has been attributed to competing pathways of folding and self-aggregation, both of which result in removing surface hydrophobic groups from contact with solvent(25) . The first part of the current study was designed to optimize recovery of active protein and to compare the refolded and native enzymes. In common with studies with other recombinant proteins, we found that a critical step was to initiate folding from an intermediate concentration of denaturant, which presumably solubilizes a conformation with exposed hydrophobic groups so that internalization of these groups can occur without protein aggregation. For rMMP-1, a redox buffer to catalyze disulfide shuffling was important; in addition, the GSSG/GSH couple, which may transiently form a charged mixed disulfide with enhanced solubility, was more effective than DTT(43) . Furthermore, agents that stabilize the folded conformation such as glycerol and calcium ions significantly affected the yield of refolded protein. Recently, Zhi et al. (44) reported that glycerol promotes renaturation of citrate synthase. Interestingly, the propeptide, which comprises roughly 20% of the mass of the proenzyme, was not necessary for folding to an active enzyme. This situation is in contrast to the proteinase subtilisin, which requires the propeptide to direct folding along a productive pathway(45, 46) , but resembles that of cathepsin D, which refolds without its propeptide(47) .
The folded recombinant protein exhibited characteristics expected of a native protein. It was catalytically active, contained significant secondary and tertiary structure as judged from CD analysis, was more compact than the unfolded protein, and the emission properties of its tryptophanyl residues suggest that they are inaccessible to solvent. Due to limited amounts of the native protein, we were unable to carry out extensive physical comparisons with the recombinant protein, but kinetic studies showed that the two preparations were virtually indistinguishable with both synthetic and natural substrates. Catalytic competence with collagen as substrate is a particularly stringent test of ``nativeness'' since both the active site domain and collagen binding domains must be properly folded for activity.
Analysis of spectroscopic and hydrodynamic properties of rMMP-1 as a
function of denaturant concentration revealed an interesting unfolding
pathway. Unfolding of rMMP-1 apparently occurs in three stages: N
I
I
U, where N and U
represent the folded and unfolded states. The first intermediate is
characterized by an increase in emission intensity and is accompanied
by a small blue shift in emission maximum. This species, while slightly
less compact than the folded protein, has at least one tryptophanyl
residue in a less polar environment. The increase in quantum yield
suggests a conformational change which removes a charged residue from
the vicinity of a tryptophanyl residue, an effect recently reported in
a study of the folding of barnase(48) . At somewhat higher
GdnHCl (2 M), the emission intensity decreased and the maximum
shifted to the red. As judged from the near and far UV CD spectra,
tertiary structure is disrupted at this stage to give I
,
which appears to fit the definition of the molten globule
state(14) . This species contains exposed hydrophobic groups as
indicated by the ANS binding experiments and also retains secondary
structure as indicated by the CD spectra. The last stage of
denaturation occurs between 2 and 4 M denaturant, and is
characterized by a sharp decrease in fluorescence intensity, a gradual
red shift in emission maximum, and loss of ellipticity at 220 nm.
These results complement and extend those of Lowry et al.(30) who investigated the effect of calcium and zinc on the refolding of 19-kDa collagenase catalytic domain. These workers found that in the absence of metal ions, the catalytic domain was totally unfolded in 1 M GdnHCl as judged by tryptophan emission. Addition of calcium and zinc stabilized the 19-kDa form of the enzyme such that unfolding occurred between 1 and 2 M GdnHCl. Full-length collagenase without calcium and zinc unfolded over a broad range of GdnHCl (1-4 M).
Loss of enzyme
activity preceded the loss of tertiary and secondary structure, an
indication that catalytic activity of rMMP-1 is more sensitive to
denaturants than is gross conformational integrity. The active site of
the enzyme is apparently more susceptible to denaturation than the
molecule as a whole. A similar phenomenon has been observed in the
unfolding reaction of several enzymes including creatine
kinase(49) , lactate dehydrogenase H(50) ,
and D-glyceraldehyde-3-phosphate
dehydrogenase(51, 52) .