Article Type : Research Article
Authors : Ayariga JA and Villafane R
Keywords : Protenase K; Epsilon 34 phage; Lipopolysaccharides; P22 Phage
Purified bacteriophage ?34 tail spike protein (?34 TSP) can
bind to Salmonella Newington (S. newington) via the binding site of the
protein, which is the O antigens of the LPS of the bacterium. We demonstrated
elsewhere that purified ?34 TSP possessed bacteria lytic property on S.
newington. The ?34 TSP has been shown via computational prediction to consist
of parallel ?-helices like that of P22 TSP. These protein moieties are among
the simplest repetitive structural elements in proteins. There exist extensive
research on the folding behaviour of ?-helix proteins, which also provides
insight on how amyloid fibrils are generated since these proteins consist of
similar parallel ?-helix motifs. One of the most significantly studied system
for investigating protein folding is the TSP from the Salmonella bacteriophage
P22. The major component of this protein is a right-handed parallel ?-helix
with 13 rungs. Initial in silico analysis of the ?34 phage TSP indicates
similar structural similarity to the P22 TSP. Our previous studies indicated
that despite the similarities of the two proteins, P22 TSP shows higher
resistance to proteases (e.g. trypsin) and heat compared to ?34 TSP. In this
study we further proved that ?34 TSP is partially sensitive to proteinase K, whereas
P22 TSP is completely resistant to this protein. Detailed analysis indicates
that specific structural motifs of ?34 TSP is insensitive to the protease,
whereas other regions of the protein showed susceptibility to it.
Salmonella has been demonstrated to cause several acute and
chronic infections in humans [1]. These bacteria is known to be responsible for
over 1.2 million illnesses in the US each year [2-5]. Salmonella species are
prone to acquiring resistance to various classes of antibiotics, this sparks
the need to look for methods of treating salmonella related infections other
than the use of antibiotics, thus calling for the application of bacteriophages
to manage, control and treat bacterial infections [6,7]. Several bacteriophages
specific to Salmonella have been studied [8-10]. ?34 phage belongs to the
P22-like phages [11-13]. The TSP of ?34 phage has been demonstrated to
possesses similar functional and structural characteristics as the P22 phage
TSP [10]. The predicted structure of ?34 TSP consists of a globular head
binding domain, a solenoid-shape parallel beta-helix domain that is used in the
LPS binding process as also found in the P22 phage TSP [9,14,15]. Phages or
phage derived components can complement host cell defense against bacteria
[3,16]. Purified bacteriophage ?34 TSP can bind to S. newington via the binding
site of the protein, which is the O antigens of the LPS of the bacterium [9].
We demonstrated elsewhere that purified ?34 TSP possessed bacteria lytic property
on S. newington [9,17]. The ?34 TSP has been shown via computational prediction
to consist of parallel ?-helices like that of P22 phage TSP [9]. These protein
moieties are among the simplest repetitive structural elements in proteins
[18]. There exist extensive research on the folding behaviour of ?-helix
proteins, which also provides insight on how amyloid fibrils are generated
since these proteins consist of parallel ?-helix motifs [19-22]. One of the
most significantly studied system for investigating protein folding is the TSP
from the Salmonella bacteriophage P22 [3,9,23]. The major component of this
protein is a right-handed parallel ?-helix with 13 rungs [24]. Initial in
silico analysis of the ?34 phage TSP indicates similar structural similarity to
the P22 phage TSP [9]. Our previous studies indicated that despite the
similarities of the two proteins, P22 phage TSP shows higher resistance to
proteases (e.g. trypsin) and heat compared to ?34 TSP [3]. Several common
proteases that have been used in most published works include chymotrypsin,
trypsin, enterokinase, proteinase K, factor Xa, Staphylococcus aureus
protease, papain etc., in proteolytic analysis, and their data are demonstrated
to be consistent with expected fragments in denatured substrates [25-29]. In
native protein, protein structural states at which proteins still retains their
tertiary conformations, most enzymes sites are shielded and hence inaccessible
to the enzyme thereby imparting resistance to the enzyme, however, if there exist
structural domains that are flexible, or linker regions that are solvent
accessible, these areas can easily be attacked by the enzymes leading to
specific cleavages and production of truncated products with unique
electrophoretic mobility. Also known as protease K or endopeptidase K, or
Tritirachium alkaline proteinase, or Tritirachium album pro-teinase is a
broad-spectrum serine protease that is capable of digesting keratin, hence the
“name proteinase K”. Belonging to the S8 family of peptidase, it has a
molecular weight of 28.9kDa. Unlike trypsin, proteinase K goes after the
peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino
acids with blocked alpha amino groups. This protease is stable over a wide pH
range (4–12), with a pH optimum of pH 8.0 [20]. Our approach here was to
unravel the native E?34 TSP while exposing it carefully and gradually to
proteinase K. To allow for comparability, samples of the TSPs were subjected to
proteolysis under non-denaturing conditions first, while to another sample, the
gradual denaturation via heating at 70 °C and 80 °C was employed for
predetermined time points and aliquots withdrawn at these time points and
treated with the enzyme. Hence, the pep-tide maps obtained when the proteins
were completely folded and hence least perturbed by the protease, juxta-posed
with peptide maps generated during denaturing conditions as well as complete
unfolded protein samples could be compared. Thus, higher structure information
such as thermostable domains could be deciphered and their stability against
heat as well as unfolding rates of the truncated products revealed. Thus, in
this study, we further proved that ?34 TSP is partially sensitive to proteinase
K, whereas P22 TSP is completely resistant to this protein. Detailed analysis
indicates that specific structural motifs of ?34 TSP is insensitive to the
protease, whereas other regions of the protein showed susceptibility to it.
The cloning and protein
expression, as well as the initial thermal characterization of E?34 TSP have
been documented previously in our laboratory [9].
In
silico disorder region prediction and PK sites
The entire amino acid
sequence of the wild-type and the E?34 TSP were submitted to the online
Predictor of Natural Disordered Regions server (http://www.pondr.com/) as well
as the secondary structure prediction server known as RaptorX
(http://raptorx.uchicago.edu) [31]. Predictions depicted amino acids that are
in disordered regions as red and those in ordered region as blue. Prediction of
PK cleave sites were carried out using the online programs PeptideCutter from
Expasy (https://www.expasy.org/resources/peptidecutter) that predicts cleavage
sites in the protein’s primary sequence.
Proteinase
K treatment
The proteinase K
treatment process followed a similar protocol [29]. Stock samples of E?34,
ME?34 TSP in solutions (10 mg/mL) were diluted to the appropriate
concentrations of 0.5 mg/mL using trisHCl buffer at pH 7.4. The protein samples
were then incubated for specific time points and at the specified temperatures
as described below. Finally, the protein samples were subjected to proteinase K
digestion. Samples reactions were immediately stopped by the addition of
protease inhibitor, then subjected to either Native PAGE or SDS-PAGE analysis.
E?34
TSP sensitivity to Proteinase K under 2% SDS at physiological temperature
To investigate the
structural dynamics of our protein under slightly denaturing conditions, we
deployed the use of the denaturing detergent, sodium dodecyl sulphate (SDS),
and denatured the protein samples for 6 h. A sample of 0.5 mg/ml of E?34 and
M?34 TSP were dialyzed against neutral buffer consisting of 50 mg/ml tris-HCl,
pH 7.4, this was followed by mixing with 2% SDS then treated with the
proteinase K enzyme at 1:20 w/w of enzyme to protein sample. The treated
samples were then incubated in a water bath at 37 °C for 6 h. Afterward, the
proteolytic reactions were stopped by the addition of protease inhibitor at a
10 µg/ml final concentration and the samples were subjected to electrophoretic
analysis using native polyacrylamide gel. Placebos received buffer instead of
the enzyme and heated controls samples also received no treatment at all except
heating of these samples in 2% SDS loading buffer at 100 °C for 5 min. Samples
were run at 100 volts for 250 min under non-denaturing running buffer
conditions. Gels were then visualized via Coomassie blue staining, and the gels
photographed using ChemiDoc XRS installed with Quantity One.
E?34
TSP sensitivity to Proteinase K under increasing concentrations of enzyme
Concentration dependent
tryptic proteolysis of E?34 TSP at 37 °C via a dose dependent treatment of our
TSP to trypsin was carried out. In this work, we measured the ability of the
enzyme to digest E?34 TSP in increasing concentrations of the enzyme (trypsin).
1 mg/mL of E?34 TSP samples were initially dialyzed against buffer consisting
of 50 mg/mL tris-HCl, pH 7.4 to remove protease inhibitors then mixed with
trypsin in differing ratios. A final concentration of 100 µg, 200 µg, 300 µg,
400 µg and 500 µg of trypsin were used to treat 1 mg of E?34 TSP. Thus, a
protein to enzyme ratio of 100:1, 100:2, 100:3, 100:4, 100:5 respectively for
each treatment sample were achieved. Then samples were incubated for 6 h under
37 °C. Afterwards samples were run in 7.5% polyacrylamide gel and the gels
photographed using ChemiDoc XRS installed with Quantity One. Experiments were
repeated thrice and the densitometric values of the resulting gels were
recorded via Image J 1.52 software and values plotted into graphs.
E?34
TSP sensitivity to Proteinase K under protease-heat combination
To learn of the unfolding kinetics of the
protein and the structural stabilities of the various domains that constitute
the protein, we subjected samples of the protein to heat at 70 °C, or 80 °C in
the absence of denaturants, and aliquots withdrawn and quenched at specified
time points (0, 2.5, 5, 10, 15, 20, 30 and 60 min). A 1:20 w/w ratio of
proteinase K to E?34 TSP sample was used for the proteolytic process. The
enzyme digestion was allowed for 30 min, and quenching was achieved via mixing
reaction samples with a protease inhibitor followed by the addition of SDS-free
gel loading buffer consisting of 50 mM Tris-HCl, 25% glycerol, and 0.01%
bromophenol blue, and SDS free running buffer consisting of 25 mM Tris-HCl, and
0.2 M glycine. Samples of triplicate experiments were electrophoresed for 60
min and stained with Coomassie blue, and then intensities of the bands that
were generated were then recorded. The densitometric values were compiled from
three replicate experiments and averaged. The mean densitometric values for the
70 °C and 80 °C heating experiments were plotted against time of heating.
For all statistical data,
values were derived from multiple measurements (from replicates of 3 or 5
experiments) and averaged, the standard deviations were evaluated using
P?values from Student's t?test (significance level ??? 0.05).
In
silico prediction of disordered regions of E?34 TSP
The entire amino acid sequence of the wildtype as well as the E?34 TSP were submitted to the online disordered region predictor and secondary structure prediction server known as PONDRs (Predictor of Natural Disordered Regions), a free online server (http://www.pondr.com/) and RaptorX (http://raptorx.uchicago.edu) respectively [31]. As shown in Figure 1, the E?34 TSP showed higher disorder as compared to the wild-type protein. This is cor-roborated by the disordered regions as in indicated by red in the Raptor X prediction in Figure 2. In the Raptor X, predictions depicted amino acids that are in disordered regions as red and those in ordered region as blue (Figure 1).
Figure 1: Disorder prediction of wild-type ?34 TSP (A) and extended ?34 TSP (B). Predictions were made using PONDRs (Predictor of Natural Disordered Regions), a free online server (http://www.pondr.com/).
To support this argument that the fusion peptide imparted disorder to the protein especially at the N-terminus, we analysed for secondary structure disorder using the Raptor X. As shown in Figure 2. The amino acids that are predicted to have order (all sequences in blue colour), and those that were disordered (all sequences in red color) are revealed (Figure 2).
Figure 2: Secondary structure prediction by Raptor X shows the entire 43 amino acid sequence (the fusion peptide) to be completely disordered out of the first 400 amino acid sequence of the protein.
Figure 3: Diagrammatic illustration of PK site in the WT ?34 TSP. The red diamond depicts putative LPS binding sites of the TSP.
With 100% disorder
prediction, the 43 amino acid sequence seems to further disrupt the succeeding
downstream sequences, the highest impact felt by the first 7 amino acids immediately
following the fusion peptide (MTDITAN) which scored above 50% disorder.
Disorder percentage tapered and faded at the 70th amino acid (A). The effect of
fusion peptide on protein stability has been well documented but no answer
exists on why most fusion peptide exert such influence on native protein’s
stability [32,33]. While we propose that the instability of our protein to
proteinase K is partly due to the unstructured fusion tag, we do not doubt the
fact that the very observed properties of the protein could be an intrinsic
behavior of the protein under those conditions, hence further biophysical
characterization is warranted.
Proteinase
K sites in wild-type ?34 TSP as compared to E?34 TSP
As shown in Figure 3, the
predicted PK sites on a denatured wild-type, as well as a native state ?34 TSP
has been shown. Prediction were carried out using Raptor X server, whereas the
tertiary structure PK sites were predicted by peptide cutter and curated
manually after identifying the secondary structure disordered regions. As
illustrated the wildtype ?34 TSP contains 300 PK sites; however the E?34 TSP
contains 311 K sites (Figure not shown). In the quaternary conformation of the
protein, the wild-type ?34 TSP is predicted to contain 3 sites as shown by the
black arrows to be solvent accessible thus are highly likely to be accessible
to PK, hence, presupposing that the protein is liable to be cleaved at these
sites. In the case of E?34 TSP in its native state, there are 14 predicted
sites, and 11 of these sites are located on the 43 fusion peptide. The
understanding of the possible cleavage sites especially on the native state of
the protein is crucial guide to analysing the peptide map generated after PK
proteolysis of the protein (Figure 3).
Proteolytic
analysis of Epsilon 34 Phage TSP indicates partial sensitivity to Proteinase K.
Partial Sensitivity of E?34 TSP in 2% SDS to PK: Shown in Figure 4, however, the treatment of our E?34 TSP in SDS to proteinase K at 37 °C for 6 h as depicted in lane 4 (EETUH) generated two protein fragments, one observed at 82 kDa and the other observed at 77 kDa, while the untreated trimers showed at the 161 kDa level. This observed significant migration difference between the un-treated sample which migrated at 161 kDa and the treated samples which migrated at 82 kDa and 77 kDa possibly indicate the sensitivity of our protein to PK. When the treated samples were heat-unfolded to their monomeric state (Figure 4, lane 5, EETH), two monomeric bands were observed at 59 kDa and 25 kDa. In lane 6, the untreated and unheated matured samples migrated at the usual size, 123 kDa, but when treated to PK as in lane 8, a 77 kDa fragment is recorded, when the treated samples of the mature protein was subjected to heat unfolding, as in lane 9, we failed to observe any band, however, the untreated samples which were unfolded via heat recorded a monomeric band at 65 kDa as depicted in lane 7 of Figure 4. P22 TSP control, which was treated to PK showed complete resistance, registering a single band as depicted in lane 10, Figure 4. This led to our assumption that even though the trimeric E?34 TSP shows sensitivity to PK, a much stable and protease resistant products resulting from the treatment were produced. This study demonstrates that E?34 TSP is possibly liable to partial proteolytic digestion by proteinase K even at physiological temperatures of 37 °C in the presence of SDS (as depicted in Figure 4 lanes 4, 5 and 8). The present data points to cleavage sites that are possibly located interiorly of the trimeric protein, with possibility of cleaving a larger fragment off the TSP than we observed in the trypsin treatment (Figure 4).
Figure 4: Native-PAGE analysis of E?34 TSP in 2 % SDS treated to PK at physiological temperature. 0.5 mg/mL of E?34 TSP and matured/rEK digested ?34 TSP (M?34 TSP) in 2 % SDS were treated with proteinase K at 37 °C for 6 h. Samples were electrophoretically analyzed using 10 % polyacrylamide gel. Lane 1, Prestained Precision Protein Standard. Lane 2, E?34 TSP untreated and unheated (EEUTUH). Lane 3, E?34 TSP untreated but heated (EEUTH). Lane 4, E?34 TSP treated but unheated (EETUH). Lane 5, E?34 TSP treated and heated (EETH). Lane 6, matured ?34 TSP (rEK digested E?34 TSP) treated but unheated (METUH). Lane 7, matured ?34 TSP (rEK digested E?34 TSP) untreated but heated (MEUTH). Lane 8, matured ?34 TSP (rEK digested E?34 TSP) treated but unheated (METUH). Lane 9, matured ?34 TSP (rEK digested E?34 TSP) treated and heated (METH). Lane 10, P22 TSP treated but unheated.
Native-PAGE analysis of
E?34 TSP in 2 % SDS treated to PK at physiological temperature. 0.5 mg/mL of
E?34 TSP and matured/rEK digested ?34 TSP (M?34 TSP) in 2 % SDS were treated
with proteinase K at 37 °C for 6 h. Samples were electrophoretically analyzed
using 10 % polyacrylamide gel. Lane 1, Prestained Precision Protein Standard.
Lane 2, E?34 TSP untreated and unheated (EEUTUH). Lane 3, E?34 TSP untreated
but heated (EEUTH). Lane 4, E?34 TSP treated but unheated (EETUH). Lane 5, E?34
TSP treated and heated (EETH). Lane 6, matured ?34 TSP (rEK digested E?34 TSP)
treated but unheated (METUH). Lane 7, matured ?34 TSP (rEK digested E?34 TSP)
untreated but heated (MEUTH). Lane 8, matured ?34 TSP (rEK digested E?34 TSP) treated
but unheated (METUH). Lane 9, matured ?34 TSP (rEK digested E?34 TSP) treated
and heated (METH). Lane 10, P22 TSP treated but unheated.
E?34 TSP sensitivity test under increasing concentrations of enzyme: E?34 TSP when unfolded to its monomeric state is 71.4 kDa, and seems to migrate at approximately 72 kDa on 7.5 % polyacrylamide gel. The sum of the molecular weight of the 43 amino acids fusion peptide and the last 65 amino acids molecular weight of the C-terminus (the trimerization domain) is approximately 11.9 kDa constituting the regions sensitive to PK, and if this molecular weight is subtracted from the 71.4 kDa molecular weight of the E?34 TSP, a 59.5 kDa fragment is resulted. We propose that PK cleaves the N-terminally fused 43-amino acid fusion peptide and the last 65 amino acid fragment of the C-terminus, but unable to affect the head binding domain or the beta-helix domain of the protein (the region not sensitive to PK) as diagrammatically illustrated in Figure 5.
Figure 5: Diagrammatical depiction of the possible sensitive regions of trimeric E?34 TSP to PK digestion (Created using Microsoft word).
The first 43 amino acids
which is N-terminally fused to the protein is predicted to be completely
unstructured (Figure 1 and 2). Hence, the unstructured nature of the fusion
peptide makes it liable to degradation by PK. Also, from solvent accessibility
and disorder assessment data generated by the Raptor X, the entire fusion
peptide scored high values and were completely disordered. In solvent accessibility
heat maps generated by the Raptor X server, the protein registered high values
for solvent accessibility at the positions S585 and I586, and these residues in
the secondary structure assessment is flanged at both sides by coils arising
from sites A582, P583, and H584, I585, S587, V588 (Data not shown), however
only sites A582 and I585 are PK cleavage sites, these all indicating a high
possibility of PK accessing and binding to these sites to cleave the fragment
off the protein (Figure 5).
The first 43 aa (amino
acid) is the fusion peptide, and is predicted to be unstructured and labial,
easily proteo-lyzed by PK (proteinase K). The sites A582 and I585 are the
possible PK cleavable sites between the ?-helix domain and the trimerization
domain. Sum of the molecular weights of 43aa and 65 aa (i.e. 585-649 aa)=11.9
kDa. The difference in molecular weights between entire E?34 TSP and PK
sensitive domains = 59.5 kDa (i.e. (71.4 – 11.9) kDa) = Region resistant to PK
at non-denaturing conditions.
Testing the effect of increasing dose of PK to E?34
TSP digestion: To understand if
increasing the concentration of PK will affect the proteolysis of E?34 TSP,
varying concentrations of PK were administered to the protein. Digested samples
were subjected to 7.5% native PAGE electrophoresis. As shown in Figure 6,
increasing concentrations of PK did not affect the amount or size of the
fragments generated (Figure 6).
Incomplete inhibitory action of the protease inhibitor PMSF on Proteinase K: To gain insight into the ability of PMSF to inhibit the proteolytic activity of PK, we subjected sample of E?34 TSP containing PMSF (10 µg/mL) to increasing dosages of PK. We observed that increasing concentrations of PK resulted in increased band intensities (Figure 7). While it was observed that all treatments (lanes 3 to 7) produced fragments (FB) that migrated close to the monomeric size, it was clear nonetheless that trimeric species populated in high amounts in all samples as indicated by the trimeric band represented by the native band (NB) indicated in Figure 7.
Figure 6: Prolonged enzymatic digestion of E?34 TSP in varying concentrations of PK. 7.5 % Native PAGE gels analysis of resulting fragment arising from dose dependent treatment of the protein to PK. Lane 1, PPPS; Pres-tained Precision Protein Standard. Lane 2, E?34 TSP unheated and untreated. Lane 3 to 7 are E?34 TSP treated to varying concentration of PK.
This trimeric band seemed to slightly increase with increasing concentrations of the enzyme (PK). This anomaly seems not to fit any feasible explanation given that approximately equal amount of TSP has been treated with the varying concentration of the enzyme. For instance, given that it was the incremental binding of PK to the TSP and yet lacking the proteolytic action due to the presence the protease inhibitor (PMSF) that led to the increasing trimeric band intensity, then the flaw will remain in the question, why did the binding of the enzyme not increase the migratory size of the trimers significantly? Another hypothesis could be that increasing concentrations of PMSF ensured that an increased amount of trimeric species were competitively protected from the protease, then if this argument is true, there should be decreasing intensities of the second fragment, which seems to hold true for lanes 3, 4, 5 and 7. The intensity of band in Lane 6 seems not to correlate with this deduction (Figure 7).
Figure 7: Proteolytic digestion of E?34 TSP in 6 h using the enzyme proteinase K (PK), at incubation temperature of 37 °C and in the presence of PMSF.
Figure 8: Limited proteolysis of E?34 TSP in 6 h under the incubation temperature of 37 °C and in the presence of PMSF using PK Five samples of 1 mg/mL of E?34 TSP each were subjected to varying concentration of PK digest for 6 h under 37 °C and in the presence of PMSF. Afterward, samples were divided into two and electrophoretically analyzed in a 7.5% SDS PAGE gel. Replicates SDS PAGE gels were analyzed using densitometry. The values were averaged and plotted into a chart. Two bands designated as native band (NB, lighter columns) and fragment band (FB, the darker columns) were plotted.
Five samples of 1mg/ml of
E?34 TSP each was subjected to varying concentration of PK digest for 6 h under
37 °C and in the presence of PMSF. Afterward, samples were electrophoretically
analysed in a 7.5% SDS PAGE gel. In here, we loaded Lane 1, PPPS; Prestained
Precision Protein Standard. Lane 2, E?34 TSP; unheated and untreated
(E?34UHUT). Lane 3, E?34 TSP; treated with 100µg of PK (E?34UHT). Lane 4, E?34
TSP; treated with 200 µg of PK (E?34UHT). Lane 5, E?34 TSP; treated with 300 µg
of PK (E?34UHT). Lane 6, E?34 TSP; treated with 400 µg of PK (E?34UHT). Lane,
E?34 TSP; treated with 500 µg of PK (E?34UHT). Lane 8, E?34 TSP; Heated and
untreated (E?34HUT). Lane 9, Blank, Lane 10, Blank.
Quantitative analysis of incomplete inhibitory action of PMSF on Proteinase K: To better, understand the mechanism by which E?34 TSP is digested by PK in the presence of PMSF as in Figure 7, the experiment was replicated and the densitometric values measured via Image J. The mean values were used in plotting a chart as shown in Figure 8. The mean densitometric values for the trimeric species band (NB) were as following; 68.2, 96.2, 127.9, 157.9 and 151.2 representing the 100, 200, 300, 400 and 500µg/mL of PK treatment to samples respectively. While the fragment band (FB) registered the mean densitometric values 112.3, 91.8, 90.1, 101.3, and 87.8 for 100, 200, 300, 400 and 500 µg/mL PK treatment to E?34 TSP samples respectively. While it is a possibility to deduce using this data, that, the increasing values as recorded in the NB were the increasing population of less stable trimeric species that were rescued in increasing amounts as PMSF increases, and yet the observed increase in trimeric band did not fully correlated with the FB band which represents the less stable species which were cleaved. The mean densitometric values for the fragment band (FB) for 100, 200, 300 and 500 µg/mL PK treatment seems to show a slight continued decrease in intensity, following our hypothesis that the less stable trimers were rescued/protected from proteolysis by increasing concentrations of the enzyme, however, the treatment 400 µg/mL seems to behave as an outlier in this model (Figure 8).
Figure 9: Native-PAGE analysis of PK proteolytic digestion of E?34 TSP after 70 °C treatment.
Proteolytic digestion of E?34 TSP at 70 °C using PK: To investigate the effect of temperature on E?34 TSP (Figure 9 and 10), we used PK digestion as a tool to scrutinize the unfolding kinetics of the protein. Here, we subjected E?34 TSP to a high temperature of 70 °C (in a water bath preset at that temperature) for varying time points. Heating of samples were done without the addition of SDS, nor any other denaturants, in this way, it provided the platform to study the effect of heat alone at 70 °C on our TSP. Heated E?34 TSP samples were cooled to room temperature, and then subjected to PK treatment. The electrophoretic mobilities of PK treated samples in all lanes 0, 2.5, 5, 10, 20, 30 and 60 (Figure 9) migrated to 77 kDa, a significant shift from the untreated E?34 TSP migration of 161 kDa. No secondary fragments were observed in all treated samples. As depicted in Figure 10, complementary to our observation (Figure 9), the plots of the mean densitometric values obtained illustrated almost similar values for some treatments, however, slight variability did exist. Beginning with a densitometric mean value of 128.58 at the 0-time point, the mean densitometric values that followed were as thus: 144.06, 132.58, 110.99, 105.52, 109.81 and 107.62 for time points 2.5, 5, 10, 20, 30 and 60 min respectively. Statistical differences were observed between means at p values less than 0.39 with the 2.5-minute treatment showing the highest difference.
Figure 10: Bar plot of residual trimers after different time points of heating at 70 °C followed by PK digestion.
Within the limits of
experimental errors, it is safe to argue for a continued margin-al reduction
density of bands with passing time, with the 60-minute treatment recording the
lowest densitometric value of 107.62. This revelation might be a pointer to a marginal
denaturation of some species of the protein at 70 °C with preponderance still
existing in trimeric state but increasing incubation time meant continual
accumulation of unfolded species which fell as substrate to PK complete
digestion, hence the decreased intensities recorded at the 60th
minute (Figure 9).
0.5 mg/mL of E?34 TSP
samples were heated at 70 °C without the addition of SDS, then were withdrawn
at set time points 0, 2.5, 5, 10, 15, 20, 30 and 60 min as indicated in lanes 3
to 9 respectively followed by PK digestion. Lane 2 served as unheated and
untreated control. Lane 1 was PPPS (prestained perfect protein standard)
(Figure 10).
This chart compares
residual E?34 TSP fragments species resulting from the treatment of the protein
to heat at 70 °C followed by PK digest. All data presented are derivative of
triplicate experiments arising from Figure 9; all values represent the mean
±SEM (n = 3, p < 0.05).
Proteolytic
digestion of E?34 TSP at 80 °C using PK
Qualitative analysis of E?34 TSP treatment to PK after 80 °C incubation: In this experiment as depicted in Figure 11, it was noticed that a single fragment band was seen from lane 3 to lane 9, which represents, 0, 2.5, 5, 10, 20, 30 and 60 min of heating of samples at 80 °C before treatment with PK. The migration of the fragment band was recorded at 77 kDa, just slightly above the monomeric band which shows a migration of 7,1360 Da, as seen in lane 10 of Figure 11. The unheated and untreated samples treatment registered its band migration at the native trimeric size as indicated in lane 2. The fragment band migrated for all treated lanes at 77 kDa and is demonstrated to have a continued reduction in intensity as the time of heating at 80 °C increased. The faintest band was recorded at the 60th minute mark as shown in lane 9 of Figure 11. Lane 10, which represents the heated but untreated samples registered a high-density band at the uppermost part of the gel, indicating aggregation, a slight band however can be noticed at the monomeric size, as indicated by the black arrow (Figure 11).
Figure 11: PK proteolysis of E?34 TSP after 80 °C heat.
Quantitative analysis of E?34 TSP treatment to PK after 80 °C incubation: As demonstrated in Figure 12, we examined the unfolding of of E?34 TSP rates as a function of time via the use of the proteolytic enzyme, PK. We quantitize and averaged the band intensities from three replica experiments. At zero time point, with a high mean densitometric value of 102.15, the proteolytic fragment band intensity decreased progressively with time to register a low of 0.14 densitometric value after 1 h of heating at 80 °C. The intervening time points 2.5, 5, 10, and 30 min registered densitometric values of 79.5, 62.3, 52.8, 32.9, and 16.4 respectively; showing that there was a progressive decrease in densities of the bands with time. Using a regressional analysis, a plot of these values seems to fit to an exponential curve with the equation; [ft] = [f0]e(-kt); for which in this graph [f0] = 143.25 (theoretical, according to the curve), though practical value obtained was 102.15 for [f0] which represents the initial intensity (which equate to the concentration) of the TSP proteolytic fragment band (FB), k = 0.105, e = mathematical constant = 2.718, Hence, to calculate for the resulting intensity or concentration of the proteolytic fragment remaining in solution at any time t; [ft] = [143.25]e(-0.105t). For which t = time of heating (0, 2.5, 5, 10, 20, 30 and 60 min as indicated in this experiment) at 80 °C before treatment with PK. This equation shows the rate of decrease of the proteolytic fragment’s intensity with respect to time and can be conveniently used to estimate the concentration of the more heat resistant and studier truncated protein fragment at any given time. The principle is based on the fact that heating our protein unfolds it, however, the sequence of unfolding follows that the most labial and unstable regions unravels first, and the most stable regions melts last, hence giving the protease access to only the open regions of the protein at a sequential manner, hence those hitherto inaccessible sites are availed to the protease to bind to and cleave as the protein unfolds. In this work, a 30-min treatment of the samples to protease was sufficient to completely proteolyze a fully unfolded protein at the said concentration. k = the rate of proteolytic degradation of E?34 TSP, which is also positively correlated to the rate of unfolding of the TSP, and was determined to be 0.105.
Figure 12: Quantitative assessment of unfolding kinetics of E?34 TSP at 80 °C using PK.
In contrast to tryptic
digest which registered k value of 0.072, PK treatment yielded a higher rate
constant of 0.105, indicating a possibility of less stability of the truncated
product formed via PK digestion as compared to the fragment produced in the
tryptic digestion. This inference gains plausibility even further from
published assessment of the importance of the trimerization domain of the P22
TSP to the stability of the protein. It has been demonstrated that the caudal
fin of the P22 TSP, which translate here as possibly the last 65 amino acids
sequence of the E?34 TSP, the domain we propose to be digested by PK, functions
as a molecular clamp in the P22 system [34]. This molecular clamp ensures thermostable
subunit association in the native trimeric P22 TSP, therefore the possibility
of similar role might not be farfetched in this E?34 TSP. The truncated product
has shown a high degree of thermal resistance, a known property of parallel
b-helix domains [35] (Figure 12).
The mean densitometric value of three replicate experiments are presented; all values represent the mean ±SEM (n = 3, p < 0.05). These values were fitted into the unfolding curve, unfolding constant (k) was found to be 0.105. [ft]= concentration of E?34 TSP at time t. [f0] = concentration of E?34 TSP at the beginning of the experiment. t= time of heating.
Trimeric protein in its
3D conformation presents complex topological surfaces and thus the native
structural features of the E?34 TSP have surfaces that are inaccessible to
solvent thus rendering PK binding and subsequent proteolysis less effective.
This sharply contrast the situation in a fully denature E?34 TSP, because all
the 311 proteinase K site along the length of the protein is readily available
for digestion. For this reason, the understanding of the exact cleavage sites
of these enzymes on E?34 TSP primary sequence has provided means to speculate
reliably the potential domains that are sensitive to the enzymes even at the
native state topology. PK was predicted by PeptideCutty program to cleave E?34
TSP in 311 sites, whereas it cleaves the wild-type ?34 TSP in 300 sites, and
these sites are distributed unevenly throughout the entire sequence. However,
upon secondary structure formation, subunit association, most of these sites
become buried and shielded from the proteolytic enzyme, hence inferring proteolytic
immunity to the protein. An exhaustive review as well as other researchers in
the field suggested a general sturdy nature of the ?-helix domain in all TSPs
and other proteins [36-38]. As reported already when PK treated samples were
heat-denatured, they migrated to approximately 59 kDa (Figure 4, lane 5), a
fragment size that corresponded to the monomeric species of E?34 TSP that is
missing two components; the 43aa fusion peptide at the N-terminal region and
the trimerization domain (caudal fin) that constitute the last 65 amino acid
sequence of the protein. This hints at a possible cleavage at the linker region
between the parallel ?-helix and the triple ?-prism of the E?34 TSP. This is
consistent with published findings concerning the robust nature of the ?
-barrel, which is known to be protease, heat and detergent resistant [37-40].
The sum of the molecular weight of the 43 amino acid fusion peptide and the
last 65 amino acids molecular weight of the C-terminus is 11880 Da, and if this
molecular weight is subtracted from the 71390 Da molecular weight of the E?34
TSP, a 59510 Da fragment is resulted. Although, this method is too crude to
propose a conclusion, yet it is suggestive of a possibility that PK might have
cleaved the N-terminally fused 43-amino acids fusion peptide and the last 65
amino acids fragment of the C-terminus (trimerization domain), but unable to
affect the head binding domain or the beta-helix domain of the protein. As a
control, P22 TSP and matured ?34 TSPs were also treated with PK. While it was
obvious from the band migration that M?34 TSP was sensitive to PK (Figure 4,
lane 6), P22 TSP indicated no sensitivity at all to this enzyme (Figure 4, lane
8). The treatment of M?34 TSP resulted in a protein fragment migrating at approximately
77 kDa while P22 TSP still maintained its usual trimeric electrophoretic
mobility at the size of 126 kDa on 7.5 % polyacrylamide gel which corresponds
to a 216 kDa in its actual molecular weight.
Given the characteristics
of E?34 TSP observed, in this study, it indicates that ?34 TSP although
previously shown to be resistant to trypsin, it is susceptible to proteinase K,
at non-denaturing conditions. Further analysis of the fragments indicated that
a studier and more heat resistant fragment was generated after proteinase K
treatment, and this fragment seems to be resistant to heat even at elevated
temperatures up to 70 °C. Nonetheless, the resulting fragment denatures at 80
°C or higher. The migration of the monomeric species of the truncated product
from PK treatment showed a characteristic match for E?34 TSP missing the fusion
peptide and the trimerization domain. The topology of the predicted trimeric
protein shows a flexible region at two PK sites located right be-tween the
LPS-binding central domain and the trimerization domain, and this could be a
prove that PK binds to and degrade this region of the protein as well as the
fusion peptide to produce a truncated product devoid of these two regions.
Interestingly, further analysis of this truncated product shows a domain
emerged to be resistant to SDS and to heat at 70 °C and hence must necessarily
be a compact and sturdy product. This study, apart from providing the
structural sculpture of the E?34 TSP, we showed that the unfolding kinetics of
this protein can be probe via protease, heat, and detergent combinations. These
simple tools can provide valuable information about unknown protein as
regarding structural flexibility or compactness. Here, E?34 TSP could be
regarded as a typical example of hybrid molecule of which an intrinsically
disordered protein (IDPs), that is the fusion peptide and structurally compact
or ordered domains (that is the matured protein devoid of the fusion peptide)
are married. The characteristic features observed in this study of E?34 TSP for
which the 43 amino acids fusion peptide is labial imparts this property of IDPs
to a rather compact and highly organized architecture formed from very
structured domains of well-defined three-dimensional folds arising from the
wild-type protein. IDPs engage in most cellular functions in which structural
plasticity presents functional advantage in terms of binding elasticity. Thus
this study has presented a novel method to produce IDPs modules for classroom
research, for bioengineering purposes, or to understand certain peculiar nature
of some proteins that are blamed for the etiology of some disease caused by
IDPs [41,42].
For all statistical data,
values were derived from multiple measurements (from replicate of 3 or 5
experiments) and averaged, the standard deviations were evaluated using P
values of Student's t test (two tailed, two samples of unequal variance,
significance level ??? 0.50).
We thank the department
of Microbiology, Faculty of Science, Mathematics and Technology, Alabama State
University for timely support with supplies and quick responses to repairs of
laboratory equipment.
All authors declare that
they have no conflicts of interest.
This article does not
contain any studies with human participants or animals performed by any of the
authors.
µL: micro liter; µM: micro
molar; AGG: Aggregates; Da: Dalton; Native-PAGE: Native Polyacrylamide gel
electrophoresis; E?34 TSP: Extended Epsilon 34 tailspike protein; Ek:
Enterokinase; ?34 TSP: Epsilon 34 tailspike protein; M?34 TSP: Matured
(enterokinase digested) ?34 TSP; HCl: Hydrochloric Acid; IDPs: Intrinsically
Disordered Proteins; KDa: Kilo Daltons; mL: millilitre; LPS:
Lipopolysaccharides; M : Molarity;
Min: Minutes; mM: milli-molar; MM: Monomers; Mr, MW: Molecular Weight; N/A: Not
applicable; NB: Native band; OD: Optical Density; PBS: Phosphate Saline Buffer;
PK: Proteinase K; PMSF: PhenylMethanesulfonyl fluoride; PONDR: Predictor of
Natural Disordered Regions; PPPS: Pre-stained perfect Protein Standard; rpm: Revolution
per minute; SDS PAGE: Sodium Dodecyl Sulphate Polyacrylamide Gel
Electrophoresis; SDS: Sodium Dodecyl Sulphate; SEM: Standard Error of Mean; TH:
Treated and Heated samples of protein; TSP: Tailspike protein; TUH: Treated but
unheated; UTH: Untreated but heated; UTUH: Untreated and Unheated