1Department of Biomedical Engineering, Campus Box 90281 and 2Department of Computer Science, Campus Box 90129, Duke University, Durham, NC 27708, USA
3 To whom correspondence should be addressed. e-mail: chilkoti{at}duke.edu
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Abstract |
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Keywords: aggregation/elastin/fusion protein/hydrophobicity/phase transition
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Introduction |
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In previous studies, we showed that the environmentally sensitive solubility of ELPs is retained upon their fusion to other proteins (Meyer and Chilkoti, 1999; Meyer et al., 2001b
). Furthermore, because the phase transition of ELPs is reversible, ELPs can be used as reversible solubility tags to purify proteins from cell lysate. We have exploited the phase transition of ELP fusion proteins, to devise a simple, non-chromatographic means of purification, which we call inverse transition cycling, whereby a recombinant ELP fusion protein of interest can be separated from other contaminating Escherichia coli biomolecules in the soluble cell lysate by sequential and repeated steps of aggregation, centrifugation and resolubilization (Meyer and Chilkoti, 1999
; Meyer et al., 2001b
). Because this purification method is carried out as a batch process, it can be easily scaled up for large-scale purification and also enables simultaneous purification of different proteins from multiple cultures.
We also observed in these studies that the Tt is modulated by the presence of the fused protein (Meyer and Chilkoti, 1999; Meyer et al., 2001b
). We observed that the Tt of a 36 kDa ELP without a fusion protein partner is
51°C, but that its fusion to two different proteins results in greatly differing Tt of the ELP. Fusion of this ELP to the C-terminus of thioredoxin, a 12 kDa protein commonly used as a carrier to increase the solubility of recombinant proteins (Lavallie et al., 1993
), results in a 9°C elevation in Tt (60°C). In contrast, fusion of the same ELP to the N-terminus of tendamistat, an 8 kDa inhibitor of porcine pancreatic
-amylase (PPA) (Wiegand et al., 1995
), results in a 16°C depression in Tt (35°C) compared with free ELP. When these components are expressed as the ternary fusion protein thioredoxinELPtendamistat (TrxELPTend), the Tt (34°C) is nearly identical to the Tt of the ELPTend binary fusion, and enzymatic cleavage of tendamistat from TrxELPTend results in a 26°C increase in Tt. Both these observations clearly suggest that the presence of tendamistat dominates the perturbation of Tt in the ternary fusion protein. We term this alteration of the Tt of an ELP upon fusion to a protein the fusion
Tt effect, [fusion
Tt = Tt (ELP fusion) Tt (ELP)], analogous to, but distinct from, the
Tt effect defined for the effect of guest residue (X) substitution in the VPGXG repeat of ELPs (Urry et al., 1991
).
In this study, we have used diverse experimental and computational approaches to elucidate the biophysical principles governing this fusion Tt effect. The hypothesis tested in this study, derived from previous observations (Meyer and Chilkoti, 1999
), is that the fusion
Tt effect is controlled by the solvent-accessible hydrophobic area within molecular proximity of the ELP; exposed hydrophobic patches on the surface of proteins fused to ELPs alter the Tt of the ELP in a manner analogous to the
Tt effect whereby the Tt is depressed in proportion to the surface hydrophobicity of a fused protein. Understanding the biophysical basis of this fusion
Tt effect is important because it may enable the
Tt for a given ELP fusion protein to be quantitatively predicted a priori, from its structure or its physico-chemical properties. Discovering such quantitative structureproperty correlations has practical implications, as well, for applications of ELP fusion proteins that exploit their phase transition behavior. For example, the ability to predict the Tt of an ELP fusion protein would be extremely useful in its purification by inverse transition cycling, because it will enable optimization of the purification protocol for a given protein without extensive trial and error.
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Materials and methods |
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A synthetic gene with SfiI-generated, compatible sticky ends encoding for a 90 pentapeptide ELP was synthesized by recursive directional ligation in pUC-19 (Meyer and Chilkoti, 2002). The characteristic ELP repeat sequence of VPGXG contained 50% Val, 30% Gly and 20% Ala at the X position in this particular ELP and the DNA sequence for this gene has been published previously (Meyer and Chilkoti, 1999
). A synthetic gene for tendamistat was assembled from three complementary sets of chemically synthesized oligonucleotides (5' phosphorylated, PAGE purified; Integrated DNA Technologies), which were designed to provide EcoRI and HindIII complementary ends. The six oligonucleotides were annealed, in equimolar amounts, in a single reaction. The annealed product was separated by agarose gel electrophoresis and a
230 bp band, corresponding to the length of the tendamistat gene, was excised and purified (Qiaex II Gel Extraction kit; Qiagen). The tendamistat gene was ligated into EcoRI/HindIII restricted and gel-purified pUC19 and transformed into XL1-Blue (Novagen) competent E.coli. Successful transformants were identified by polymerase chain reaction (PCR) and subsequent DNA sequencing.
The tendamistat gene was inserted into a modified pET-32a expression vector (Novagen), which contains the thioredoxin gene. pET-32a was modified as follows: the S-tag peptide-coding region of the pET-32a vector was removed by replacement of the MscI/NcoI fragment with two alternate DNA cassettes, each encoding for a SfiI restriction site and the thrombin recognition site. The alternate ternary ELP constructs expressed from the two versions of the modified pET-32a vector allowed cleavage with thrombin either between thioredoxin and the ELP or between tendamistat and the ELP (Figure 1). The pUC19 plasmid containing the tendamistat gene was digested with EcoRI and HindIII and agarose gel purified to isolate the 230 bp fragment. The modified pET-32a expression vector was digested with EcoRI and HindIII, treated with calf intestinal phosphatase (CIAP; Gibco, BRL), and ligated with the purified tendamistat EcoRI/HindIII insert. Ternary ELP fusion proteins were created by digestion with SfiI of the modified pET-32a plasmid containing the gene for the thioredoxintendamistat binary fusion, dephosphorylation of the digested vector with CIAP, followed by ligation of an insert containing the ELP gene with SfiI-compatible sticky ends. Standard molecular biology techniques were employed for all DNA manipulations (Ausubel et al., 1995
), and details regarding pET-32a DNA modifications and fusion protein construction have been published previously (Meyer and Chilkoti, 1999
).
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BFP and GFP fusions with ELP were produced as follows. Plasmid DNA containing the genes for BFP (pQBIT7-BFP) and GFP (pQBIT7-GFP) were obtained from Qbiogene. Plasmids were digested with XbaI and AlwnI, and the first 723 bp of the BFP and GFP genes were isolated and purified from a low melting point agarose gel. A linker cassette with AlwnI and SalI compatible sticky ends encoding for the remaining 35 bp of the BFP and GFP genes was created from oligonucleotides (Integrated DNA Technologies). The modified pET25b vector containing the ELP gene was digested with XbaI and SalI, treated with CIAP, and purified from a low melting point agarose gel. Assembly of the fusion protein was accomplished by a ternary ligation of the 723 bp fragments of the BFP and GFP genes, the linker cassettes, and digested and purified pET25b vector. A pictorial representation of this cloning scheme and the sequence of the linker cassettes can be found in the supplementary material. Proper fusion protein assembly was confirmed by DNA sequencing.
The CAT gene was retrieved from its plasmid (provided by Invitrogen) by PCR and TA cloning (Invitrogen), in which NdeI and SalI restriction sites were also incorporated 5' and 3' to the gene, respectively. The gene was excised from the TA vector using NdeI and SalI and was purified from agarose gel. The modified pET25b vector, previously described, was digested with NdeI and SalI, and the fusion protein was assembled by ligation of the CAT gene with the restricted pET25b vector containing the ELP gene. Correct fusion protein assembly was confirmed by DNA sequencing. The expressed peptide sequences for all the fusion protein constructs can be seen in Figure 1.
Expression and purification
The ternary ELP fusion proteins (Trx'ELPTend and TrxELP'Tend, where ' denotes the location of a thrombin cleavage site) were expressed from BL21trxB(DE3) E.coli (Novagen). The binary fusions of ELP to the C-terminus of BFP, GFP, CAT and Trx were expressed from BLR(DE3) E.coli (Novagen). All proteins were expressed in 1 l cultures of E.coli in CircleGrowTM media (Qbiogene, Carlsbad, CA, USA), supplemented with 100 µg/ml ampicillin. These 1 l cultures were inoculated with E.coli cells from 10 ml of a starter culture (250 ml flask containing 50 ml of medium supplemented with 100 µg/ml ampicillin) that was inoculated from frozen (80°C) DMSO stocks and grown overnight. The 1 l cultures were grown either to an OD600 of 0.8, induced with 1 mM isopropyl -thiogalactopyranoside, and grown for an additional 3 h before being harvested or grown without induction for 24 h. Cultures were harvested by centrifugation at 4°C, resuspended in low ionic strength buffer (
1/25 culture volume), and lysed by ultrasonic disruption at 4°C. The lysate was centrifuged at
20 000 g at 4°C for 15 min to remove insoluble matter.
Each ELP fusion protein was purified from soluble E.coli lysate using inverse transition cycling, a protein purification technique that we have previously developed (Meyer and Chilkoti, 1999). In a typical purification, the ionic strength of the soluble lysate was increased to cause aggregation of the ELP in the cell lysate at room temperature, and the aggregated ELP was separated from soluble E.coli proteins by centrifugation. The pellet containing the ELP coacervate was resuspended in cold PBS and centrifuged at 4°C to remove insoluble contaminants. This procedure of thermal cycling and centrifugation was repeated (usually three times) until the ELP was determined to be
95% pure of E.coli contamination by visualization of Coomassie and/or copper-stained SDSPAGE gels. Protein concentration was determined by UVvisible spectroscopy using extinction coefficients at 280 nm calculated from the primary amino acid sequence with the software program Protean (DNA Star).
Binary ELP fusion proteins were also obtained by enzymatic cleavage of two different TrxELPTend ternary fusion proteins, TrxELP'Tend and Trx'ELPTend, using thrombin (Novagen). TrxELP was obtained from the enzymatic cleavage of TrxELP'Tend, which contains a thrombin cleavage site located between the ELP and the tendamistat. ELPTend was obtained from the cleavage of a different ternary protein, Trx'ELPTend containing a thrombin cleavage located between thioredoxin and the ELP. Free ELP-A was obtained by enzymatic cleavage of ELP binary fusion protein (Figure 1), and free ELP-B was obtained by expression of the ELP with the N-terminal and C-terminal linker peptides used in ELP binary fusions (Figure 1). All proteins were cleaved overnight at room temperature from solutions at 100 µM fusion protein using 10 U thrombin per µmole of fusion protein, and the cleavage products were purified by another round of inverse transition cycling.
Characterization of inverse temperature transition of ELP fusion proteins
The inverse transition temperatures of ELPs and ELP fusion proteins were measured on a Cary 300 UVvisible spectrophotometer equipped with a multicell thermoelectric temperature controller (Varian Instruments) by the change in optical density (OD) at 350 nm as the temperature was increased from 25 to 60°C at a heating rate of 1°C/min (Meyer and Chilkoti, 1999). The inverse transition temperature (Tt) was defined as the temperature at which the OD reached 50% of its maximum value.
The effect of PPA binding on the Tt of TrxELPTend, TrxELP and ELPTend was determined from turbidity measurements as a function of solution temperature. PPA (Roche) was obtained as a slurry in 3.2 M ammonium sulfate. Samples of 10 µM TrxELPTend, ELPTend and TrxELP were mixed in PBS with or without the presence of 10 µM PPA. Fusion protein concentrations were limited to 10 µM because of the approximate 12 µM solubility limit of PPA. Samples without PPA were supplemented with ammonium sulfate such that the buffer for all the samples was PBS with 320 mM ammonium sulfate.
Computer modeling
Protein Data Bank (PDB) files were selected from a representative group of 500 high-resolution structures (Lovell et al., 2003) (http://kinemage.biochem.duke.edu/databases/top500. php). Nineteen PDB files were discarded from the data set because they were structures of either short peptides or subunits of multi-domain proteins. The PDB file names of the proteins are listed in the supplementary material. Total and hydrophobic surface area calculations were performed using the program PROBE (Word et al., 1999
) and a contact sphere with a 1.4 Å radius. The probe sphere was rolled over the outside of the protein and a contact dot was placed on the atomic surface where the water-sized probe contacted the protein without simultaneously intersecting the van der Waals shell of another atom. The contact surface was considered hydrophobic when the dot contacted any atom within the following residues having non-polar side chains: Ala, Cys, Ile, Leu, Phe, Trp, Tyr or Val, and the exposed hydrophobic surface area was calculated as a fraction of the hydrophobic dots relative to the total number of dots. PDB files 1gfl, 1bfp, 2trxA, 1noc, 1bvn and 1bvn were used to calculate the surface characteristics of GFP, BFP, Trx, CAT, tendamistat and PPA, respectively.
ELP-functionalized gold colloids
Hydrogen tetrachloroaurate(III)hydrate [HAuCl4.xH2O (99.99%)], 11-mercaptoundecanoic acid (MUA) and 1-undec anethiol (UDT) were purchased from Aldrich. The ELP used for these studies was a 71 kDa polypeptide with the repetitive pentapeptide sequence (ValProGlyXaaGly)180 where Xaa was Val, Ala and Gly in the ratio of 5:2:3. This ELP has twice the chain length of the ELP used in the fusion proteins but has an identical composition.
All glassware used for preparation of gold colloids was thoroughly washed with aqua regia (3:1 HNO3:HCl), rinsed extensively with distilled water and then dried in an oven at 100°C for 2 h. Colloidal gold was prepared by sodium citrate reduction of HAuCl4 as reported earlier (Weisbecker et al., 1996). Ten milliliters of a 1 mg/ml solution of HAuCl4.xH2O were added with vigorous stirring to 180 ml of boiling water in a 500 ml round-bottomed flask fitted with a reflux condenser. After boiling resumed, 10 ml of a 10 mg/ml solution of sodium citrate in water were quickly added with continued stirring. The solution was boiled for another 20 min and then allowed to cool to room temperature, which resulted in the formation of a red suspension of colloidal gold. The suspension was filtered using a 0.22 µm filter (Corning, Corning, NY) and stored at 4°C until further use. The diameter of the gold colloids was determined to be
13 nm by transmission electron microscopy, as previously described (Nath and Chilkoti, 2001
).
The surface of the gold colloids was modified to be hydrophilic or hydrophobic by formation of a self-assembled monolayer (SAM) of a COOH-terminated thiol (MUA) or a CH3-terminated thiol (UDT), respectively, on the surface of the gold colloid (Bain et al., 1989; Weisbecker et al., 1996
). A suspension of the gold colloids was dialyzed overnight against 1 mM NaOH solution to remove excess citrate and chlorides from the solution. SAMs were prepared on the gold colloids by overnight incubation of a 1:1 (v/v) mixture of an aqueous suspension of colloidal gold and a 0.2 mM solution of MUA or UDT in absolute ethanol. Unreacted MUA and ethanol were removed from the COOH-functionalized gold colloids by centrifugation of the suspension at 14 000 g for 15 min, discarding the supernatant followed by resuspension of the colloids in 10 mM sodium phosphate buffer, pH 7.2 (PB). This step was repeated three times to ensure complete removal of the unreacted thiol and ethanol. Unreacted UDT and ethanol were removed from the CH3-functionalized gold colloidal suspension by dialysis in distilled water; centrifugation of hydrophobic gold colloids was avoided because it causes their irreversible aggregation.
COOH-functionalized gold colloids were incubated with 1 mg/ml ELP in PB for 2 h, and then washed twice by centrifugation at 14 000 g with PB and finally resuspended in 0.6 ml of PB for further studies. CH3-terminated gold colloids were incubated with 1 mg/ml ELP for 2 h. Unbound ELP was removed using centrifugal ultrafiltration (Centricon Millipore, Bedford, MA, USA) through a 100 kDa molecular weight cut-off filter.
Temperature-dependent behavior of ELP-functionalized gold colloids
The temperature-dependent aggregation of ELP-modified gold colloids was monitored by measuring the extinction spectrum as a function of temperature on a UVvisible spectrophotometer (Cary 300Bio; Varian Instruments), equipped with a thermoelectrically controlled multi-cell holder and temperature probe. The temperature was varied in 5°C increments over 1040°C. A thermocouple was used to monitor the temperature of the solution in the cuvette. The extinction spectra were collected between 350 and 750 nm, and the normalized integrated extinction (NIE) between 600 and 750 nm was used as an indicator of aggregate formation. The NIE is defined as follows: NIE(T) = (BT A)/A, where A is the initial integrated extinction between 600 and 750 nm at 10°C before commencement of thermal cycling, and BT is the integrated extinction between 600 and 750 nm at temperature T (Nath and Chilkoti, 2001).
Surface plasmon resonance (SPR)
For SPR measurements, glass coverslips were cleaned and a thin layer of Cr (20 Å) was thermally deposited under vacuum, followed by gold (500 Å). A carboxyl and methyl terminated SAM was prepared on the gold-coated glass coverslips by overnight incubation in a 1 mM solution of MUA and UDT, respectively, in absolute ethanol.
SPR studies were performed on a BiacoreX instrument (Biacore AB, Uppsala, Sweden) on SAM-functionalized gold substrates on glass, which were mounted on empty BiacoreX cassettes using water-insoluble double-sided sticky tape (3M Inc.). The ELP was adsorbed onto a COOH-terminated and CH3-terminated SAMs by perfusing a 1.0 mg/ml solution of the ELP (14.0 µM) at 1.0 µl/min, in 10 mM PB, pH 7.2 for 100 min at room temperature.
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Results and discussion |
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TrxELPTend fusion proteins containing the 90 pentapeptide ELP gene (TrxELP'Tend and Trx'ELPTend, where ' indicates the location of thrombin cleavage site) were expressed and purified by inverse transition cycling. A portion of each ternary fusion protein was treated with thrombin to digest the TrxELP'Tend and Trx'ELPTend fusions into (TrxELP+Tend) and (Trx+ELPTend), respectively. Figure 2 shows the turbidity profiles for 25 µM solutions of Trx'ELPTend, TrxELP'Tend, their thrombin-cleaved products, and free ELP-A. Clearly, the location of the thrombin cleavage site does not alter the Tt, because both Trx'ELPTend and TrxELP'Tend, indicated by closed and open triangles, respectively, have overlapping turbidity profiles and a Tt of 34°C. Thrombin cleavage of both TrxELPTend variants reveals the individual contributions of thioredoxin and tendamistat to the altered Tt of the ternary fusion protein. Cleavage of the ternary fusion protein TrxELP'Tend to liberate tendamistat results in the binary TrxELP protein with a Tt of 60°C, which is 9°C higher than that of the free ELP. Liberation of thioredoxin from the ternary fusion protein Trx'ELPTend results in the binary fusion ELPTend having a Tt of 35°C, which is 16°C lower than that of the free ELP. The Tt of ELPTend (35°C) is very similar to the ternary fusion proteins (34°C), indicating that tendamistat has the dominant effect on the Tt of the ternary fusion proteins.
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In a separate experiment (data not shown), we determined that free tendamistat in equimolar solution (i.e. after thrombin cleavage from the ternary fusion without subsequent purification steps) has no effect on the Tt of TrxELP, and likewise, free thioredoxin in solution has no effect on the Tt of ELPTend. Thus, the close proximity of fused proteins and not merely their presence in solution is responsible for the perturbation of the ELP Tt. These results led us to hypothesize that the molecular basis of these dramatic perturbations in Tt are connected with the interfacial properties of tendamistat and thioredoxin.
This same ELP was also fused to the C-terminus of Trx, GFP, BFP and CAT with the peptide linker sequence encoding for an oligohistidine tag and a thrombin cleavage site shown in Figure 1A. Figure 3 shows the turbidity profiles of these fusion proteins as well as the turbidity profile of the free ELP-B at 25 µM in PBS. Free ELP-B (with the binary fusion linker peptide shown in Figure 1A) exhibits a Tt of 48°C. Fusion of Trx, GFP and BFP resulted in the elevation of Tt to 54, 52 and 51°C, respectively, while fusion of CAT resulted in a depression to 36°C. Aggregation of Trx, BFP, GFP and CAT was completely reversible upon cooling after heating to 65°C. Fusion of CAT results in a broadening of the turbidity profile of the CATELP fusion protein relative to the free ELP-B; the cause of this remains a mystery.
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Minor changes in amino acid sequence in the peptide linkers adjacent to but not explicitly part of the folded fused protein or the ELP can have significant effects on the ELP Tt. Figures 2 and 3 demonstrate that the addition of 23 amino acids including an oligohistidine sequence to the N-terminus of the ELP results in a 3°C depression in the ELP transition temperature. TrxELP fusion proteins expressed from two different constructs with two different peptide sequences at the N- and C-termini of the ELP also exhibit two different Tts. TrxELP expressed as a binary fusion protein exhibited a Tt of 54°C (Figure 3) while TrxELP cleaved from the ternary fusion protein TrxELP'Tend had a Tt of 60°C (Figure 2) (Meyer and Chilkoti, 1999). Examination of the amino acid composition of these two different expression products yields clues as to the source of this perturbation. Table I shows the leader and trailer sequences immediately flanking the ELP for two different sources of free ELP and TrxELP, their experimentally measured Tts at 25 µM in PBS, and their calculated hydrophobicity from the Urry hydrophobicity scale (Urry et al., 1991
; Urry, 1997
).
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The calculated hydrophobicities of the linker peptides qualitatively correlate with the experimentally measured transition temperatures of both the free ELP and TrxELP having different leader and trailer sequences. For both proteins (ELP and TrxELP) the lower measured Tt and calculated hydrophobicity are consistent with the assumption that the oligohistidine tag in the leader sequence is a major contributor to the hydrophobicity to that linker sequence. Although His is not considered to be particularly hydrophobic on many amino acid hydrophobicity scales (Fauchere and Pliska, 1983; Radzicka and Wolfenden, 1988
; Roseman, 1988
), Urry indicates that at pH 8.0, where His is largely uncharged, His is the fourth most hydrophobic amino acid (Urry, 1992
, 1997
) on the ELP hydrophobicity scale. Because the pKa of His falls somewhere between 6.0 and 7.0 (Bundi and Wuthrich, 1979
; Matthew et al., 1985
), we expect the majority of histidines to be uncharged and hydrophobic at our working pH of 7.4. We have, therefore, used the uncharged values for histidine hydrophobicity in our calculations from the Urry scale. With this assumption, we observe that the hydrophobicity of the leader and trailer peptide sequence is consistent with the differences in the measured Tts of free ELPs and TrxELP fusion proteins containing different linker peptides.
Functionalized gold colloids as a probe of the effect of proximity of solid interfaces on the ELP Tt
We chose to use gold colloids as a mimic of the interfacial properties of proteins for the following reasons. First, they can be synthesized in size ranges that approximate that of proteins (5 nm and larger). Secondly, the interfacial properties of gold colloids can be easily modified by the formation of SAMs of alkanethiols; surfaces can be created via formation of SAMs that are hydrophobic, hydrophilic or chemically reactive, and these surface properties of gold can be further tuned by the formation of mixed SAMs. Finally, as shown previously, the phase transition of ELPs adsorbed or covalently conjugated to gold colloids can be conveniently monitored colorimetrically by the SPR of gold colloids (Kreibig and Vollmer, 1995
). This is because aggregation of ELP-modified gold colloids, owing to the hydrophilichydrophobic phase transition of the ELP, results in a dramatic change in color of the colloidal suspension from red to violet, and monitoring the wavelength shift of the extinction spectrum or the intensity at a fixed wavelength provides a simple read-out of the interfacial phase transition behavior of ELPs (Nath and Chilkoti, 2001
). Hence, these properties of gold colloids suggested their use as a simple model system, within the context of this study, to examine the effect of interfacial hydrophobicity (at a dimensional scale roughly commensurate with that of proteins) on the phase transition behavior of ELPs. Because the size of the gold colloids is roughly twice that of most of these fusion proteins, we used an ELP to functionalize the colloids that has twice the MW (71 kDa) of the ELP in the fusion proteins. This ELP has the same chemical composition and exhibits a similarly sharp inverse transition as the ELP in the fusion proteins. The only relevant difference in its properties from the shorter ELP is that it has a Tt that is 10°C lower (at 25 µM in PBS), which is a direct consequence of its larger MW (Meyer and Chilkoti, 2002
). Because these ELPs are so similar in their solution transition behavior and have identical compositions, we expect similar surface transition behavior as well.
We adsorbed the ELP onto gold colloids that were modified with either a COOH-terminated SAM or a CH3-terminated SAM. These SAMs were chosen to make the surface of the gold colloids hydrophilic (COOH-terminated SAM) or hydrophobic (CH3-terminated SAM). Figure 4A shows the extinction spectrum of an aqueous suspension of gold colloids, functionalized with a CH3-terminated SAM and adsorbed ELP, as a function of increasing solution temperature. Below a solution temperature of 20°C, the extinction spectrum does not vary with temperature. Above 20°C, however, the extinction spectra exhibit a shift in the extinction maximum towards higher wavelengths and an increase in the absolute intensity at wavelengths above 600 nm. The NIE, the area under the peak from 600 to 750 nm, is shown in Figure 4B for the adsorbed ELP on the COOH-terminated SAM on colloidal gold (circles) and CH3-terminated SAM (squares). The ELP adsorbed onto the COOH-terminated SAM exhibits a sharp and reversible phase transition similar to its behavior in solution, with a Tt of 30°C. In contrast, ELP adsorbed onto the CH3-terminated SAM exhibits significantly different thermal behavior. First, its Tt is depressed by
10°C compared with the COOH-terminated SAM, and secondly, the phase transition is irreversible. The Tt is defined by the onset of the transition from the baseline of the NIE because, unlike turbidity measurements, the maximum NIE measured for each of the surfaces does not necessarily represent a saturation in the spectral changes of the colloids as a function of temperature. The decrease in the Tt, when the surface presented to the ELP by the gold colloids is changed from hydrophilic to hydrophobic, is consistent with the hypothesis that an increase in interfacial hydrophobicity lowers the Tt of an ELP.
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The fusion Tt effect
Analogous to the means by which the proximity of a hydrophobic interface such as a hydrophobic gold colloid can perturb the Tt of an ELP, we hypothesize that covalently attached proteins and peptides alter the Tt of an ELP by a similar mechanism: fused proteins provide a surface in close proximity to the ELP, and the presence of hydrophobic residues on that surface depresses the ELP Tt. If this hypothesis is valid, then fused proteins of differing hydrophobic character should exhibit differing effects on the ELP Tt, and deliberate alterations in the surface hydrophobicity of fused proteins should perturb this effect on the Tt of the fused ELP.
We quantified the total and fraction hydrophobic solvent-accessible surface area (SAShydrophobic) of ELP fusion proteins using PROBE (Word et al., 1999). Using this program, the surface of a protein is interrogated by a rolling sphere with a 1.4 Å radius, corresponding to the radius of a water molecule, and the surface character is mapped according to its hydrophobicity. The surface is considered to be hydrophobic when the probe contacts residues having the following non-polar side chains: Ala, Cys, Ile, Leu, Phe, Trp, Tyr or Val. The fusion
Tt effect for each protein was determined by taking the Tt of the fusion protein and subtracting the Tt of the most appropriate free ELP control. All Tts were measured at 25 µM in PBS. For GFP, BFP, Trx and CAT, which were all expressed as binary fusions, an ELP Tt of 48°C corresponding to the ELP Tt expressed with the N-terminal linker peptide was used. For ELPTend, which was produced from the enzymatic cleavage of Trx'ELPTend, a Tt of 51°C, corresponding to an ELP lacking the His-tag linker peptide, was subtracted to compute
Tt for these ELP fusion proteins.
Figure 5 shows the fusion Tt of the binary ELP fusion proteins GFP, BFP, Trx, CAT and tendamistat as a function of SAShydrophobic and these data exhibit a linear correlation between fusion
Tt and SAShydrophobic with a slope of 96°C (fraction SAShydrophobic)1 and a
2 error of 20.4. Attempts to correlate the fusion
Tt effect with the total SAShydrophobic rather than the fraction SAShydrophobic resulted in substantially higher correlation errors (
2 = 350) suggesting that Tt is far more closely correlated with the composite surface characteristics of the fused protein rather than the total hydrophobic surface area.
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To gain a better understanding of the relative degrees of hydrophobicity for each of these proteins within a broader context, we calculated the fraction hydrophobic surface area for a group of nearly 500 proteins with high quality crystal structures. Figure 6 shows the fraction hydrophobic surface area for this representative group and highlights the values for the proteins of interest. Tendamistat and CAT are grouped at the hydrophobic end of this representative group, with tendamistat being among the most hydrophobic of all the proteins examined, while GFP, BFP and Trx are grouped at the opposite, hydrophilic end. The PPAtendamistat complex has an intermediate surface hydrophobicity in the database of approximately 500 proteins. From these calculations, we hypothesize that ELP fusion proteins containing tendamistat would exhibit a higher Tt upon binding to PPA.
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To experimentally test this hypothesis, we deliberately altered the surface properties of the TendELP fusion proteins by binding tendamistat to PPA and measuring the effect of complexation on the Tt of the fusion protein. Tendamistat, a strong competitive inhibitor of PPA, is a small protein (8 kDa) with a very large fraction of exposed hydrophobic surface (43%) (Wiegand et al., 1995). Tendamistat contacts PPA over a large surface area (
1330 Å2), and its high affinity for PPA is derived largely from the exclusion of water from much of the hydrophobic interface that contacts PPA (Wiegand et al., 1995
). Figure 7A shows the tertiary structure of tendamistat including its large solvent-exposed, hydrophobic PPA binding site. PPA, its binding partner, is a considerably larger protein (55 kDa) that hydrolyzes the
-1,4-glucan link in starch to form maltose (Wiegand et al., 1995
). Figure 7B shows the exposed solvent-accessible surface of the PPAtendamistat complex and the relative sizes of the two proteins. Upon binding, solvent is excluded not only from the partially hydrophobic binding pocket of PPA but also from the highly hydrophobic binding ridge in tendamistat, and the resulting tendamistatPPA complex has a hydrophobic surface area of 28%.
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These results also highlight the potential importance of electrostatics on the fusion protein Tt. Previous studies have clearly shown that the Tts of ELPs and ELP fusion proteins are depressed by the addition of salts (Urry, 1993; Meyer et al., 2001b
). This is also evident by comparing the Tts of TrxELPTend and Trx'ELP measured from the turbidity profiles in Figures 2, 3 and 8. The relatively low Tts in Figure 8 result from the addition of 320 mM ammonium sulfate from the PPA storage solution to maintain the activity of the PPA as recommended by the supplier (Roche). Ammonium sulfate solutions have ionic strengths three times greater than solutions having equivalent molar concentrations of NaCl. The amplified depression in fusion protein Tt with relatively small amounts of ammonium sulfate is consistent with the shielding of surface charges on fused proteins and the effect of multivalent anions on the inverse phase transition of the ELP itself. While ammonium sulfate causes a depression in Tt for both TrxELPTend and Trx'ELP, the magnitude of the depression is significantly higher for Trx'ELP. TrxELPTend and ELPTend (data not shown) exhibit a 10°C depression in Tt upon the addition of 320 mM ammonium sulfate, but Trx'ELP exhibits a 29°C depression. This observation suggests that electrostatics likely play a larger role in the aggregation behavior of TrxELP fusion proteins compared with TendELP fusions.
Because hydrophobic surfaces only account for negative changes in Tt, the fraction SAShydrophobic alone cannot explain the increase in the Tt of an ELP upon fusion to a protein. BFP, GFP and Trx cause an elevation in the ELP Tt upon their fusion. Previous studies have clearly shown that incorporation of polar, hydrophilic and charged amino acids at the fourth guest residue position in the VPGXG repeat of ELPs elevate the Tt of ELP. Charged moieties produce the most dramatic increases in ELP Tt, with the charged forms of Lys, Asp and Glu causing the greatest increase in ELP Tt when substituted at the guest residue position (Urry et al., 1991; Urry, 1997
). In fact, the phosphorylation of a single Ser residue in an ELP of 81 amino acids increases the Tt of an ELP by 12°C (Pattanaik et al., 1991
; Urry, 1992
). By analogy, we suggest the presence of these solvent-accessible polar and charged groups on the surface of the fusion partner competes with the negative effect of hydrophobic surfaces. Thus, the Tt of a particular ELP fusion protein likely arises from combination of the negative effects of hydrophobic surface area and positive effects of charged and/or polar surface area with charged residues being of particular importance. Although tendamistat and Trx have very similar calculated (DNA star) pIs of 4.4 and 4.5, respectively, the inverse transition behavior of TrxELP, which has a substantially smaller fraction SAShydrophobic, is more heavily dominated by its charged/polar groups, as evident by its high salt sensitivity and its positive shift in Tt relative to free ELP at 25 µM in PBS.
Proposed mechanism of the fusion Tt effect
Urry and colleagues have shown that the local environment of the ELP chain, and the degree of hydrophobic hydration, can be directly modulated by substitution of natural or unnatural amino acids for the Val that is normally present in native elastin; the Tts of ELPs are reduced by the incorporation of hydrophobic residues and the Tt is increased by polar and charged residues at the fourth, guest residue (X), position of the repeat sequence, VPGXG. The magnitude of this effect (the Tt effect) is dependent not only on the mole fraction but also on the hydrophobicity of the guest residue (Urry et al., 1991
; Urry, 1992
, 1997
). The fusion
Tt effect reported here is analogous to, but distinct from, the
Tt effect (Urry et al., 1991
; Urry, 1992
, 1997
) in which the hydrophobicity of the guest residues within the ELP chain modulates the Tt; in contrast, the fusion
Tt effect is one in which the ELP Tt is modulated by hydrophobic moieties not within the ELP chain but those closely associated with it.
As suggested by the molecular dynamics simulations of Daggett and colleagues (Li et al., 2001a,b; Li and Daggett, 2003
) and the microwave dielectric relaxation measurements of Urry et al. (1997
), the release of water molecules of hydrophobic hydration from the ELP with increasing temperature is the dominant molecular contributor to providing the thermodynamic driving force for the ELP phase transition. We believe that the interfacial waters of hydration solvating hydrophobic patches within molecular proximity to an ELP as well as the waters of hydrophobic hydration surrounding the ELP itself constitute the total number of waters that act in unison in response to an increase in the solution temperature. These surfaces include, but are not limited to, hydrophobic patches on the surfaces of fused folded proteins, short hydrophobic peptides fused to ELPs, or other hydrophobic surfaces within molecular proximity (i.e. hydrophobic gold colloids). Hence, with increasing temperature, the water molecules of hydrophobic hydration solvating the ELP chain as well as any closely associated with a hydrophobic surface are released to bulk. That the Tt of the ELP fusion protein is lowered relative to the ELP is simply a consequence of the release of the additional water molecules of hydrophobic hydration contributed by the fusion partner, which provides an additional gain in entropy per ELP chain upon their release to bulk.
Likewise, charged residues compete for waters of hydrophobic hydration (Urry, 1992). Fusion proteins having a low fraction SAShydrophobic and a greater fraction of charged residues exhibit a positive shift in ELP Tt consistent with this competition for waters of hydrophobic hydration. The smaller gain in entropy for this class of fusion proteins upon desolvation, relative to the ELP, is then reflected in the greater Tt of the fusion protein. An implicit set of assumptions that underlie this analysis are: (i) the ELP phase transition in a fusion protein is a first-order phase transition; (ii) the
H of the phase transition for the ELP fusion protein is similar in magnitude to that of the ELP and is weakly endothermic (Luan et al., 1990
, 1992
), so that the change in enthalpy is not the dominant factor in modulating the Tt; (iii) the conformational entropy of the ELP upon fusion is not significantly different from that of the ELP.
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Conclusions |
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The effect that fused proteins have on the Tt of ELPs is likely a complex one with charged and polar surface residues elevating and hydrophobic residues depressing Tt. Studies currently under way will further explore the effect of electrostatics on fusion protein Tt. These studies will help identify the optimal ELP tag and environmental conditions for protein purification based on the physico-chemical properties of the fusion partner. Finally, we note that aside from the insights it provides on the molecular origins of the fusion Tt effect, the altered Tt of TendELP upon binding of PPA is also interesting because it is the first demonstration, to our knowledge, of molecular recognition-mediated modulation of an ELP phase transition. We believe that modulation of an ELP fusion protein by ligand binding is likely to be useful for diverse applications in biotechnology including protein purification, ligand capture and biocatalysis.
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Acknowledgements |
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References |
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Bain,C.D. et al. (1989) J. Am. Chem. Soc., 111, 321335.[ISI]
Bundi,A. and Wuthrich,K. (1979) Biopolymers, 18, 285297.[ISI]
Fauchere,J.L. and Pliska,V. (1983) Eur. J. Med. Chem., 18, 369375.[ISI]
Kreibig,U. and Vollmer,M. (1995) Optical Properties of Metal Clusters. Springer-Verlag, New York.
Lavallie,E.R. et al. (1993) Biotechnology, 11, 187193.[ISI][Medline]
Li,B. and Daggett,V. (2003) Biopolymers, 68, 121129.[CrossRef][ISI][Medline]
Li,B. et al. (2001a) J. Am. Chem. Soc. 123, 1199111998.[CrossRef][ISI][Medline]
Li,B. et al. (2001b) J. Mol. Biol. 305, 581592.[CrossRef][ISI][Medline]
Lovell,S.C. et al. (2003) Proteins: Struct. Func. Genet., 50, 437450.[CrossRef][ISI]
Luan,C.-H. et al. (1990) Biopolymers, 29, 16991706.[ISI][Medline]
Luan,C.-H. et al. (1992) Biopolymers, 32, 12511261.[ISI][Medline]
Matthew,J.B. et al. (1985) CRC Crit. Rev. Biochem., 18, 91197.[ISI][Medline]
Meyer,D.E. and Chilkoti,A (1999) Nat. Biotechnol., 17, 11121115.[CrossRef][ISI][Medline]
Meyer,D.E. and Chilkoti,A. (2002) Biomacromolecules, 3, 357367.[CrossRef][ISI][Medline]
Meyer,D.E. et al. (2001a) Cancer Res., 61, 15481554.
Meyer,D.E. et al. (2001b) Biotechnol. Prog., 720728.
Nath,N. and Chilkoti,A. (2001) J. Am. Chem. Soc., 123, 81978202.[CrossRef][ISI][Medline]
Pattanaik,A. et al. (1991) Biochem. Biophys. Res. Commun., 178, 539545.[ISI][Medline]
Pflugrath,J.W. et al. (1986) J. Mol. Biol. 189, 383386.[ISI][Medline]
Radzicka,A. and Wolfenden,R. (1988) Biochemistry, 27, 16641670.[ISI]
Roseman,M.A. (1988) J. Mol. Biol., 200, 513522.[ISI][Medline]
Urry,D.W. (1988) J. Protein Chem., 7, 134.[ISI][Medline]
Urry,D.W. (1992) Prog. Biophys. Mol. Biol., 57, 2357.[CrossRef][ISI][Medline]
Urry,D.W. (1993) Angew. Chem. Int. Ed. Engl., 32, 819841.[ISI]
Urry,D.W. (1997) J. Phys. Chem., 101, 1100711028.[ISI]
Urry,D.W. et al. (1985) Biopolymers, 24, 23452356.[ISI][Medline]
Urry,D.W. et al. (1991) J. Am. Chem. Soc., 113, 43464348.[ISI]
Urry,D.W. et al. (1997) J. Am. Chem. Soc. 119, 11611162.[CrossRef][ISI]
Weisbecker,C.S et al. (1996) Langmuir, 12, 37633772.[CrossRef][ISI]
Wiegand,G. et al. (1995) J. Mol. Biol., 247, 99110.[CrossRef][ISI][Medline]
Word,J.M. et al. (1999) J. Mol. Biol., 285, 17111733.[CrossRef][ISI][Medline]
Received June 5, 2003; accepted October 21, 2003 Edited by Valerie Daggett