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Published online before print September 30, 2004, 10.1110/ps.04902704
Protein Science (2004), 13:2899-2908. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Insertion of the cytochrome b5 heme-binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome’s architecture

Jane A. Knappenberger, Christina M. Kraemer-Pecore1 and Juliette T.J. Lecomte

Department of Chemistry and the Center for Biomolecular Structure and Function, The Pennsylvania State University, University Park, Pennsylvania 16802, USA

Reprint requests to: Juliette T.J. Lecomte, Chemistry Department, The Pennsylvania State University, 152 Davey Laboratory, University Park, PA 16802, USA; e-mail: jtl1{at}psu.edu; fax: (814) 863-8403.

(RECEIVED May 28, 2004; FINAL REVISION August 9, 2004; ACCEPTED August 10, 2004)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 Electronic supplemental material
 References
 
Under native conditions, apocytochrome b5 exhibits a stable core and a disordered heme-binding region that refolds upon association with the cofactor. The termini of this flexible region are in close proximity, suggesting that loop closure may contribute to the thermodynamic properties of the apocytochrome. A chimeric protein containing 43 residues encompassing the cytochrome loop was constructed using the cyanobacterial photosystem I accessory protein E (PsaE) from Synechococcus sp. PCC 7002 as a structured scaffold. PsaE has the topology of an SH3 domain, and the insertion was engineered to replace its 14-residue CD loop. NMR and optical spectroscopies showed that the hybrid protein (named EbE1) was folded under native conditions and that it retained the characteristics of an SH3 domain. NMR spectroscopy revealed that structural and dynamic differences were confined near the site of loop insertion. Variable-temperature 1D NMR spectra of EbE1 confirmed the presence of a kinetic unfolding barrier. Thermal and chemical denaturations of PsaE and EbE1 demonstrated cooperative, two-state transitions; the stability of the PsaE scaffold was found only moderately compromised by the insertion, with a {Delta}Tm of 8.3°C, a {Delta}Cm of 1.5 M urea, and a {Delta}{Delta}G° of 4.2 kJ/mole. The data implied that the penalty for constraining the ends of the inserted region was lower than the ~6.4 kJ/mole calculated for a self-avoiding chain. Extrapolation of these results to cytochrome b5 suggested that the intrinsic stability of the folded portion of the apoprotein reflected only a small detrimental contribution from the large heme-binding domain.

Keywords: protein structure/folding; stability and mutagenesis

Abbreviations: {Delta}{delta}, change in chemical shift in ppm • bp, base pair • CD, circular dichroism • DQF-COSY, double-quantum-filtered correlated spectroscopy • EbE1, chimeric protein whose N- and C-terminal stretches are composed of residues from PsaE and whose intervening residues come from cytochrome b5 • EDTA, ethylenediaminetetraacetic acid • IPTG, isopropylthio-{beta}-D-galactoside • LB, Luria-Bertani medium • MRE, molar residual ellipticity • NOE, nuclear Overhauser effect • NOESY, two-dimensional nuclear Overhauser effect spectroscopy • OD600, optical density at 600 nm • PAGE, polyacrylamide gel electrophoresis • PDB, Protein Data Bank • PMSF, phenylmethylsulfonyl fluoride • PsaE, photosystem I accessory protein E • S7002, Synechococcus sp. PCC 7002 • SDS, sodium do-decylsulfate • SH3, Src homology domain 3 • TOCSY, totally correlated two-dimensional spectroscopy • Tris, tris(hydroxymethyl)aminomethane


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 Electronic supplemental material
 References
 
In the fashion of many ligand-binding proteins, the water-soluble domain of rat hepatic cytochrome b5 undergoes induced refolding upon association with its heme cofactor. In the holoprotein state, the three-dimensional structure is well represented by a narrow ensemble of conformations. As illustrated in Figure 1Go, the fold contains six helices and five strands (Mathews et al. 1979). The heme is fastened in its cavity by coordination bonds ligating the Fe ion to the imidazole N{varepsilon} atoms of His 39 (end of {alpha}2) and His 63 (beginning of {alpha}4). Two hydrophobic regions, termed core 1 and core 2, are also apparent in the cytochrome structure. The residues of core 1 are located in the central part of the amino acid sequence; they establish most of the protein-heme contacts and therefore play a functional role. In contrast, the residues comprising core 2 are found within the N- and C-terminal segments. Core 2 does not require the heme group to fold correctly (Moore and Lecomte 1993) and is thought to serve a structural purpose. In the apoprotein, core 1 can be viewed as a large, flexible loop inserted into a stable scaffold (Falzone et al. 1996). In previous studies aimed at analyzing the structural determinants of apocytochrome b5, an abridged version of the protein was prepared that contained core 2 and a short linker in place of the heme-binding segment (Constans et al. 1998). The main tertiary features of core 2 appeared to be specified by the shortened sequence, in support of a certain degree of structural independence of the two regions. In the present work, we focus on the heme-binding segment and its role in the apocytochrome’s architecture.



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  		Figure 1. Ribbon structures of (A) Synechococcus sp. PCC 7002 photo-system I accessory protein E (S7002 PsaE) (1psf [PDB] ; Falzone et al. 1994a) and (B) the water-soluble domain of bovine microsomal cytochrome (1cyo [PDB] ; Durley and Mathews 1996). Dashed lines indicate the points b5 at which the loops in the parent proteins begin and end. The chimeric protein EbE1 contains the folded scaffold of PsaE (portion below the dashed lines) and the heme-binding region of cytochrome (portion b5 above the dashed lines). Figure 1Go was prepared with MOLSCRIPT (Kraulis 1991).

 
Because a compromise between conformational flexibility and stability often needs to be achieved for a protein’s proper function, it is of interest to establish the effect of local disorder on the thermodynamic properties of naturally occurring and designed sequences containing unstructured loops. The thermal and chemical denaturations of apocytochrome b5 from various sources are cooperative, two-state processes (Pfeil 1993; Constans et al. 1998; Storch et al. 1999a,b; Cowley et al. 2002). Specifically, the Gibbs free energy of denaturation for rat microsomal apocytochrome b5 is only ~6 kJ/mole at room temperature and neutral pH, such that a small fraction of the molecules sample the unfolded state under those conditions. By comparison, many examples of water-soluble proteins the size of core 2 exist that are more stable than apocytochrome b5. It is not known whether the low stability is intrinsic to the folded core or due to the unstructured segment.

A theoretical framework is available to predict the entropic effect of disordered loops on fold stability. The contribution is associated with constraining the ends of a chain and is evaluated at


(1)

where k is a scaling factor equal to 3/2 in the three-dimensional freely-jointed case (Jacobson and Stockmayer 1950), l is the linking number or number of peptide bonds between the beginning and the end of the loop, c is a constant related to bond length and distance of approach, and R is the gas constant (Chan and Dill 1989). Several experimental studies have indeed shown that the introduction of flexible segments in certain regions of proteins can lead to marked thermodynamic destabilization of the fold. This is the case with the addition of just four Gly residues into the EF loop of the tenth fibronectin type III domain of human fibronectin (FNfn10) (Batori et al. 2002) and that of four Gly or four Thr residues into two long {beta}{alpha} loops in yeast phosphoglycerate kinase (yPGK) (Collinet et al. 2001). Replacement of a flexible region in bovine {alpha}-lactalbumin with the D helix from equine lysozyme results in a chimera of increased stability with respect to the wild-type protein (Tada et al. 2002) in an illustration of the reverse process, that is, the conversion of a disordered element into a rigid one. These specific instances suggest a quantifiable destabilization associated with restraining the ends of an inserted or elongated loop.

In contrast to the above findings, a number of cases exist for which the effects are significantly smaller than predicted. In the fibronectin example, 4-Gly insertions can also be accommodated without consequence at different locations in the protein (Batori et al. 2002). Likewise, yPGK tolerates 4-Gly and 4-Thr insertions in one of its short loops (Collinet et al. 2001). Up to 13 additional residues in a loop of a truncated version of chymotrypsin inhibitor-2 (CI2) affect stability and folding rates only weakly (Ladurner and Fersht 1997). Oftentimes, larger loops also fail to comply with the Jacobson-Stockmayer Gaussian model. Insertions of 60–80 random amino acids into the lck SH2 domain do not markedly affect the protein’s thermodynamic stability or ability to bind a phosphotyrosyl peptide (Scalley-Kim et al. 2003). Likewise, some E. coli RNase HI mutants retain enzymatic activity despite the insertion of 120–130 random, unstructured residues (Doi et al. 1997). These combined results indicate that the consequence of loop elongation is dependent upon the nature and site of the insertion. In light of this, it is not yet possible to predict what effect a particular loop will have at a particular position.

In cytochrome b5, the ends of the segment encompassing the disordered region rejoin after a span of 43 residues. According to equation 1Go and with k = 2.41 and c = 4.28 (Chan and Dill 1989) derived from ribonuclease T1 experimental data (Pace et al. 1988) rather than the freely-jointed chain model, the entropic penalty for end joining is expected to be on the order of 80 J/mole/K, which corresponds to a Gibbs free energy of 24 kJ/mole at room temperature. This large destabilization is comparable to the measured difference between apo- and holoprotein denaturation free energies (Pfeil 1993). To explore whether a scaffold is necessarily destabilized to this extent by supporting the cytochrome b5 core 1 region, this portion of the sequence was inserted in place of an existing 14-residue loop in Synechococcus sp. PCC 7002 (S7002) PsaE. The PsaE scaffold was chosen for its small size and SH3 domain structure (Fig. 1Go; Falzone et al. 1994a). The target insertion points are in the turn connecting the C and D strands. Naturally occurring PsaEs display variability in this region, which suggests that the site is tolerant of alterations and can be used for engineering purposes. In addition, the {alpha}-spectrin and p85{alpha} subunit of phosphatidylinositol 3 kinase SH3 domain proteins have previously been subjected to loop swapping, circular permutation, and loop elongation (Viguera and Serrano 1997; Martínez et al. 1999).

Using the same Eq. 1Go parameters as above to anticipate the entropic penalty of loop closure, the replacement of 14 residues by 43 residues should lead to a decrease in stability ({Delta}{Delta}G°) of ~6.4 kJ/mole. The thermodynamic stability of SH3 domains is in the range of 12–21 kJ/mole (Lim et al. 1994; Filimonov et al. 1999; Martínez et al. 1999), with PsaE at the lower limit (Mayer et al. 1999). As a result, if the cytochrome b5 loop destabilizes the PsaE scaffold by the calculated {Delta}{Delta}G°, a noticeable effect should be detected in the equilibrium populations of molecules sampling the native and denatured conformations under native conditions.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 Electronic supplemental material
 References
 
The primary structures of the wild-type proteins used in this work (S7002 PsaE and the soluble domain of rat hepatic cytochrome b5) and the hybrid protein are shown in Figure 2Go. The portion of cytochrome b5 to be inserted into PsaE and the insertion points were chosen by inspection of the three-dimensional structures (Fig. 1Go). The resulting polypeptide was called EbE1 to reflect that the N and C termini of the protein are comprised of residues from PsaE, the intervening portion is taken from cytochrome b5, and it is the first such chimera constructed. The gene encoding the 98-residue EbE1 was prepared using the overlapping oligonucleotides method (Gupta et al. 1968). The purity of the protein was determined by SDS-PAGE to be greater than 95%. Electrospray ionization mass spectrometry confirmed the high purity and returned an observed mass of 10,929.6 ± 0.3 Da, consistent with the expected theoretical mass of 10930.0 Da.



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  		Figure 2. Primary structures of rat hepatic cytochrome b5 (Cyt b5), S7002 PsaE (PsaE), and the resulting chimeric protein EbE1. The heme-binding region of cytochrome b5 and the three-dimensional scaffold provided by PsaE into which the loop was inserted are shown in capital letters. EbE1 is so named because its N and C termini are comprised of residues from PsaE, the intervening residues come from cytochrome b5, and it is the first such chimera. To facilitate the comparison of EbE1 to PsaE and cytochrome b5, the original amino acid numbering was conserved in both portions of the hybrid. The letter E was appended to the residues originating from PsaE and the letter b to those from the cytochrome.

 
The structural properties of EbE1 were studied by optical methods. The far-UV CD spectra of PsaE and the hybrid protein are shown in Figure 3Go. Unlike PsaE, EbE1 did not exhibit positive ellipticity values between 220 nm and 228 nm. However, inspection of the spectra for the folded and unfolded states of both proteins (Fig. 3Go) revealed a comparable change in ellipticity over this wavelength range upon unfolding. Although not typical of {beta}-sheet proteins, the native state spectra in Figure 3Go are characteristic of SH3 domains (Lim et al. 1994; Viguera et al. 1994; Knapp et al. 1996; Renzoni et al. 1996; Bousquet et al. 2000; Okishio et al. 2003). In the absence of {alpha}-helical structure, CD bands in the region of 225 nm have been attributed to aromatic side chains (Schmid 1989). In SH3 domains, the positive ellipticity in the region of 220 nm stems from interactions among clustered aromatic residues (Bousquet et al. 2000; Okishio et al. 2003). PsaE and EbE1 contain Tyr 16-Trp 17-Tyr 18, Phe 40, and Phe 60 (Fig. 2Go) and have the potential for distinct clustering.



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  		Figure 3. Far-UV CD spectra of native (a, black) and denatured (b, light gray) PsaE and native (c, gray) and denatured (d, dark gray) EbE1 at pH 7.4 in 20 mM phosphate buffer. Native spectra were obtained at 25°C, denatured at 95°C. Both proteins show similar loss of ellipticity upon heating.

 
NMR spectroscopy was used to characterize the hybrid further. Figure 4Go presents the 1H spectra of PsaE and EbE1, which indicated that the hybrid was folded under normal conditions of pH and temperature. Well-resolved features that were nearly identical in the PsaE and EbE1 spectra included the two upfield peaks and a cluster of resonances downfield from 9.4 ppm. Examination of correlation data confirmed that one {gamma}-CH3 of Val 38E and the {delta}-CH3 of Ile10E gave rise to the upfield peaks, in agreement with the PsaE spectrum (Falzone et al. 1994b). The downfield-shifted peaks corresponded to the backbone NHs of Lys 9E, Ala 25E, Asp 28E, Phe 60E, Ala 61E, and Glu 62E, as well as Trp 17E indole N{varepsilon}H. This latter peak was shifted upfield by 0.48 ppm compared to its position in PsaE, and signified a subtle alteration in the environment of Trp 17. Fluorescence spectra of the two proteins were collected under native conditions to confirm the change. The emission maximum of EbE1 was blue-shifted by 2 nm and its intensity decreased by 15% compared to PsaE (data not shown), in support of a minor local perturbation and in agreement with the NMR and far-UV CD data. Trp 17 is positioned such that its N{varepsilon}H is less than 4 Å away from the 14-residue loop replaced in the creation of the hybrid protein, and the environment adjustment was not surprising.



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  		Figure 4. 1H NMR spectra of (A) PsaE (25°C [pH 7.2]) and (B) EbE1 (19°C [pH 7.2], 20 mM phosphate buffer) in 90% 1H2O/10% 2H2O. Many of the resolved features are nearly identical as shown by the connecting dashed lines.

 
Backbone chemical shifts can be used qualitatively to assess structural similarity. Figure 5Go presents a comparison of EbE1 to PsaE and, to gauge the significance of the deviations, EbE1 to random coil. The absolute value of {Delta}{delta} for random coil and EbE1 ranged from 0 ppm to 1.65 ppm, whereas |{Delta}{delta}| for PsaE and EbE1 ranged only from 0 ppm to 0.12 ppm. Significant spectral alterations (either shift or broadening) on going from PsaE to EbE1 were observed in the proximity of the foreign cytochrome b5 portion of the molecule at the level of Arg 39E, Phe 40E, and Asn 41E. These residues comprise the final three on the N-terminal side of the insertion. Noticeable changes near the foreign loop and minimal differences elsewhere have been reported for circular permutants of another SH3 domain with varying loop lengths (Viguera and Serrano 1997). Similar results were obtained for Asn 56E through Asn 59E on the C-terminal side of the EbE1 loop. As mentioned above, the environment surrounding Trp 17 has been affected, and this was reflected in {Delta}{delta}’s observed for backbone signals in the PsaE and EbE1 backgrounds. On average, the pronounced chemical shift similarity of the two proteins indicated that EbE1 closely resembled PsaE.



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  		Figure 5. Chemical shift differences between PsaE and EbE1 and random coil and EbE1, plotted as a function of PsaE residue number. Squares represent backbone NH ({blacksquare}, PsaE–EbE1; {square}, random coil–EbE1), and triangles represent C{alpha}H ({blacktriangleup}, PsaE–EbE1; {triangleup}, random coil–EbE1). The hatched region marks the location of the CD and cytochrome b5 loop within the PsaE scaffold. Several backbone signals of EbE1 were not detected in the region preceding and following the loop. Chemical shift differences between the two proteins are otherwise small (see Supplemental Material), indicating that the backbone protons are in nearly identical fold and environment.

 
Two-dimensional homonuclear NMR experiments were analyzed to inspect the details of the EbE1 fold. The quality of the data is illustrated in Figure 6Go with the footprint region of the EbE1 NOESY data. The {beta}-strand secondary structure of the hybrid protein was determined by following scalar (NHi to C{alpha}Hi, obtained from COSY and TOCSY spectra) and dipolar (C{alpha}Hi to NHi+1) connectivities; its topology is summarized in Figure 7Go. EbE1 and PsaE had many common long-range NOEs, including the cross-strand effects establishing the register of the sheet. Among the characteristic pairwise C{alpha}H interactions were Ile 10E to Leu 65E and Val 8E to Val 67E; these confirmed that PsaE strands A and E were antiparallel as in the original protein (Falzone et al. 1994a). Other NOEs between strands A and B, B and C, C and D, D and E, and E and A were also preserved, in support of the SH3 fold. However, cross-strand NOEs between Arg 39E and Thr 57E were not found, which verified that the three-dimensional structure leading into and out of the loop had been altered.



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  		Figure 6. Footprint region of the NOESY spectrum of EbE1 obtained in 90% 1H2O/10% 2H2O at 19°C (pH 7.2). Many of the NOEs observed for EbE1 are identical to those obtained for PsaE (Falzone et al. 1994a). The {beta}-strand secondary structure of the hybrid protein was determined by following scalar (NHi to {alpha}Hi, obtained from COSY and TOCSY spectra) and dipolar ({alpha}Hi to NHi+1) connectivities. Lines connect cross-peaks corresponding to strands in the SH3 fold (dashed black, {beta}A, 6 to 11; dashed gray, {beta}B, 21 to 28; solid gray, {beta}D, 58 to 62; solid black, {beta}E, 64 to 69). Cross-peaks are identified either with the number i for NHi-{alpha}Hi effects or with the numbers i/i+1 for sequential {alpha}Hi-NHi+1 effects.

 


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  		Figure 7. Layout of the {beta}-sheet structure of PsaE and EbE1. Black lines symbolize the loops contained in the two proteins. Solid black lines, loops common to PsaE and EbE1; dashed black line, CD loop of PsaE replaced by the heme-binding region of cytochrome b5. Gray lines symbolize inter-residue NOE cross-peaks; solid single lines, NOEs observed in both proteins; solid double lines, NOEs observed in PsaE but not EbE1; dotted lines, NOEs observed in PsaE that were obscured by overlap in EbE1.

 
The NMR signals contributed by the cytochrome b5 portion of the hybrid protein were not well resolved, and a minimal number of assignments were obtained. Interestingly, multiple sets of cross-peaks were detected for the Val 61b-Gly 62b-His 63b stretch, and this segment of the protein therefore adopted at least two conformations exchanging slowly on the chemical shift time scale. The chemical shifts for one of these conformations agreed well with those previously determined for apocytochrome b5 (Falzone et al. 2001), whereas the second set of observed chemical shifts differed by approximately 0.1 ppm.

One-dimensional 1H NMR spectra of EbE1 were collected as a function of temperature to monitor the loss of structure upon heating (Fig. 8Go). Resolved peaks broadened and decreased in height throughout the heating process, but chemical shifts remained unchanged. This indicated that the N {rightleftharpoons} U equilibrium was slow with respect to the chemical shift time scale, and implied the existence of an energetic barrier to unfolding. The spectrum of EbE1 obtained upon returning the sample to room temperature exhibited some broad features underneath the sharp, native signals, in a hint that a portion of the sample had been denatured irreversibly via aggregation.



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  		Figure 8. 1H NMR spectra of EbE1 in 2H2O as a function of temperature: (A) 21°C, (B) 25°C (first heating), (C) 29°C, (D) 37°C, (E) 45°C, (F) 25°C (after cooling).

 
The structural and variable temperature data demonstrated that the PsaE scaffold could tolerate the long insertion, and EbE1 was therefore deemed suitable to inspect the thermodynamic consequences of loop replacement. The thermal denaturation of PsaE and EbE1 was followed by far-UV CD spectroscopy and absorbance spectroscopy. The data were fit to a two-state equilibrium-unfolding model and yielded Tms for EbE1 and PsaE of 52.5 ± 0.3°C and 60.8 ± 0.2°C, respectively. The results are shown in Figure 9Go and summarized in Table 1Go. Because the difference in folded and unfolded signal was small and the transition for PsaE was not fully reversible, values for the Gibbs free energy difference obtained from curve fitting were not accurate. However, the presence of a native baseline in the EbE1 unfolding curve was consistent with a moderate destabilization compared to PsaE.



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  		Figure 9. Thermal denaturation of EbE1 and PsaE followed by absorbance and CD spectroscopy. Open symbols represent data ({triangleup}, EbE1 CD; {circ}, EbE1 UV-Vis; {lozenge}, PsaE CD; {square}, PsaE UV-Vis), and solid lines represent the result of fitting to a two-state model as described in the Materials and Methods section. Parameters obtained from the best fit of the data are given in Table 1Go.

 

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  		Table 1. Thermodynamic parameters for the denaturation of PsaE and EbE1
 
In contrast to thermal denaturation, the urea-induced process was reversible, and the data (Fig. 10Go) could be used for quantitative purposes. Fitting of the EbE1 urea unfolding curves to a two-state model yielded a standard Gibbs free energy of unfolding of 10.27 ± 0.14 kJ/mole. The midpoint of the unfolding transition occurred at ~3.2 M urea, and the m value was 3.2 ± 1.0 kJ/mole/M. In comparison, the standard Gibbs free energy of unfolding obtained for PsaE was 14.47 ± 0.03 kJ/mole, with a midpoint of 4.74 M urea and an m value of 3.04 ± 0.10 kJ/mole/M (data are summarized in Table 1Go). As the m parameter is generally associated with the amount of nonpolar surface area exposed upon unfolding of the protein (Creighton 1993), obtaining similar values for two proteins that differed only in the size of a solvent-exposed loop was expected. The numbers above evaluated the difference in the thermodynamic stabilities of the two proteins ({Delta}{Delta}G°) at ~4.2 kJ/mole.



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  		Figure 10. Urea denaturation of EbE1 and PsaE followed by absorbance and CD spectroscopy. Open symbols represent data ({triangleup}, EbE1 CD; {circ}, EbE1 UV-Vis; {lozenge}, PsaE CD; {square}, PsaE UV-Vis), and solid lines represent the result of fitting to a two-state model as described in Materials and Methods. Parameters obtained from the best fit of the data are given in Table 1Go.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 Electronic supplemental material
 References
 
The denaturation experiments indicated that the destabilization caused by replacing the 14-residue CD loop of PsaE with the 43-residue heme-binding loop of cytochrome b5 was small. The structural deviations imposed on the original SH3 domain by the insertion, though modest, involved the disruption of H-bonds between the C-terminal end of the {beta}C strand and the N-terminal end of the {beta}D strand; these disruptions were likely to contribute an enthalpic component to the observed thermodynamic effect.

There are several difficulties in comparing the experimental {Delta}{Delta}G° with theoretical estimates of –T{Delta}{Delta}S°. First, given the structural distortions at the base of the EbE1 loop, there is some uncertainty in the linking numbers that should be used in equation 1Go. Second, the enthalpic contribution could not be obtained accurately from the thermal denaturation data. The third difficulty, and the most daunting, rests with the scaling factor k in equation 1Go. The freely-jointed three-dimensional chain has a k of 1.5 (Jacobson and Stockmayer 1950), but excluded volume effects are expected to raise this value (Redner 1980; Chan and Dill 1989). Calculations for a self-avoiding chain in a face-centered cubic lattice return k = 2.16 (Redner 1980). The location of the loop within the polymer also influences the anticipated extent of destabilization (Chan and Dill 1989). In apocytochrome b5, the flexible region spans the central portion of the primary structure, whereas the CD loop of PsaE is closer to the C than the N terminus. This discrepancy in loop position would decrease the estimated {Delta}{Delta}S°. Interestingly, surveys of structures contained in the PDB show that the probability of contact between two amino acids, i and j, decreases as |i – j|k, where k is 1.5 – 1.64 (Berezovsky et al. 2000; Dokholyan and Shakhnovich 2001). However, experimental data collected on disulfide-containing proteins suggest k values close to 2.4 and 2.5 (Chan and Dill 1989; Darby and Creighton 1993), whereas certain iso-1-cytochrome c loops yield a higher value of 4.2 (Hammack et al. 2001).

As discussed by Bowler and coworkers (Hammack et al. 2001), several sequence characteristics impinge on the scaling factor. For example, a rise in proline content will increase the persistence length of the chain and its k value, whereas an increase in glycine content will have the opposite effect (Schimmel and Flory 1967), with composition having the greatest impact for loops with l < 40 (Conrad and Flory 1976). Regarding the PsaE-to-EbE1 change, the pro-line content is practically constant, rising from 0% to 2%, but the glycine content decreases from 29% to 12%. The CD loop and the heme-binding loop should therefore be treated with different k’s, reflecting a stiffer chain in the latter case. Residual structure can also affect the dependence of {Delta}S° on loop length; it may either facilitate closure by compacting the chain in a favorable conformation, or hinder it by increasing the persistence length. Although no regular structure is observed in the CD loop (Falzone et al. 1994a), the 43-residue cytochrome segment forms a couple of helical turns in its wild-type context (Falzone et al. 1996). Whether or not a similar conformation was sampled by the EbE1 loop could not be ascertained.

Overall, the calculated entropic destabilization relying on the same k value for the 14- and 43-residue loops ranged between 4 and 11 kJ/mole; these estimates are of limited usefulness considering the above reservations. Regardless of the agreement between calculated and observed values, the EbE1 insertion is of little thermodynamic consequence, and this chimera adds to the list of proteins for which loop elongation is minimally perturbing.

The results obtained with EbE1 lead to a better understanding of the properties of the parent proteins. In photo-system I, the CD loop of PsaE plays a functional role; it makes contact with PsaC (another extrinsic protein) and the stromal loops of PsaA and PsaB. These interactions naturally result in a conformational rearrangement compared to the family of solution structures (Fromme et al. 2001), but there is no alteration of the H-bond network within the resilient scaffold. Interestingly, the thermodynamic stability of a related PsaE protein (Nostoc sp. PCC 8009) with a 7-rather than 14-residue CD loop is similar to that reported here (Mayer et al. 1999). The ability of PsaE to accommodate 7–36 extra residues without extreme consequences also supported that this particular insertion location was ideal.

Extrapolation of the EbE1 results to the architecture of the cytochrome can be contemplated in light of the above. If the CD loop has a minor effect on its own scaffold, then its removal and replacement with a foreign loop should inform principally on the effect of the latter. Assuming that the PsaE and core 2 backgrounds are equally receptive of the heme-binding region, the observations made for EbE1 lend support to the view that core 2 has a low intrinsic stability. We conclude that the thermodynamics of the folded portion of apocytochrome b5 need not be severely affected by the size of the heme-binding loop. Additional work is in progress to test the hypothesis of limited interdependence of flexible and fixed regions and to identify the features that lead to strong coupling between them.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
Electronic supplemental material
 References
 
EbE1 gene construction
The pET3d vector containing the gene for PsaE from the cyanobacterium Synechococcus sp. strain PCC 7002 was cut using BamHI and NcoI, and the ends were not dephosphorylated. The vector was purified using a 1% SeaPlaque gel. The gene for the hybrid protein was constructed using overlapping oligonucleotides, with 12-bp overhangs. DNA oligos ranging in length from 43 to 57 nucleotides were ordered from Operon Technologies. The oligos were purified on a denaturing polyacrylamide gel. The DNA was eluted out of the crushed gel with TE buffer and recovered using EtOH precipitation. Next, the oligos were phosphorylated using polynucleotide kinase. The oligos were then annealed by heating the mixture up to 90°C and slowly cooling to room temperature. The nicks were then sealed using T4 DNA ligase. The completed insert was purified on a 3% NuSieve, 1% SeaKem gel, run onto a DEAE membrane, eluted with TE buffer, and recovered by EtOH precipitation. The vector and insert were ligated using T4 DNA ligase, and the resulting plasmid was transformed into competent E. coli DH5{alpha} cells. The plasmid was sequenced by the Penn State Nucleic Acid Facility.

Protein overexpression and purification
The plasmid for the hybrid protein was used to transform competent E. coli BL21(DE3) cells (Novagen), which were grown in LB medium at 37°C. Expression of the protein was induced with IPTG when the OD600 was ~0.8. The cells were harvested 4.5 h postin-duction and subjected to three rounds of lysing by sonication in the presence of PMSF. The majority of the protein was found to be in the soluble portion of the cell. The protein was purified on a DEAE Sephacel anion exchange column (Amersham Pharmacia Biotech) using a buffer of 50 mM Tris and 1 mM EDTA at pH 7.5. The fractions containing the protein were then concentrated and purified on a Sephadex G-50 Fine column (Amersham Pharmacia Biotech) using a buffer of 50 mM Tris, 1 mM EDTA, and 20 mM NaCl at pH 7.5. The purity of the protein was determined to be >95% by SDS-PAGE, and the appropriate fractions were exchanged into pH 7.5, 20 mM phosphate buffer. Although the protein is pure as determined by SDS-PAGE, it runs anomalously in the denaturing gel, appearing to weigh ~19 kDa. EbE1 did not bind hemin chloride specifically.

The pET3d plasmid harboring the gene for PsaE was transformed into competent BL21(DE3) cells, which were grown in LB medium at 37°C. Expression was induced when the OD600 reached 0.8. Cells were harvested 5–6 h postinduction and stored at –20°C until used. Resuspended cells were subjected to three rounds of sonication. The vast majority of the protein was insoluble. The inclusion bodies were solubilized with 8 M urea, and the protein was renatured/purified on a Sephadex G-50 Fine column (Amersham Pharmacia Biotech) using a buffer of 50 mM Tris and 1 mM EDTA at pH 7.5. The purity of the protein was determined to be >95% by SDS-PAGE. Protein concentration was determined using extinction coefficients derived from amino acid composition, 11040 M–1cm–1 at 278 nm for both proteins.

Optical spectroscopy
All CD spectra were obtained on an Aviv 62DS CD spectropolarimeter. Data were collected using cuvettes with either a 2-mm or a 1-cm pathlength. Far-UV spectra were obtained at 25°C (pH 7.4) using a spectral bandwidth of 2.5 nm. Absorbance spectra were recorded on an Aviv 14-DS spectrophotometer. Fluorescence data were collected on a Jobin Yvon-SPEX Fluorolog-3 fluorimeter (Instruments S.A.). Sample concentration was such that the absorbance at 280 nm was less than 0.08 au. Emission spectra spanned 285 nm to 450 nm for 280-nm excitation and 300 nm to 450 nm for 295-nm excitation.

Thermal denaturation
Thermal denaturation was monitored by far-UV CD and absorbance spectroscopy. For the CD experiments, protein samples ranged in concentration from 10 to 40 µM at pH 7.4 in 20 mM phosphate buffer. The temperature was increased from 25°C to 91°C in steps of 2°C. Reversibility was determined by cooling the sample using the same equilibration times and temperature steps as used for the heating curve. EbE1 exhibited ~90%–95% reversibility, and PsaE only ~40%–50%. CD spectra were collected between 300 nm and 200 nm with an averaging time of 5 sec at each temperature; alternatively, ellipticity was monitored at a single wavelength (221 nm for PsaE or 222 nm for EbE1) and an averaging time of 30 sec. In each case, the total equilibration time was 5 min. For the absorbance measurements, 1 cm cells were used, and the protein concentration was ~100 µM. Spectra were collected from 320 nm to 250 nm with the same heating/cooling schedule. The absorbance at 295 nm was chosen for data analysis.

Thermal denaturation data were fit using the program NFIT (University of Texas, Galveston) or SAVUKA (D.G. Lambright and O. Bilsel) to a Gibbs-Helmholtz equation for a two-state process:


(2)

where {Delta}H°Tm represents the enthalpy of denaturation at the mid-point temperature (Tm). {Delta} C°p values in Table 1Go are the results of fitting with this equation, and their accuracy is limited. Throughout the experiment, the observed signal at any temperature T (Y(T)) is a function of the fractional population in the native (FN(T)) and unfolded (FU(T)) states and the spectral properties of these states (YN(T) and YU(T)):


(3)

The fractional population of the native state is given by:


(4)

where KU(T) is the equilibrium constant for unfolding. This constant is related to {Delta}G°(T) via –RTlnKU(T). The unfolding profiles obtained by the two optical methods were coincident (Fig. 9Go) in support of the two-state treatment.

Urea denaturation
Urea denaturations were performed using ultra-pure urea (MP Biomedicals), which was purified further on a mixed-bed resin column (Bio Rad). The urea concentration in the pre-titration sample was determined on a Leica Abbe Mark II Refractometer (Pace 1986; Pace et al. 1989). For the CD experiments, appropriate volumes of pH 7.4, 9–10 M urea, 20 mM phosphate buffer, 40 µM protein were titrated into a 1-cm cuvette initially containing 2 mL of 20 mM phosphate buffer, 40 µM protein at pH 7.4. Data were collected at 25°C. The titration was controlled by a Hamilton Microlab titrator with a syringe volume of 500 µL. Equilibration time was 5 min, and data were collected at 221 nm every 2 sec for 400 sec with 2.5-nm bandwidth and 2-sec averaging time. The root mean square value for the 400 sec of data collection was used for fitting purposes. For the absorbance measurements, protein concentration was 40–60 µM. Near-UV spectra were collected from 320 nm to 250 nm, with a 1-nm step size, 5-sec averaging time, and 2.5-nm bandwidth. The absorbance at 295 nm was chosen for data analysis. The same titrator and conditions were used.

Chemical denaturation data were processed using the fitting program SAVUKA, and the free energy of folding was determined based on the equation


(5)

where {Delta}G°H2O is the free energy of folding in water, {Delta}G°D is the free energy of folding at a denaturant concentration of D, and m is the dependence of the free energy on denaturant concentration. In this case, [D] plays the role of T in equations 3Go and 4Go. Posttitration pH and urea concentration were checked to ensure that the titration had proceeded correctly. Reversibility of the chemical denaturation was determined by taking a highly concentrated protein sample in ~8–9 M urea and diluting with 20 mM phosphate buffer at pH 7.4. Both proteins exhibited a high degree of reversibility. Here as well, the unfolding profiles obtained by the two optical methods were coincident (Fig. 10Go) and justified the application of the two-state treatment.

NMR spectroscopy
All experiments were performed on a Bruker DRX 600 spectrometer (Bruker BioSpin) on samples in which the protein concentration was 1–1.5 mM. The solvent for H2O samples was 90% 1H2O/10% 2H2O. The pH for all samples was in the range of 7.0 to 7.5, and was not corrected for isotope effect. 1D spectra were collected using presaturation of the water line. NOESY, COSY, and TOCSY experiments were performed as described (Falzone et al. 1994a). The mixing times were 100 msec (NOESY) and 45 msec (TOCSY). A total of 2 k data points were collected in each dimension, with a spectral width of 8 kHz.

Concentration dependence of EbE1 properties
The concentration dependence of EbE1 conformational properties was examined by NMR and optical methods. 1D 1H-NMR spectra were collected for samples ranging in concentration from 50 µM to 2 mM. The only detectable difference in the spectra was a slight sharpening of lines for lower concentrations (data not shown), presumably owing to the decreased viscosity of the sample. The far-UV CD spectra showed no concentration dependence from 10 µM to 40 µM. Hence, throughout the concentration range employed in this study, the oligomeric state of the protein appeared invariant. All data were consistent with a monomer.


   Electronic supplemental material
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Electronic supplemental material
References
 
A table of 1H chemical shifts for the chimeric protein EbE1.


   Footnotes
 
1 Present address: Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington, VT 05405, USA. Back

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04902704.

Supplemental material: see www.proteinscience.org


   Acknowledgments
 
This work was supported by NIH grant GM-54217. We thank Dr. C.J. Falzone and Ms. N.L. Scott for assistance and insight. SAVUKA was a kind gift from Dr. O. Bilsel.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Electronic supplemental material
 References
 
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