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Published online before print November 22, 2006, 10.1110/ps.062557707
Protein Science (2007), 16:14-19. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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Solution structure of a small protein containing a fluorinated side chain in the core

Gabriel Cornilescu1, Erik B. Hadley2, Matthew G. Woll2, John L. Markley1, Samuel H. Gellman2, and Claudia C. Cornilescu1

1 NMRFAM, Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA
2 Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA

(RECEIVED September 13, 2006; FINAL REVISION October 6, 2006; ACCEPTED October 10, 2006)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
We report the first high-resolution structure for a protein containing a fluorinated side chain. Recently we carried out a systematic evaluation of phenylalanine to pentafluorophenylalanine (Phe -> F5-Phe) mutants for the 35-residue chicken villin headpiece subdomain (c-VHP), the hydrophobic core of which features a cluster of three Phe side chains (residues 6, 10, and 17). Phe -> F5-Phe mutations are interesting because aryl–perfluoroaryl interactions of optimal geometry are intrinsically more favorable than either aryl–aryl or perfluoroaryl–perfluoroaryl interactions, and because perfluoroaryl units are more hydrophobic than are analogous aryl units. Only one mutation, Phe10 -> F5-Phe, was found to provide enhanced tertiary structural stability relative to the native core (by ~1 kcal/mol, according to guanidinium chloride denaturation studies). The NMR structure of this mutant, described here, reveals very little variation in backbone conformation or side chain packing relative to the wild type. Thus, although Phe -> F5-Phe mutations offer the possibility of greater tertiary structural stability from side chain–side chain attraction and/or side chain desolvation, the constraints associated with the native c-VHP fold apparently prevent the modified polypeptide from taking advantage of this possibility. Our findings are important because they complement several studies that have shown that fluorination of saturated side chain carbon atoms can provide enhanced conformational stability.

Keywords: pentafluorophenylalanine; backbone thioester exchange; villin headpiece; NMR; 1H/19F and 19F/1H heteronuclear NOE


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Proteins are biochemical workhorses, performing a wide range of tasks that are essential for life. A remarkable breadth of activity is achieved by varying the sequential arrangement of a relatively small number of {alpha}-amino acid building blocks (~20), i.e., a relatively small number of side chain functionalities. Advances in genetic manipulation and heterologous expression have enabled the practical use of engineered proteins as medicines, industrial agents, and research tools, but the composition of manufactured non-natural proteins has to date been limited largely to the proteinogenic building blocks because of constraints associated with the biosynthetic machinery. Incorporation of non-proteinogenic side chains can substantially expand the range of protein properties and functions (Wang and Schultz 2005), and the development of new strategies for protein synthesis promises to provide large-scale access to polypeptides containing non-proteinogenic subunits in the near future (Dawson and Kent 2000; Muir 2003; Wang and Schultz 2005). This prospect creates a need for fundamental knowledge regarding the effects of replacing natural side chains with non-proteinogenic analogs on the structure and other properties of folded proteins.

Fluorinated side chains have emerged as particularly interesting protein engineering tools (Marsh 2000; Yoder and Kumar 2002; Jäckel and Koksch 2005), because replacement of C–H bonds with C–F bonds causes only a modest change in molecular volume but can induce dramatic changes in molecular interactions (Smart 2001; Dunitz 2004; Lai and Kool 2004). For example, saturated hydrocarbons and their perfluorocarbon analogs are immiscible, which indicates a strong preference for homointeraction over heterointeraction. Benzene and hexafluorobenzene, on the other hand, interact very favorably with one another; the 1:1 mixture has a higher melting point than does either pure substance (Williams 1993). The favorability of the C6H6/C6F6 interaction apparently reflects attractive quadrupolar interactions (West et al. 1997), among other factors, since adjacent C6H6 and C6F6 molecules stack face-to-face in the mixed solid, while such face-to-face interactions are absent in either pure solid. Both saturated and aliphatic fluorocarbon units are more hydrophobic than their hydrocarbon analogs, which represents an additional potential source of protein conformational stabilization (Smart 2001; Dunitz 2004; Lai and Kool 2004). On the other hand, replacing H with F results in a small increase in molecular volume, which could lead to steric repulsions in the folded state. Incorporation of fluorinated residues into polypeptides has usually resulted in conformational stabilization (Holmgren et al. 1998; Dawson and Kent 2000; Bilgicer et al. 2001; Tang et al. 2001; Butterfield et al. 2002; Muir 2003; Lee et al. 2004, 2006; Wang and Schultz 2005; Jäckel et al. 2006); however, most efforts to date have focused on fluorination of saturated carbon atoms, and the impact of aromatic fluorination has received little attention (Butterfield et al. 2002). None of the prior studies has provided high-resolution structural characterization of folded fluorine-containing polypeptides.

We recently undertook a systematic evaluation of phenylalanine-to-pentafluorophenylalanine (Phe -> F5-Phe) mutants (M.G. Woll, E.B. Hadley, S. Mecozzi, and S.H. Gellman, in prep.), for the 35-residue chicken villin headpiece subdomain (c-VHP) (McKnight et al. 1997; Vardar et al. 1999; Vermeulen et al. 2004; Chiu et al. 2005), the hydrophobic core of which features a cluster of three Phe side chains (residues 6, 10, and 17). Of the seven possible F5-Phe replacement patterns at the core positions, only Phe10 -> F5-Phe was found to confer enhanced tertiary structural stability relative to the all-Phe core. Here we describe the solution structure of the stabilized F5-Phe-containing cVHP mutant, based on NMR analysis. In addition to the Phe10 -> F5-Phe mutation, all five of the lysine residues in cVHP were conservatively mutated to arginine (Fig. 1). Global replacement of the Lys residues in cVHP with Arg was required for implementation of a new method that we used to compare mutant conformational stabilities; stabilization of the tertiary structure by the Phe10 -> F5-Phe mutation was demonstrated in the context of the Lys -> Arg replacements (M.G. Woll, E.B. Hadley, S. Mecozzi, and S.H. Gellman, in prep.). The structural results described here provide the first high-resolution structural information on the packing of a fluorinated side chain within a folded protein.


Figure 1
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  		Figure 1. Sequence comparison for cVHP and the F5-Phe-containing mutant. For the latter sequence, residues indicated with bold letters represent points of divergence from the wild type (cVHP).

 

   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 Acknowledgments
 References
 
Resonance assignments and secondary structure
Assignments were made for ~90% of the backbone and side chains using PIPP/STAPP software (Garrett et al. 1991). The TALOS program (Cornilescu et al. 1999) used chemical shifts and sequence information to provide 29 pairs of {varphi}/{psi} backbone torsion angle restraints and to identify the secondary structure elements, confirmed by the NOE patterns characteristic of {alpha}-helical secondary structure (Fig. 2). Distance restraints of 1.9 Å and 2.9 Å were used to represent the HN–O and N–O non-bonded distances, respectively, for the 12 inferred helical hydrogen bonds (Wüthrich 1986). Peak intensities from the NOE experiments were translated into a continuous distribution of interproton distance restraints, with a 40% distance uncertainty applied to take into account spin diffusion.


Figure 2
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  		Figure 2. Summary of secondary structure elements; local NOE connectivities vs. the amino acid sequence of the F5-Phe-containing mutant of cVHP.

 
1H/19F and 19F/1H heteronuclear NOE (19F-HOESY) measurements with a 400-msec mixing time were used to obtain constraints for the F5-Phe residue (spectra with 140 msec and 240 msec mixing times were also recorded but showed less or weaker peaks). All (19F-HOESY) spectra were acquired on a 500 mHz Bruker QNP probe (>90% of the peaks were assigned). Cross-peak intensities were divided into four bins, and the corresponding distance constraints were assigned proportionally to the 1H NOEs (strongest and weakest intensities corresponded to 2.0 Å and 5.0 Å, respectively).

Our attempts to measure residual dipolar couplings (RDCs) resulted in either no quadrupolar splitting of the water signal due to interaction with the aligning medium for liquid crystalline positively charged bicelles (DMPC:DHPC:CTAB, 15:5:1; 5% w/v) (Ramirez and Bax 1998) or in unfolding of the peptide in 5% w/v C12E5 polyethylene glycol (PEG)/hexanol mixture with a surfactant to alcohol ratio of 0.96 (Ruckert and Otting 2000) and Helfrich lamellar phase, i.e., 5% w/v cetylpyridinium bromide (CPBr)/n-hexanol (Barrientos et al. 2000).

Structure determination
Simulated annealing, based on the described structural restraints, was carried out using the torsion angle molecular dynamics and the internal variable dynamics module (Schwieters and Clore 2001) of Xplor-NIH (Schwieters et al. 2003). Table 1 shows the structural statistics of the final structures. The target function minimized comprises the experimental NMR restraints (NOE-derived interproton distances and torsion angles), a repulsive van der Waals potential for the non-bonded contacts (Nilges et al. 1988), a torsion angle database potential of mean force (Clore and Kuszewski 2002), and a radius of gyration restraint (Kuszewski et al. 1999). The 19F5-Phe side chain topology and parameters were generated using The Dundee PRODRG2 Server (Schuettelkopf and van Aalten 2004).


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  		Table 1. Structural statistics

 
To establish crucial contacts involving the F5-Phe side chain, we obtained 18 heteronuclear NOE restraints (seven of which were long-range) using 2D 1H/19F and 19F/1H heteronuclear NOE (19F-HOESY) spectra. A total of 675 conventional NOE-derived interproton distance restraints (45 of which were long-range) were used for the structure calculations, along with the restraints mentioned above.

Superposition of the 20 lowest energy structures of the cVHP mutant is shown in Figure 3A. The tertiary fold consists of three {alpha}-helices connected by loops: helix {alpha}1 (residues 4–9), helix {alpha}2 (residues 14–18), and helix {alpha}3 (residues 23–30). The RMSD (residues 3–31) among the 20 lowest energy structures is 0.23 Å for the backbone atoms and 0.80 Å for all heavy atoms. The calculated structures show well-defined side chain conformations in the hydrophobic core of the protein. Broad rotameric restraints were derived from statistics of 100 structures calculated with torsion angle database potentials (Clore and Kuszewski 2002). More precisely, the energetically favorable rotameric combinations were deduced from the sterically allowed configurations in the cVHP fold in conjunction with statistics of the database of high resolution structures used to derive those potentials. For example, the presence in more than 95% of the cVHP calculated structures of a –60°/180° (±30°) Leu {chi}1 can be used to constrain its {chi}2 to 180°/60° (±30°), respectively. To eliminate any possible bias caused either by rotameric constraints or by ambiguities in the 19F NOE assignments, we repeated the structure calculation with these constraints removed. The resulting structures did not show any major change in the conformations or packing of the core Phe residues 6, 10, and 17.


Figure 3
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  		Figure 3. (A) Ensemble of the 20 lowest energy backbone structures of the F5-Phe-containing mutant of cVHP, based on NMR (left). The same ensemble of structures rotated by (–90°) about the vertical axis (right). (B) Stereo view of best-fit superposition (RMSD = 0.66 Å) of the heavy backbone atoms (residues 3–31) of the NMR structure of the Phe10 -> F5-Phe variant of m-cVHP and the X-ray structure of the His27 -> Asn mutant of cVHP (19). Side chains for the NMR structure are shown in thick green for F5-Phe10, thick cyan for Phe6 and Phe17, or thin cyan for all others; side chains for X-ray structure shown in thick blue for Phe6, Phe10, and Phe17, or thin blue for all others.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 Acknowledgments
 References
 
The overall backbone fold is remarkably invariant among the several X-ray and NMR structures of VHP-family proteins solved to date (McKnight et al. 1997; Vardar et al. 1999; Vermeulen et al. 2004; Chiu et al. 2005), including ours. Best-fit superposition of the heavy backbone atoms from residues 3–31 of our structure with the corresponding residues from a recently published 1 Å-resolution X-ray structure (Chiu et al. 2005) results in an RMSD of 0.66 Å (Fig. 3B). The differences between the two sequences consist of the Phe10 -> F5-Phe mutation along with the five conservative Lys -> Arg mutations in our version and a His27 -> Asn mutation in the crystallized version (residue 27 is situated in the middle of helix {alpha}3 with its side chain pointing away from the hydrophobic core). The similarity between these two structures indicates that the five Lys -> Arg mutations we made to facilitate the thermodynamic comparisons do not exert a significant effect on the VHP tertiary fold.

Since the atomic radius of fluorine is only 0.27 Å greater than that of hydrogen, replacement of hydrogen by fluorine is often regarded as an isosteric substitution. The effect of the extra steric bulk created by the Phe10 -> F5-Phe substitution appears to be minor compared to the more important changes in the electrostatic properties of the aromatic ring. Our structural data show that Phe10 -> F5-Phe mutation exerts very little effect on the cVHP tertiary structure, even though fluorination of the Phe10 side chain stabilizes the cVHP fold by 1 kcal/mol, according to guanidinium chloride denaturation studies (M.G. Woll, E.B. Hadley, S. Mecozzi, and S.H. Gellman, in prep.). The similarity between the polypeptide conformations anchored by the Phe/Phe/Phe and Phe/F5-Phe/Phe cores, and the diminution in conformational stability arising from all other Phe -> F5-Phe mutations suggest that the VHP fold has little capacity to realize the potential for improved side chain–side chain interactions provided by introduction of a fluoroaryl ring. We initially anticipated that a F5-Phe-containing mutant might depart modestly from the native cVHP fold to gain stability from an energetically favorable face-to-face juxtaposition of Phe and F5-Phe side chains; indeed, we suspected that the small size of this folding unit might make it particularly amenable to minor changes in side chain packing (fewer compensating changes required than in a larger folded domain). The structural results described here, however, suggest that such a conformational adjustment would entail an energetic cost that cannot be recouped from the Phe/F5-Phe interaction itself. The enhanced conformational stability and native-like fold of the Phe10 -> F5-Phe mutant suggest that conferring greater stability by design on cVHP, and perhaps other protein folding patterns, will require careful selection and positioning of side chains that can increase stability in the native tertiary context. It will be interesting to see whether the apparent resistance of the VHP tertiary structure to backbone rearrangement/core repacking in response to incorporation of non-natural side chains proves to be general. This prospect should influence other efforts to design folding units that contain non-proteinogenic side chains.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
Acknowledgments
 References
 
Protein expression and purification
Peptides were synthesized on solid phase using a Synergy automated synthesizer (Applied Biosystems model 432A loaded with 9-fluorenylmethoxycarbonyl [Fmoc]-Phe-Wang polystyrene resin). Synthetic cycles were completed with a standard coupling time of 30 min using O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, four equivalents) and DMF as solvent. Three equivalents of Fmoc amino acid were used for each coupling cycle. Deprotection steps used 20% piperidine in DMF for 15 min. Peptides were cleaved and/or deprotected by stirring with (CF3CO2H:H2O:triisopropylsilane, 90:5:5, v/v/v) (2 mL/25 µmol) for 4 h followed by precipitating into cold diethyl ether. The precipitate was collected by centrifugation/decantation prior to purification. Peptides were purified by reverse-phase HPLC and characterized by analytical HPLC and matrix-assisted laser desorption ionization (MALDI) mass spectrometry.

Characterization data
m-cVHP
HPLC: C18 preparative column (25 x 250 mm), flow rate 15 mL/min, gradient of 30%–40% B solvent (CH3CN:CF3COOH, 100:0.1, v/v) in A (H2O:CF3CO2H, 100:0.1, v/v) over 20 min, retention time of 17.2 min. MALDI-TOF [M + H] + calculated 4196.1, observed 4198.7.

m-cVHP (F10f5-F)
HPLC: C18 preparative column (25 x 250 mm), flow rate 15 mL/min, gradient of 30%–40% B solvent (CH3CN:CF3COOH, 100:0.1, v/v) in A (H2O:CF3CO2H, 100:0.1, v/v) over 20 min, retention time of 15.8 min. MALDI-TOF [M + H] + calculated 4286.1, observed 4287.1.

NMR measurements
NMR data were acquired at 25°C on 280 µL samples at pH 5.0 containing either 4.0 mM of unlabelled protein or 2 mM of selectively 15N-[Ala, Phe, Leu]-labeled protein, 10 mM sodium phosphate, and 0.1 mM NaN3 in either 93% H2O/7% 2H2O or 99.9% 2H2O. [1H–15N] HSQC, [1H–13C] HSQC, COSY, TOCSY, and 2D NOESY (t mix = 140 msec) spectra were collected on the unlabeled sample. Significant resonance overlap, as expected for a largely {alpha}-helical protein, allowed assignment of only ~40% of the proton resonances for the unlabeled sample (Fig. 4). [1H–15N] HSQC, 2D HNCACB, 2D HNCO, 3D HNHA, and 3D HNHB spectra were acquired on the selectively 15N-[Ala, Phe, Leu]-labeled sample. NMR experiments were performed on Varian INOVA 600 and 900 mHz, Bruker AVANCE 500 and 600 mHz spectrometers. Varian INOVA 600 mHz and Bruker AVANCE 600 mHz were equipped with cryogenic probes. NMR data were processed using the NMRPipe package (Delaglio et al. 1995).


Figure 4
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  		Figure 4. 1H–15N HSQC of data for the F5-Phe-containing mutant of cVHP (unlabeled sample).

 
Accesion numbers
Coordinates have been deposited at the Protein Data Bank (PDB ID 2JM0) and NMR data at BMRB (accession no. 15000).


   Footnotes
 
Reprint requests to: Claudia C. Cornilescu, NMRFAM, Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA; e-mail: cclaudia{at}nmrfam.wisc.edu; fax: (608) 262-3759.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062557707.


   Acknowledgments
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
References
 
We thank Charles Schwieters and Marius Clore for helpful suggestions on incorporating F5-Phe into the XPLOR-NIH analysis; Prof. C.J. McKnight for providing NMR coordinates in advance of publication; and Charles G. Fry, Mark E. Anderson, Ed S. Mooberry, and William M. Westler for helpful discussions on 19F heteronuclear NOE. This research was supported in part by NIH grant R01 GM-61238 (S.H.G.). M.G.W. was the recipient of a fellowship from the Organic Division of the American Chemical Society, supported by Eli Lilly and Company. This study made use of the National Magnetic Resonance Facility at Madison, which is supported by NIH grants P41RR02301 (Biomedical Research Technology Program, NCRR) and P41GM66326 (NIGMS). Equipment in the facility was purchased with funds from the University of Wisconsin, the NIH (P41GM66326, P41RR02301, RR02781, RR08438), the NSF (DMB-8415048, OIA-9977486, BIR-9214394), and the USDA.


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 Introduction
 Results
 Discussion
 Materials and methods
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