Erschienen in: Chemistry - A European Journal ; 15 (2009), 31. - S. 7628-7636 https://dx.doi.org/10.1002/chem.200802136 Position-Dependent Effects of Fluorinated Amino Acids on the Hydrophobic Core Formation of a Heterodimeric Coiled Coil Mario Salwiczek,[a] Sergey Samsonov,[b] Toni Vagt,[a] Elisabeth Nyakatura,[a] Emanuel Fleige,[a] Jorge Numata,[c] Helmut Cçlfen,[d] M. Teresa Pisabarro,*[b] and Beate Koksch*[a] Dedicated to Professor Klaus Burger on the occasion of his 70th birthday Abstract: Systematic model investiga- to evaluate the impact of fluorinated cal ultracentrifugation) and theoretical tions of the molecular interactions of amino acid substitutions within differ- (MD simulations and MM-PBSA free fluorinated amino acids within native ent hydrophobic protein microenviron- energy calculations) methods. The protein environments substantially im- ments. The structural and thermody- coiled coil environment imposes posi- prove our understanding of the unique namic stability of the dimers were ex- tion-dependent conformations onto the properties of these building blocks. A amined by applying both experimental fluorinated side chains and thus affects rationally designed heterodimeric (CD spectroscopy, FRET, and analyti- their packing and relative orientation coiled coil peptide (VPE/VPK) and towards their native interaction part- nine variants containing amino acids ners. We find evidence that such pack- Keywords: amino acids · fluorine · with variable fluorine content in either ing effects exert a significant influence helical structures · molecular position a16 or d19 within the hydro- on the contribution of fluorine-induced dynamics phobic core were synthesized and used polarity to coiled coil folding. Introduction [a] M. Salwiczek,+ T. Vagt,+ E. Nyakatura,+ E. Fleige,+ Prof. Dr. B. Koksch+ The widespread interest in peptides and proteins as highly Department of Biology, Chemistry, and Pharmacy potent pharmaceuticals[1] as well as bio-inspired materials[2] Freie Universit Berlin motivates attempts towards the de novo design of peptides Institute of Chemistry and Biochemistry–Organic Chemistry and proteins with superior properties such as chemical and Takustrasse 3, 14195 Berlin (Germany) [3] Fax : (+49)30-838-55644 metabolic resistance as well as thermodynamic stability. E-mail: [email protected] Moreover, endowing these biomolecules with novel func- [b] S. Samsonov,# Dr. M. T. Pisabarro# tions that are not carried out by natural proteins[4] is perhaps Structural Bioinformatics, BIOTEC TU Dresden one of the most interesting, albeit challenging prospects in Tatzberg 47–51, 01307 Dresden (Germany) protein engineering.[5] To this end, continuous efforts are Fax : (+49)351-463-40087 E-mail: [email protected] made to expand the repertoire of genetically encoded amino [c] J. Numata acids through manipulation of the translational machinery [6,7] Department of Biology, Chemistry, and Pharmacy in vitro and in vivo. Also, pure synthetic and semisynthet- Freie Universit Berlin ic approaches, that is, the direct chemical modification of Institute of Chemistry and Biochemistry–Crystallography protein functional groups[8] as well as solid-phase peptide Takustrasse 6, 14195 Berlin (Germany) synthesis,[9] native chemical ligation,[10] and expressed pro- [d] Dr. H. Cçlfen tein ligation[11] enable the incorporation of non-natural Max Planck Institute of Colloids and Interfaces Am Mhlenberg 1, 14476 Potsdam-Golm (Germany) amino acids into peptide and protein sequences. In this con- [+] Experimental part of this work. text, fluorinated amino acids have increasingly gained recog- [#] Theoretical part of this work. nition as analytical probes and modulators for protein struc- ture and stability.[12] 7628 Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-mcivuevve2dq8 Organic molecules containing CÀF bonds display unique vides two very well defined recognition surfaces.[32] Its pri- properties[13] that account for their ever-growing importance mary structure is based on a repetitive pattern of seven [14] in medicinal chemistry. Most prominent amongst these is amino acids, the heptad repeat (abcdefg)n. Along the helical a pronounced enhancement in steric size upon fluorination surface, the hydrophobic positions a and d and the mostly of alkyl groups that is combined with the very low polariza- polar positions b, c, and f point in opposite directions. The a bility of the fluorine atom. This often, although not general- and d residues of the interacting helices are packed in a ly,[15] leads to a manifold increase in hydrophobicity and zipper-like fashion to form the hydrophobic core while all thus improves membrane permeability.[16] It has been antici- the other heptad positions are solvent exposed. The perfect pated that global replacement of hydrophobic amino acids interactions within the hydrophobic core provide the basis in hydrophobic domains with fluorinated analogues would for a stable fold and drive oligomerization. In consequence accordingly stabilize the structure of proteins. As summar- the peptides associate to form a slightly left-handed super- ized in a recent review[17] this has been proven to be a suc- helix. In dimeric coiled coils positions e and g are preferably cessful concept for the design of hyperstable a-helical coiled populated by charged residues that further contribute to sta- coils. Along with enhanced self-association behavior, some bility and control the specificity of folding by forming inter- of these peptides display an increase in membrane-binding helical salt bridges. Following this primary structure code affinity[18,19] that lead to the design of fluorinated peptides coiled coils of different length and oligomerization specifici- with enhanced antimicrobial activity.[20, 21] The attempt to- ty can be designed de novo.[32] Because the packing of the wards a global replacement of leucine residues by fluorinat- hydrophobic side chains in a parallel coiled coil, a against a’ ed analogues within globular proteins, however, resulted in and d against d’, is not equivalent in terms of relative side reduced thermodynamic stability.[22, 23] In these cases, addi- chain orientation (see below),[33] a parallel design as present- tional mutations were needed to compensate for the disad- ed below can be used to study the impact of fluorination vantageous effects.[24] It was also shown that fluorination of within two different hydrophobic microenvironments. aromatic side chains within proteins does not generally en- The model system VPE/VPK was designed to provide the hance secondary structure formation.[25, 26] These findings environment for specific interactions between a fluorinated suggest that properties other than hydrophobicity may also and a non-fluorinated peptide. The peptide model fulfils two play an important role in directing the interactions of fluo- important criteria: 1) specificity for one distinct orientation rine within native protein environments. Though a weak of the peptide strands within the dimer and 2) heterodimeri- electron donor and thus poor hydrogen-bond acceptor,[27] zation. Figure 1 illustrates the design of the model peptide. carbon-bound fluorine has been shown to participate in fa- The amino acid composition of the hydrophobic core is in- vorable multipolar interactions within native protein envi- spired by the GCN4 transcription factor, which has already ronments.[28] It is also important to note that despite the fact been extensively characterized at high resolution.[34] Here, that specific fluorine–fluorine interactions are able to pro- valine in all a and leucine in all d positions provide for a mote ordered self-association,[17] it has been proposed that parallel orientation of the peptide strands in the coiled coil they may also result in misfolding.[22,29] In addition, our pre- dimer. vious studies suggest that hydrophobic interactions in pro- Most important for the purpose of the study, heterodime- teins may be severely disturbed by fluorine-induced polari- rization is required to guarantee that the observed effects ty.[29] In summary, it still seems rather difficult to predict the trace back to a single fluoroamino acid substitution per impact of fluorination on the structure and activity of pep- dimer. This condition is accomplished by introducing e–g’ tides and proteins. To further investigate the impact of fluo- and g–e’ pairs that engage in favorable electrostatic interac- rine substitution in native protein environments, we de- tions in the heterodimer but would repel one another in signed a heterodimeric a-helical coiled coil peptide contain- both possible homodimers. The fully natural VPE peptide ing one fluorinated amino acid at either of two positions was then used as a template to screen the interactions with within the hydrophobic core, which are different in terms of different fluorine-containing variants of the complementary side chain packing. We find that the effect of fluorine-in- interaction partner VPK. duced polarity highly depends on the microenvironment of As mentioned above, the packing characteristics of the a the substitution. and d positions in parallel coiled coils are different (see Figure 7). Therefore, the VPK strand contains the fluorinat- ed amino acids either at position a16 or d19, which allows Results and Discussion evaluating the impact of fluorination within two different hydrophobic microenvironments. Peptides containing leu- Peptide design: The aim of this study was