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The Influence of Commonly Used Tags on Structural Monatshefte für Chemie - Chemical Monthly (2019) 150:913–925 https://doi.org/10.1007/s00706-019-02401-x ORIGINAL PAPER The infuence of commonly used tags on structural propensities and internal dynamics of peptides Maria Bräuer1 · Maria Theresia Zich1 · Kamil Önder2 · Norbert Müller1,3 Received: 11 January 2019 / Accepted: 19 February 2019 / Published online: 29 April 2019 © The Author(s) 2019 Abstract The infuence of biotin and fuorescein tags attached to the N-terminus of peptides on their structural propensities is assessed by NMR methods. While the small peptides investigated are highly mobile with no uniquely preferred conformation, the introduction of the tags, in particular hydrophobic ones, clearly shows an infuence on NMR parameters such as chemical shifts and relaxation properties, which are not restricted to the nearby residues, but also afect distant parts. Thus, long-range efects on structural propensities become evident and are cause for concern with respect to the interpretation of weak interac- tion tests, which rely upon the assumption that tags do not exert infuence on intermolecular interactions. Graphical abstract Keywords Peptides · NMR spectroscopy · Conformation · Fluorescence tags · Afnity tags Introduction cloned sequences to facilitate purifcation of overexpressed recombinant proteins or to improve solubility or to facilitate The covalent attachment of tag groups to peptides and folding of the target protein [1, 2]. Tags are also employed proteins for either purifcation, immobilization, or spec- in Western blotting, enzyme-linked immunosorbent assay troscopic detection is a widely applied practice in biomo- (ELISA), or other biological assays [3]. Probably, the most lecular research. For example, afnity tags are included in common application of tags is the labelling of biomolecules with fuorescent dyes, for analytical purposes based on fuo- rophore-based techniques including confocal microscopy, Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s0070 6-019-02401 -x) contains fuorescence resonance energy transfer (FRET), lumines- supplementary material, which is available to authorized users. cence resonance energy transfer (LRET), or microscale thermophoresis (MST) [4–7]. * Norbert Müller It is often tacitly assumed that such tags have no or neg- [email protected] ligible infuence on the secondary and tertiary structures 1 Institut für Organische Chemie, Johannes Kepler Universität of the fused peptides or proteins as well as on their con- Linz, Altenbergerstraße 69, 4040 Linz, Austria formational dynamics. Especially small tags, like hexa- 2 ProComCure BioTech GesmbH, 5000 Anif, Austria histidine, FLAG, Strep II, or chitin-binding protein (CBP), 3 are assumed to have no or only slight impact on protein Faculty of Science, University of South Bohemia, Branišovská 31, 37005 České Budějovice, Czech Republic function and, therefore, often remain attached at the N- or Vol.:(0123456789)1 3 914 M. Bräuer et al. C-termini of proteins, even during structure determination compound does not infuence the binding behaviour. This studies. Larger afnity tags including glutathione-S-trans- procedure was used in the development of a high-through- ferase (GST) and maltose-binding protein (MBP), although put fuorescence polarization anisotropy assay. Although increasing solubility [8] need to be removed before struc- NMR experiments had earlier confrmed that labelled and ture determination using X-ray or NMR and have also been unlabelled peptides were binding in the same manner, they found to modify intermolecular interactions signifcantly [9]. also revealed small changes in the chemical shifts, difer- While afnity tags introduced by recombinant DNA methods ential broadening of several peaks, and a slightly higher can be removed, e.g., by breaking linker sequence encoded binding afnity of the fuorescein isothiocyanate (FITC)- between the tag and the target protein using specifc pro- labelled peptide derivative [26]. As reasons for the changes, teases [3], tags introduced via chemical derivatization, like the authors suggested additional interactions of the protein most fuorescence tags, are normally not removed. Although with either the hydrophilic fuorescein moiety or the ali- small tags are claimed to have minimal or no impact on the phatic aminohexanoic (Ahx) linker. The introduction of a secondary structure of bio-macromolecules, there are some spacer between the tag and the frst amino acid of the pep- reports of opposite experiences using fuorescent as well as tide sequence is usually considered sufcient to prevent the afnity tags like fuorescein, biotin [10–13], or hexa-histi- infuences of the bulky fuorophore on conformation. Short dine [14–17]. Most reports about tags afecting structures linker sequences can also prevent side reactions upon the or functions can be found for hexa-histidine tagged bio- acidic cleavage of peptides produced by solid-phase peptide macromolecules, probably because it is the most frequently synthesis from the resin, as the cleavage reaction using tri- used tag for the purifcation of recombinant proteins [18]. fuoroacetic acid (TFA) can lead to reactions following the The infuence of short afnity tags like hexa-histidine and Edman degradation pathway [27], resulting in the formation FLAG on crystallisation [14, 19] and on the dynamics in the of fuorescein thiohydantoin including a cleavage of the frst picosecond range of His-tagged myoglobin used in infrared amino acid [28]. vibrational echo spectroscopy [15] are prominent examples During our studies of antimicrobial, conformationally of such reports. labile peptides, some signifcant diferences in the NMR Conversely, not many reports for efects of biotinylation spectroscopic properties between tagged and native pep- on structure and function could be found in the literature. tides were observed. Here, we report experimental results It is frequently used to attach peptides to solid surfaces as, concerning a dodecapeptide attached to an FITC tag and for example, in surface plasmon resonance [20–22]. Biotin two 22 amino acid peptides attached to biotin. For all pep- labelling at a specifc lysine residue of human lysozyme did tides, NMR experiments with and without a tag were per- not show any alteration of structure or efects on the binding formed, and their secondary structure propensities as well behaviour [10]. Only a slight decrease in enzymatic activity as local order parameters were compared on the basis of of the biotinylated sample was observed and explained by chemical shifts, scalar coupling constants, and relaxation the steric hindrance of the biotin moiety preventing binding parameters. in spatial proximity to the introduced tag. Another recently In spite of the lower number of signals, the interpreta- published report about a structure modifcation owed to the tion of peptide NMR data is often less straightforward than biotinylation of aminoacyl-tRNA describes altered growth for structured proteins. The problem with structure deter- behaviour at higher temperatures (37 °C), lower heat stabil- mination of peptides using solution state NMR is the high ity, and less efciency than the unlabelled counterpart [11]. fexibility of the molecules leading to the co-existence of a Fluorescein is frequently used as fuorophore in fuores- plethora of diferent conformers in solution. Therefore, the cence microscopy, immunofuorescence assays, and fow experimental NMR parameters represent weighted averages cytometry or fuorescence polarization [23]. Fluorescent of several conformers rather than a small number of well- tags apparently can impair the functionality of DNA mis- defned global folds. The structural propensities are highly match repair proteins when attached at the C-terminus [24]. dependent on the environment of the peptide; in polar sol- Studies on MreB, the actin homologue in bacteria, which is vents the contributions of intramolecular hydrogen bonds are essential for cell wall synthesis, showed that an alpha helix minor. Owed to the high fexibility of small peptides, crystal- reported in an earlier study was actually induced by a fuo- lisation is accompanied by conformational selection, which rescent tag [25]. is obscuring or biasing the structural information relevant For fuorescence polarization assays, especially in the in solution. Solution NMR is considered a most efective context for high-throughput screening, it has been sug- technique for the assessment of peptide conformation and gested to determine a competitive binding curve between the conformational dynamics [29, 30]. labelled probe and the corresponding unlabelled molecule The peptides investigated are part of a larger quest for [23]. If the binding curve fts, indicating similar afnity and antibacterial activity [31], they are: A0 having the sequence competitivity, it can be assumed that the tag of the labelled RKKRLKLLKRLL, with fA0 bearing a fuorescein tag 1 3 915 The infuence of commonly used tags on structural propensities and internal dynamics of peptides (with an Ahx spacer) at the N-terminus; S2 having the amino not deviate much from zero, with exception of the backbone acid sequence RIQALYSKQPKLVERILTKNEQ; and P1 nitrogen atoms. The major conformation according to the re- having the sequence DSLDIAELVMELEDEFGTEIPD, referenced secondary chemical shifts appears to be random with bioS2 and bioP1 respectively being biotinylated at the coil or dynamic. Remarkably, some of the largest deviations N-terminus.
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