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1.  Fragmentation Follows Structure: Top-Down Mass Spectrometry Elucidates the Topology of Engineered Cystine-Knot Miniproteins 
PLoS ONE  2014;9(10):e108626.
Over the last decades the field of pharmaceutically relevant peptides has enormously expanded. Among them, several peptide families exist that contain three or more disulfide bonds. In this context, elucidation of the disulfide patterns is extremely important as these motifs are often prerequisites for folding, stability, and activity. An example of this structure-determining pattern is a cystine knot which comprises three constrained disulfide bonds and represents a core element in a vast number of mechanically interlocked peptidic structures possessing different biological activities. Herein, we present our studies on disulfide pattern determination and structure elucidation of cystine-knot miniproteins derived from Momordica cochinchinensis peptide MCoTI-II, which act as potent inhibitors of human matriptase-1. A top-down mass spectrometric analysis of the oxidised and bioactive peptides is described. Following the detailed sequencing of the peptide backbone, interpretation of the MS3 spectra allowed for the verification of the knotted topology of the examined miniproteins. Moreover, we found that the fragmentation pattern depends on the knottin’s folding state, hence, tertiary structure, which to our knowledge has not been described for a top-down MS approach before.
PMCID: PMC4193770  PMID: 25303319
2.  Combinatorial Optimization of Cystine-Knot Peptides towards High-Affinity Inhibitors of Human Matriptase-1 
PLoS ONE  2013;8(10):e76956.
Cystine-knot miniproteins define a class of bioactive molecules with several thousand natural members. Their eponymous motif comprises a rigid structured core formed by six disulfide-connected cysteine residues, which accounts for its exceptional stability towards thermic or proteolytic degradation. Since they display a remarkable sequence tolerance within their disulfide-connected loops, these molecules are considered promising frameworks for peptide-based pharmaceuticals. Natural open-chain cystine-knot trypsin inhibitors of the MCoTI (Momordica cochinchinensis trypsin inhibitor) and SOTI (Spinacia oleracea trypsin inhibitor) families served as starting points for the generation of inhibitors of matriptase-1, a type II transmembrane serine protease with possible clinical relevance in cancer and arthritic therapy. Yeast surface-displayed libraries of miniproteins were used to select unique and potent matriptase-1 inhibitors. To this end, a knowledge-based library design was applied that makes use of detailed information on binding and folding behavior of cystine-knot peptides. Five inhibitor variants, four of the MCoTI family and one of the SOTI family, were identified, chemically synthesized and oxidatively folded towards the bioactive conformation. Enzyme assays revealed inhibition constants in the low nanomolar range for all candidates. One subnanomolar binder (Ki = 0.83 nM) with an inverted selectivity towards trypsin and matriptase-1 was identified.
PMCID: PMC3795654  PMID: 24146945
3.  A sensitive method for rapid detection of alkyl halides and dehalogenase activity using a multistep enzyme assay 
AMB Express  2012;2:51.
A method for the detection of haloalkane conversion to the corresponding alcohols by haloalkane dehalogenases is described. It is based on a multistage enzyme reaction which allows for the analysis of alkyl halides in buffered systems. Irreversible hydrolytic dehalogenation catalyzed by haloalkane dehalogenase DhaA from Rhodococcus erythropolis transfers an alkyl halide into a corresponding alcohol that is further oxidized by alcohol oxidase AOX from Pichia pastoris yielding a respective aldehyde and hydrogen peroxide easily detectable via the horseradish peroxidase catalyzed oxidation of chromogenic molecules. Due to its high sensitivity (0.025 mM, 0.43 ppm for 1,3-dibromopropane), low expenditure and the ability of handling a large number of samples in parallel, this method is an attractive alternative to existing procedures for the monitoring of both haloalkanes and dehalogenases.
PMCID: PMC3487978  PMID: 23006907
Alcohol oxidase; Haloalkane dehalogenase; Haloalkanes; Horseradish peroxidase; Multistage enzyme reaction
4.  Knottin cyclization: impact on structure and dynamics 
Present in various species, the knottins (also referred to as inhibitor cystine knots) constitute a group of extremely stable miniproteins with a plethora of biological activities. Owing to their small size and their high stability, knottins are considered as excellent leads or scaffolds in drug design. Two knottin families contain macrocyclic compounds, namely the cyclotides and the squash inhibitors. The cyclotide family nearly exclusively contains head-to-tail cyclized members. On the other hand, the squash family predominantly contains linear members. Head-to-tail cyclization is intuitively expected to improve bioactivities by increasing stability and lowering flexibility as well as sensitivity to proteolytic attack.
In this paper, we report data on solution structure, thermal stability, and flexibility as inferred from NMR experiments and molecular dynamics simulations of a linear squash inhibitor EETI-II, a circular squash inhibitor MCoTI-II, and a linear analog lin-MCoTI. Strikingly, the head-to-tail linker in cyclic MCoTI-II is by far the most flexible region of all three compounds. Moreover, we show that cyclic and linear squash inhibitors do not display large differences in structure or flexibility in standard conditions, raising the question as to why few squash inhibitors have evolved into cyclic compounds. The simulations revealed however that the cyclization increases resistance to high temperatures by limiting structure unfolding.
In this work, we show that, in contrast to what could have been intuitively expected, cyclization of squash inhibitors does not provide clear stability or flexibility modification. Overall, our results suggest that, for squash inhibitors in standard conditions, the circularization impact might come from incorporation of an additional loop sequence, that can contribute to the miniprotein specificity and affinity, rather than from an increase in conformational rigidity or protein stability. Unfolding simulations showed however that cyclization is a stabilizing factor in strongly denaturing conditions. This information should be useful if one wants to use the squash inhibitor scaffold in drug design.
PMCID: PMC2659701  PMID: 19077275

Results 1-4 (4)