Hideo Iwai

Improved molecular replacement by density- and energy-guided protein structure optimization

Molecular replacement procedures, which search for placements of a starting model within the crystallographic unit cell that best account for the measured diffraction amplitudes, followed by automatic chain tracing methods, have allowed the rapid solution of large numbers of protein crystal structures. Despite extensive work, molecular replacement or the subsequent rebuilding usually fail with more divergent starting models based on remote homologues with less than 30% sequence identity. Here we show that this limitation can be substantially reduced by combining algorithms for protein structure modelling with those developed for crystallographic structure determination. An approach integrating Rosetta structure modelling with Autobuild chain tracing yielded high-resolution structures for 8 of 13 X-ray diffraction data sets that could not be solved in the laboratories of expert crystallographers and that remained unsolved after application of an extensive array of alternative approaches. We estimate that the new method should allow rapid structure determination without experimental phase information for over half the cases where current methods fail, given diffraction data sets of better than 3.2 Å resolution, four or fewer copies in the asymmetric unit, and the availability of structures of homologous proteins with >20% sequence identity.

Highly efficient protein trans‐splicing by a naturally split DnaE intein from Nostoc punctiforme

Protein trans‐splicing by the naturally split intein of the gene dnaE from Nostoc punctiforme (Npu DnaE) was demonstrated here with non‐native exteins in Escherichia coli. Npu DnaE possesses robust trans‐splicing activity with an efficiency of >98%, which is superior to that of the DnaE intein from Synechocystis sp. strain PCC6803 (Ssp DnaE). Both the N‐ and C‐terminal parts of the split Npu DnaE intein can be substituted with the corresponding fragment of Ssp DnaE without loss of trans‐splicing activity. Protein splicing with the Npu DnaEN is also more tolerant of amino acid substitutions in the C‐terminal extein sequence.

Circular β‐lactamase: stability enhancement by cyclizing the backbone

We have cyclized the polypeptide backbone of β‐lactamase with a short peptide loop as a novel method for protein stabilization, using intein‐mediated protein ligation. Successful cyclization was proven by mass spectrometry and subsequent re‐linearization by proteolytic cleavage, as well as by resistance against carboxypeptidase. Under the conditions of the experiment, no disulfide bond is present. The circular form of β‐lactamase was found to be significantly more stable against irreversible aggregation upon heating than the linear form. The circular form could be purified from the linear one either by this heat treatment or by a his‐tag which became exopeptidase‐resistant by cyclization. The increased stability of the circular form is probably due to the decreased conformational entropy in the unfolded state and in the intermediate states. While the introduction of additional disulfide bonds for protein stabilization follows the same rationale, the cyclization strategy may disturb the structure less and thus constitute a general method for stabilizing those proteins with N‐ and C‐termini in close proximity.

Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies

Segmental isotopic labeling of proteins using protein ligation is a recently established in vitro method for incorporating isotopes into one domain or region of a protein to reduce the complexity of NMR spectra, thereby facilitating the NMR analysis of larger proteins. Here we demonstrate that segmental isotopic labeling of proteins can be conveniently achieved in Escherichia coli using intein-based protein ligation. Our method is based on a dual expression system that allows sequential expression of two precursor fragments in media enriched with different isotopes. Using this in vivo approach, unlabeled protein tags can be incorporated into isotopically labeled target proteins to improve protein stability and solubility for study by solution NMR spectroscopy.

Segmental isotopic labeling of multi-domain and fusion proteins by protein trans -splicing in vivo and in vitro

Segmental isotopic labeling is a powerful labeling technique for reducing nuclear magnetic resonance (NMR) signal overlap, which is associated with larger proteins by incorporating stable isotopes into only one region of a protein for NMR detections. Segmental isotopic labeling can not only reduce complexities of NMR spectra but also retain possibilities to carry out sequential resonance assignments by triple-resonance NMR experiments. We described in vivo (i.e., in Escherichia coli) and in vitro protocols for segmental isotopic labeling of multi-domain and fusion proteins via protein trans-splicing (PTS) using split DnaE intein without any refolding steps or α-thioester modification. The advantage of PTS approach is that it can be carried out in vivo by time-delayed dual-expression system with two controllable promoters. A segmentally isotope-labeled protein can be expressed in Escherichia coli within 1 d once required vectors are constructed. The total preparation time of a segmentally labeled sample can be as short as 7–13 d depending on the protocol used.

Solution structure of DnaE intein from Nostoc punctiforme: Structural basis for the design of a new split intein suitable for site‐specific chemical modification

Naturally split DnaE intein from Nostoc punctiforme (Npu) has robust protein trans‐splicing activity and high tolerance of sequence variations at the splicing junctions. We determined the solution structure of a single chain variant of NpuDnaE intein by NMR spectroscopy. Based on the NMR structure and the backbone dynamics of the single chain NpuDnaE intein, we designed a functional split variant of the NpuDnaE intein having a short C‐terminal half (C‐intein) composed of six residues. In vivo and in vitro protein ligation of model proteins by the newly designed split intein were demonstrated.

Formation of Nano-Bio-Complex as Nanomaterials Dispersed in a Biological Solution for Understanding Nanobiological Interactions

Information on how cells interface with nanomaterials in biological environments has important implications for the practice of nanomedicine and safety consideration of nanomaterials. However, our current understanding of nanobiological interactions is still very limited. Here, we report the direct observation of nanomaterial bio-complex formation (other than protein corona) from nanomaterials dispersed in biologically relevant solutions. We observed highly selective binding of the components of cell culture medium and phosphate buffered saline to ZnO and CuO nanoparticles, independent of protein molecules. Our discoveries may provide new insights into the understanding of how cells interact with nanomaterials.

In vivo and in vitro protein ligation by naturally occurring and engineered split DnaE inteins.

Background Protein trans-splicing by naturally occurring split DnaE inteins is used for protein ligation of foreign peptide fragments. In order to widen biotechnological applications of protein trans-splicing, it is highly desirable to have split inteins with shorter C-terminal fragments, which can be chemically synthesized. Principal Findings We report the identification of new functional split sites in DnaE inteins from Synechocystis sp. PCC6803 and from Nostoc punctiforme. One of the newly engineered split intein bearing C-terminal 15 residues showed more robust protein trans-splicing activity than naturally occurring split DnaE inteins in a foreign context. During the course of our experiments, we found that protein ligation by protein trans-splicing depended not only on the splicing junction sequences, but also on the foreign extein sequences. Furthermore, we could classify the protein trans-splicing reactions in foreign contexts with a simple kinetic model into three groups according to their kinetic parameters in the presence of various reducing agents. Conclusion The shorter C-intein of the newly engineered split intein could be a useful tool for biotechnological applications including protein modification, incorporation of chemical probes, and segmental isotopic labelling. Based on kinetic analysis of the protein splicing reactions, we propose a general strategy to improve ligation yields by protein trans-splicing, which could significantly enhance the applications of protein ligation by protein trans-splicing.

Influence of the pK a value of the buried, active-site cysteine on the redox properties of thioredoxin-like oxidoreductases

Thioredoxin constitutes the prototype of the thiol‐disulfide oxidoreductase family. These enzymes contain an active‐site disulfide bridge with the consensus sequence Cys‐Xaa‐Xaa‐Cys. The more N‐terminal active‐site cysteine is generally a strong nucleophile with an abnormal low pK a value. In contrast, the more C‐terminal cysteine is buried and only little is known about its effective pK a during catalysis of disulfide exchange reactions. Here we have analyzed the pK a values of the active‐site thiols in wild type thioredoxin and a 400‐fold more oxidizing thioredoxin variant by NMR spectroscopy, using selectively 13Cβ‐Cys‐labeled proteins. We find that the effective pK a of the buried cysteine (pK b) of the variant is increased, while the pK a of the more N‐terminal cysteine (pK N) is decreased relative to the corresponding pK a values in the wild type. We propose two empirical models which exclusively require the knowledge of pK N to predict the redox properties of thiol‐disulfide oxidoreductases with reasonable accuracy.

Segmental Isotopic Labeling of a Central Domain in a Multidomain Protein by Protein Trans-Splicing Using Only One Robust DnaE Intein†

Many proteins, including cellular signaling proteins, cellsurface receptors, and modular enzymes, are constructed from individual domains and connecting linkers. Structural analysis by NMR spectroscopy or by X-ray crystallography often focuses on self-contained domains excised from full-length proteins so as to reduce the molecular weight to a manageable size for high-quality NMR spectra or to improve crystallization by removing disordered regions. Such minimization often neglects functional aspects of a domain in an intact protein and may not represent all aspects of the structure– function relationship in the full-length context. While the structure determination of individual, isolated domains has been tremendously accelerated—in part through several structural genomics consortia—understanding of domain– domain interactions within a multidomain protein is lacking behind. NMR spectroscopy is an ideal tool for characterizing such, often transient, interactions. However, NMR spectroscopic analysis of large proteins often suffers from signal overlap. This problem becomes even more profound when proteins contain recurring modular domains and/or highly disordered regions. One solution to this overlap problem is segmental isotopic labeling that enables the exclusive incorporation of NMR active, stable isotopes into selected domains or parts of larger proteins, thereby reducing the complexity of the NMR spectra. In the past years, we have advanced segmental isotopic labeling procedures by discovering and engineering robust protein splicing domains (inteins) and by developing in vivo methods with the time-delayed dual expression system. While segmental isotopic labeling of Nor Cterminal halves of proteins by protein ligation of two fragments has become a well-established routine procedure, labeling of a middle domain or segment in a larger protein still poses significant challenges. Native chemical ligation (NCL) or expressed protein ligation (EPL) could be used for such a three-fragment ligation, however, the requirement for isotope-enriched peptides with a protected N-terminal cysteine residue is not cost-effective and the protection/deprotection steps to create a reactive N-terminal cysteine could be laborintensive. One promising approach to ligate three polypeptide fragments is to use protein trans-splicing (PTS) with two split inteins (denoted as Int1 and Int2) because PTS does not require any cumbersome steps to create an N-terminal cysteine needed for EPL (Figure 1a). 10, 11] For this approach, however, the two split inteins must be orthogonal without any cross-reactivity and have high splicing efficiency. Segmental labeling of a central region of a protein by three-fragment ligation was elegantly realized with the two orthogonal split inteins, PI-PfuI and PI-PfuII. However, the artificially split inteins of PI-PfuI and PI-PfuII required tedious refolding and optimization steps to restore the splicing activity because the fusion proteins with the split intein fragments were insoluble. 12] Since refolding can also result in inactive proteins, avoiding any refolding steps would be highly beneficial. This can be achieved with naturally occurring split inteins, such as DnaE inteins from cyanobacteria, which usually do not require any refolding step to induce PTS. 6, 8, 13,14] Particularly, DnaE intein from Nostoc punctiforme (Npu) could be ideally suited based on the combination of its robust splicing activity, high tolerance of sequence variations at the splicing junctions, and its high solubility. For three-fragment ligation, however, the DnaE inteins cannot be directly used because the domain or fragment inserted in the middle of the precursor protein containing both Nand C-intein (IN/IC) could result in cyclization or polymerization (Figure 1b). 14, 15] Herein, we develop a novel strategy for three-fragment protein ligation that overcomes the cyclization/polymerization problem but still utilizes only one robust DnaE intein to achieve higher yields. To circumvent the inevitable cyclization using only one split intein (Figure 1b), we exploited engineered NpuDnaE inteins with the same sequence but different split sites (Figure 1c). Shortening, [*] A. S. Aranko, Dr. H. Iwa Research Program in Structural Biology and Biophysics Institute of Biotechnology, University of Helsinki P.O. Box 65, Helsinki 00014 (Finland) Fax: (+ 358)9-1915-9541 E-mail: hideo.iwai@helsinki.fi Homepage: http://www.biocenter.helsinki.fi/bi/iwai/ A. E. L. Busche, Dr. M. Talebzadeh-Farooji, Dr. F. Bernhard, Prof. Dr. V. D tsch Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance and Cluster of Excellence Frankfurt Macromolecular Complexes (CEF), University of Frankfurt 60438 Frankfurt (Germany) [] These authors contributed equally to this work.