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Featured researches published by Thomas Steiner.


The EMBO Journal | 2002

Crystal structures of transcription factor NusG in light of its nucleic acid- and protein-binding activities

Thomas Steiner; Jens T. Kaiser; Snezan Marinkovic; Robert Huber; Markus C. Wahl

Microbial transcription modulator NusG interacts with RNA polymerase and termination factor ρ, displaying striking functional homology to eukaryotic Spt5. The protein is also a translational regulator. We have determined crystal structures of Aquifex aeolicus NusG showing a modular design: an N‐terminal RNP‐like domain, a C‐terminal element with a KOW sequence motif and a species‐specific immunoglobulin‐like fold. The structures reveal bona fide nucleic acid binding sites, and nucleic acid binding activities can be detected for NusG from three organisms and for the KOW element alone. A conserved KOW domain is defined as a new class of nucleic acid binding folds. This module is a close structural homolog of tudor protein–protein interaction motifs. Putative protein binding sites for the RNP and KOW domains can be deduced, which differ from the areas implicated in nucleic acid interactions. The results strongly argue that both protein and nucleic acid contacts are important for NusGs functions and that the factor can act as an adaptor mediating indirect protein–nucleic acid associations.


PLOS ONE | 2008

Synthetic Biology of Proteins: Tuning GFPs Folding and Stability with Fluoroproline

Thomas Steiner; Petra Hess; Jae Hyun Bae; Birgit Wiltschi; Luis Moroder; Nediljko Budisa

Background Proline residues affect protein folding and stability via cis/trans isomerization of peptide bonds and by the Cγ-exo or -endo puckering of their pyrrolidine rings. Peptide bond conformation as well as puckering propensity can be manipulated by proper choice of ring substituents, e.g. Cγ-fluorination. Synthetic chemistry has routinely exploited ring-substituted proline analogs in order to change, modulate or control folding and stability of peptides. Methodology/Principal Findings In order to transmit this synthetic strategy to complex proteins, the ten proline residues of enhanced green fluorescent protein (EGFP) were globally replaced by (4R)- and (4S)-fluoroprolines (FPro). By this approach, we expected to affect the cis/trans peptidyl-proline bond isomerization and pyrrolidine ring puckering, which are responsible for the slow folding of this protein. Expression of both protein variants occurred at levels comparable to the parent protein, but the (4R)-FPro-EGFP resulted in irreversibly unfolded inclusion bodies, whereas the (4S)-FPro-EGFP led to a soluble fluorescent protein. Upon thermal denaturation, refolding of this variant occurs at significantly higher rates than the parent EGFP. Comparative inspection of the X-ray structures of EGFP and (4S)-FPro-EGFP allowed to correlate the significantly improved refolding with the Cγ-endo puckering of the pyrrolidine rings, which is favored by 4S-fluorination, and to lesser extents with the cis/trans isomerization of the prolines. Conclusions/Significance We discovered that the folding rates and stability of GFP are affected to a lesser extent by cis/trans isomerization of the proline bonds than by the puckering of pyrrolidine rings. In the Cγ-endo conformation the fluorine atoms are positioned in the structural context of the GFP such that a network of favorable local interactions is established. From these results the combined use of synthetic amino acids along with detailed structural knowledge and existing protein engineering methods can be envisioned as a promising strategy for the design of complex tailor-made proteins and even cellular structures of superior properties compared to the native forms.


Proceedings of the National Academy of Sciences of the United States of America | 2007

Chymotryptic specificity determinants in the 1.0 A structure of the zinc-inhibited human tissue kallikrein 7.

Mekdes Debela; Petra Hess; Viktor Magdolen; Norman M. Schechter; Thomas Steiner; Robert Huber; Wolfram Bode; Peter Goettig

hK7 or human stratum corneum chymotryptic enzyme belongs to the human tissue kallikrein (hKs) serine proteinase family and is strongly expressed in the upper layers of the epidermis. It participates in skin desquamation but is also implicated in diverse skin diseases and is a potential biomarker of ovarian cancer. We have solved x-ray structures of recombinant active hK7 at medium and atomic resolution in the presence of the inhibitors succinyl-Ala-Ala-Pro-Phe-chloromethyl ketone and Ala-Ala-Phe-chloromethyl ketone. The most distinguishing features of hK7 are the short 70–80 loop and the unique S1 pocket, which prefers P1 Tyr residues, as shown by kinetic data. Similar to several other kallikreins, the enzyme activity is inhibited by Zn2+ and Cu2+ at low micromolar concentrations. Biochemical analyses of the mutants H99A and H41F confirm that only the metal-binding site at His99 close to the catalytic triad accounts for the noncompetitive Zn2+ inhibition type. Additionally, hK7 exhibits large positively charged surface patches, representing putative exosites for prime side substrate recognition.


Journal of Biological Chemistry | 2007

Open and closed structures of the UDP-glucose pyrophosphorylase from Leishmania major.

Thomas Steiner; Anne-Christin Lamerz; Petra Hess; Constanze Breithaupt; Stephan Krapp; Gleb Bourenkov; Robert Huber; Rita Gerardy-Schahn; Uwe Jacob

Uridine diphosphate-glucose pyrophosphorylase (UGPase) represents a ubiquitous enzyme, which catalyzes the formation of UDP-glucose, a key metabolite of the carbohydrate pathways of all organisms. In the protozoan parasite Leishmania major, which causes a broad spectrum of diseases and is transmitted to humans by sand fly vectors, UGPase represents a virulence factor because of its requirement for the synthesis of cell surface glycoconjugates. Here we present the crystal structures of the L. major UGPase in its uncomplexed apo form (open conformation) and in complex with UDP-glucose (closed conformation). The UGPase consists of three distinct domains. The N-terminal domain exhibits species-specific differences in length, which might permit distinct regulation mechanisms. The central catalytic domain resembles a Rossmann-fold and contains key residues that are conserved in many nucleotidyltransferases. The C-terminal domain forms a left-handed parallel β-helix (LβH), which represents a rarely observed structural element. The presented structures together with mutagenesis analyses provide a basis for a detailed analysis of the catalytic mechanism and for the design of species-specific UGPase inhibitors.


Biological Chemistry | 2004

Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code

Nediljko Budisa; Prajna Paramita Pal; Stefan Alefelder; Petra Birle; Tatjana Krywcun; Marina Rubini; Waltraud Wenger; Jae Hyun Bae; Thomas Steiner

Abstract The expanded genetic code in combination with sitedirected mutagenesis was used to probe spectroscopic and structural roles of tryptophan (Trp) residues in Aequorea victoria green fluorescent proteins (avGFPs). Nine different halogen-, chalcogen-, and methyl-containing Trp isosteric analogues and surrogates were incorporated into avGFPs containing indole moieties in, and outside of, the chromophore, by the use of the selective pressure incorporation method. Such isosteric replacements introduced minimal local geometry changes in indole moieties, often to the level of single atomic exchange (atomic mutation) and do not affect three-dimensional structures of avGFPs but induce changes in spectral properties. Our approach offers a new platform to re-evaluate issues like resonance transfer, mechanisms of chromophore formation and maturation, as well as the importance of local geometry and weak sulphuraromatic interactions for avGFP spectral properties and structural stability. The library of novel tailor-made avGFP mutants and variants generated in this work has demonstrated not only the potentials of the expanded genetic code to study spectroscopic functions, but also a new approach to generate tailor-made proteins with interesting and useful spectral properties.


Medicine and Science in Sports and Exercise | 2011

Does hemoglobin mass increase from age 16 to 21 and 28 in elite endurance athletes

Thomas Steiner; Jon Peter Wehrlin

PURPOSE It is unclear if hemoglobin mass (Hbmass) and red cell volume (RCV) increase in endurance athletes with several years of endurance training from adolescence to adulthood. The aim of this study, therefore, was to determine with a controlled cross-sectional approach whether endurance athletes at the ages of 16, 21, and 28 yr are characterized by different Hbmass, RCV, plasma volume (PV), and blood volume (BV). METHODS BV parameters (CO rebreathing), VO(2max) and other blood, iron, training, and anthropometric parameters were measured in three endurance athlete groups AG16 (n = 14), AG21 (n = 14), and AG28 (n = 16) as well as in three age-matched control groups (<2 h endurance training per week): CG16 (n = 16), CG21 (n = 15), and CG28 (n = 16). RESULTS In AG16, body weight-related Hbmass (12.4 ± 0.7 g·kg(-1)), RCV, BV, and VO(2max) (66.1 ± 3.8 mL·kg·(-1)min(-1)) were lower (P < 0.001) than those in AG21 (14.2 ± 1.1 g·kg(-1), 72.9 ± 3.6 mL·kg·(-1)min(-1)) and AG28 (14.6 ± 1.1 g·kg(-1), 73.4 ± 6.0 mL·kg·(-1)min(-1)). Results for these parameters did not differ between AG21 and AG28 and among the control groups. VO(2max), PV, and BV were higher for AG16 than for CG16 (12.0 ± 1.0 g·kg(-1), 58.9 ± 5.0 mL·kg·(-1)min(-1)) but not Hbmass and RCV. CONCLUSIONS Our results suggest that endurance training has major effects on Hbmass and RCV from ages 16 to 21 yr, although there is no further increase from ages 21 to 28 yr in top endurance athletes. On the basis of our findings, an early detection of the aptitude for endurance sports at age 16 yr, solely based on levels of Hbmass, does not seem to be possible.


Scandinavian Journal of Clinical & Laboratory Investigation | 2011

Comparability of haemoglobin mass measured with different carbon monoxide-based rebreathing procedures and calculations

Thomas Steiner; Jon Peter Wehrlin

Abstract Background. Measurements of haemoglobin mass (Hbmass) with the carbon monoxide (CO) rebreathing method provide valuable information in the field of sports medicine, and have markedly increased during the last decade. However, several different approaches (as a combination of the rebreathing procedure and subsequent calculations) for measuring Hbmass are used, and routine measurements have indicated that the Hbmass differs substantially among various approaches. Therefore, the aim of this study was to compare the Hbmass of the seven most commonly used approaches, and then to provide conversion factors for an improved comparability of Hbmass measured with the different approaches. Methods. Seventeen subjects (healthy, recreationally active, male, age 27.1 ± 1.8 y) completed 3 CO-rebreathing measurements in randomized order. One was based on the 12-min original procedure (COoriginal), and two were based on the 2-min optimized procedure (COnew). From these measurements Hbmass for seven approaches (COoriginalA-E; COnewA-B) was calculated. Results. Hbmass estimations differed among these approaches (p < 0.01). Hbmass averaged 960 ± 133 g (COnewB), 981 ± 136 g (COnewA), 989 ± 130 g (COoriginalE), 993 ± 126 g (COoriginalA,D), 1030 ± 130 g (COoriginalB), and 1053 ± 133 g (COoriginalC). Procedural variations had a minor influence on measured Hbmass. Conclusions. The relevant discrepancies between the CO-rebreathing approaches originate mainly from different underlying calculations for Hbmass. Provided Hbmass enabled the development of conversion factors to compare average Hbmass values measured with different CO-rebreathing approaches. These factors can be used to develop reasonable Hbmass reference ranges for both clinical and athletic purposes.


Journal of Applied Physiology | 2016

Commentaries on Viewpoint: Time for a new metric for hypoxic dose?

Grégoire P. Millet; Franck Brocherie; Olivier Girard; Jon Peter Wehrlin; Severin Troesch; Anna Hauser; Thomas Steiner; Juha E. Peltonen; Heikki Rusko; Keren Constantini; Timothy J. Fulton; Daniel G. Hursh; Tyler J. Noble; Hunter L. Paris; Chad C. Wiggins; Robert F. Chapman; Benjamin D. Levine; Vasantha H. Kumar; Walter Schmidt

TO THE EDITOR: The proposal by our well-respected colleagues (2) to introduce a new metric—incorporating the altitude elevation and the total exposure duration, termed “kilometer hours”—for better describing the “hypoxic dose” is decidedly a step forward. By only quantifying the “external” stress, this metric presents several limitations: It suggests a linear relationship between altitude elevation and saturation decrease [but the Fick curve is curvilinear (3)] or that it applies to all athletes irrespectively of their training background [but elite endurance athletes suffer the largest decrease in V̇O2max (1)], altitude experience [but elite athletes who have had previous hypoxic exposure better adapt to hypoxic condition (4)], or type of hypoxia [but hypobaric vs. normobaric hypoxia induces larger desaturation (5)]. The large intersubject variability in the physiological responses to a given “hypoxic dose” implies that the magnitude of the stimulus rather than the altitude elevation should instead be considered. We therefore propose a new metric based on the sustained duration at a given arterial saturation level. Hence, desaturation levels in normoxia (exercise-induced arterial hypoxemia) or in hypoxia (3) predict the decrement in V̇O2max in hypoxia and therefore the ̇amplitude of the “hypoxic stimulus.” This metric termed “saturation hours” is defined as %·h (98/s 1) h 100, where s is the saturation value (in %) and h the time (in hours) sustained at any second level. Practically, with the development of new sport gears incorporating the oximeter inside the textile, this metric will readily be measured without any disturbances to individuals.


Structure | 2013

An Autoinhibited State in the Structure of Thermotoga maritima NusG

Johanna Drögemüller; Christian M. Stegmann; Angshuman Mandal; Thomas Steiner; Björn M. Burmann; Max E. Gottesman; Birgitta M. Wöhrl; Paul Rösch; Markus C. Wahl; Kristian Schweimer

NusG is a conserved regulatory protein interacting with RNA polymerase (RNAP) and other proteins to form multicomponent complexes that modulate transcription. The crystal structure of Thermotoga maritima NusG (TmNusG) shows a three-domain architecture, comprising well-conserved amino-terminal (NTD) and carboxy-terminal (CTD) domains with an additional, species-specific domain inserted into the NTD. NTD and CTD directly contact each other, occluding a surface of the NTD for binding to RNAP and a surface on the CTD interacting either with transcription termination factor Rho or transcription antitermination factor NusE. NMR spectroscopy confirmed the intramolecular NTD-CTD interaction up to the optimal growth temperature of Thermotoga maritima. The domain interaction involves a dynamic equilibrium between open and closed states and contributes significantly to the overall fold stability of the protein. Wild-type TmNusG and deletion variants could not replace endogenous Escherichia coli NusG, suggesting that the NTD-CTD interaction of TmNusG represents an autoinhibited state.


Journal of Applied Physiology | 2017

Individual hemoglobin mass response to normobaric and hypobaric "live high-train low": A one-year crossover study.

Anna Hauser; Severin Troesch; Jonas J. Saugy; Laurent Schmitt; Roberto Cejuela-Anta; Raphael Faiss; Thomas Steiner; Neil Robinson; Grégoire P. Millet; Jon Peter Wehrlin

The purpose of this research was to compare individual hemoglobin mass (Hbmass) changes following a live high-train low (LHTL) altitude training camp under either normobaric hypoxia (NH) or hypobaric hypoxia (HH) conditions in endurance athletes. In a crossover design with a one-year washout, 15 male triathletes randomly performed two 18-day LHTL training camps in either HH or NH. All athletes slept at 2,250 meters and trained at altitudes <1,200 meters. Hbmass was measured in duplicate with the optimized carbon monoxide rebreathing method before (pre) and immediately after (post) each 18-day training camp. Hbmass increased similarly in HH (916-957 g, 4.5 ± 2.2%, P < 0.001) and in NH (918-953 g, 3.8 ± 2.6%, P < 0.001). Hbmass changes did not differ between HH and NH (P = 0.42). There was substantial interindividual variability among subjects to both interventions (i.e., individual responsiveness or the individual variation in the response to an intervention free of technical noise): 0.9% in HH and 1.7% in NH. However, a correlation between intraindividual ΔHbmass changes (%) in HH and in NH (r = 0.52, P = 0.048) was observed. HH and NH evoked similar mean Hbmass increases following LHTL. Among the mean Hbmass changes, there was a notable variation in individual Hbmass response that tended to be reproducible.NEW & NOTEWORTHY This is the first study to compare individual hemoglobin mass (Hbmass) response to normobaric and hypobaric live high-train low using a same-subject crossover design. The main findings indicate that hypobaric and normobaric hypoxia evoked a similar mean increase in Hbmass following 18 days of live high-train low. Notable variability and reproducibility in individual Hbmass responses between athletes was observed, indicating the importance of evaluating individual Hbmass response to altitude training.

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Jon Peter Wehrlin

Indiana University Bloomington

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Anna Hauser

Indiana University Bloomington

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Grégoire P. Millet

Indiana University Bloomington

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Severin Troesch

Indiana University Bloomington

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Franck Brocherie

Indiana University Bloomington

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Julien Rysman

Université libre de Bruxelles

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