Content
2008
Interaction of tim23 with tim50 is essential for protein translocation by the mitochondrial tim23 complex
18.11.2008
The TIM23 complex is the major translocase of the mitochondrial inner membrane responsible for the import of essentially all matrix proteins and a number of inner membrane proteins.
Tim23 and Tim50, two essential proteins of the complex, expose conserved domains into the intermembrane space which
interact with each other. Here, we describe in vitro reconstitution of this interaction using recombinantly expressed and purified intermembranespace domains of Tim50 and Tim23. We
established two independent methods, chemical crosslinking and surface plasmon resonance, to track their interaction. In addition, we identified mutations in Tim23 which abolish its interaction with Tim50 in vitro. These mutations also destabilized the interaction
between the two proteins in vivo leading to defective import of preproteins via the TIM23 complex and to cell death at
higher temperatures. This is the first study to describe the reconstitution of the Tim50-Tim23 interaction in vitro and to
identify specific residues of Tim23 that are vital for the interaction with Tim50.
Characterization of TIC110, a channel-forming protein at the inner envelope membrane of chloroplasts, unveils a response to Ca2+ and a stromal regulatory disulfide bridge
05.11.2008
Tic110 has been proposed to be a channel-forming protein at the inner envelope of chloroplasts whose function is essential for the import of proteins synthesized in the cytosol. Sequence features and topology determination experiments presently summarized suggest that Tic110 consists of six transmembrane helices. Its topology has been mapped by limited proteolysis experiments in combination with mass spectrometric determinations and cysteine modification analysis. Two hydrophobic transmembrane helices located in the N-terminus serve as a signal for the localization of the protein to the membrane as shown previously. The other amphipathic transmembrane helices are located in the region comprised of residues 92-959 in the pea sequence. This results in two regions in the intermembrane space localized to form supercomplexes with the TOC machinery and to receive the transit peptide of preproteins. A large region also resides in the stroma for interaction with proteins such as molecular chaperones. In addition to characterizing the topology of Tic110, we show that Ca2+ has a dramatic effect on channel activity in vitro and that the protein has a redox-active disulfide with the potential to interact with stromal thioredoxin.
Semisynthesis of a Glycosylphosphatidylinositol-Anchored Prion Protein
13.10.2008
Angewandte Chemie,
2008,
DOI: 10.1002/anie.200802161,
Volume 47, Issue 43, pages 8215–8219,
published on 13.10.2008
Angew. Chemie, online article
Angew. Chemie, online article
Proteins are often modified posttranslationally by glycosylation and lipidation. Glycosylphosphatidylinositol (GPI) anchors combine both types of modification and link many proteins to the cell surface. Advances in solid-phase peptide synthesis (SPPS) and recombinant protein engineering, in combination with the development of native chemical ligation (NCL) and expressed protein ligation (EPL), have resulted in numerous total syntheses and semisyntheses of proteins. These approaches facilitate access to homogeneous glycoand lipoproteins, which serve as defined molecular probes to elucidate the effects of glycosylation and lipidation on the biophysical properties of proteins. Synthetic GPI glycans and lipidated GPI anchors have emerged as valuable tools
that allow for the precise dissection of their biological relevance in infectious and metabolic diseases. Efforts towards the assembly of chemically defined GPI-anchored proteins have focused on model studies; no synthetic GPIanchored protein has been reported to date.
Import of preproteins into the chloroplast inner envelope membran
14.08.2008
The chloroplast inner envelope membrane contains many integral proteins which differ in the number of a-helices that anchor the protein into the bilayer. For most of these proteins it is not known which pathway they engage to reach their final localisation within the membrane. In yeast mitochondria, two distinct sorting/insertion
pathways have been described for integral inner membrane
proteins, involving the Tim22 and Tim23 translocases.
These routes involve on the one hand a conservative sorting,
on the other hand a stop-transfer pathway. In this study
we performed a systematic characterisation of the import
behaviour of seven inner envelope proteins representing
different numbers of predicted a-helices. We investigated
their energy dependence, import rate, involvement of components of the chloroplast general import pathway and distribution between soluble and membrane fractions. Our results show the existence of at least two different families of inner envelope proteins that can be classified due to the occurrence of an intermediate processing form. Each of the proteins we investigated seems to use a stop-transfer
pathway for insertion into the inner envelope.
Rheology
04.08.2008
What is rheology? Most people are familiar with the basics of rheology from experience with diarrhea or perhaps rheostats. The word rheology was invented in 1929 to name the discipline of a society engaged in the study of how materials deform in
response to forces. It was inspired by a quote by Heraclitus: “παντα
ρει” translated as “everything flows”. Indeed everything does flow, but
to different extents depending on how much force is applied, in what
direction, and for how long. For materials more complex than simple
springs, where a spring constant relates force to elongation, the goal
of rheology is to provide quantitative parameters that define how a material will deform as a function of force,time and spatial orientation.
Thirty years of protein translocation into mitochondria: Unexpectedly complex and still puzzling
12.07.2008
Biochim Biophys Acta,
2008,
1793(1),
33-41
published on 12.07.2008
BBA - Molecular Cell Research, online article
BBA - Molecular Cell Research, online article
Mitochondria are essential organelles of the eukaryotic cells that are made by expansion and division of pre-existing mitochondria. The majority of their protein constituents are synthesized in the cytosol. They are transported into and put together within the organelle. This complex process is facilitated by several protein translocases. Here we summarize current knowledge on these sophisticated molecular machines that mediate recognition, transport across membranes and intramitochondrial sorting of many hundreds of mitochondrial proteins.
The Actinome of Dictyostelium discoideum in Comparison to Actins and Actin-Related Proteins from Other Organisms
09.07.2008
Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps). To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group). According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8) as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures
suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that
the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes.
The structure of a folding intermediate provides insight into differences in immunoglobulin amyloidogenicity
02.07.2008
Folding intermediates play a key role in defining protein folding
and assembly pathways as well as those of misfolding and aggregation. Yet, due to their transient nature, they are poorly accessible to high-resolution techniques. Here, we made use of the intrinsically slow folding reaction of an antibody domain to characterize its major folding intermediate in detail. Furthermore, by a single point mutation we were able to trap the intermediate in equilibrium and characterize it at atomic resolution. The intermediate
exhibits the basic b-barrel topology, yet some strands are distorted.
Surprisingly, two short strand-connecting helices conserved
in constant antibody domains assume their completely
native structure already in the intermediate, thus providing a
scaffold for adjacent strands. By transplanting these helical elements into b2-microglobulin, a highly homologous member of the
same superfamily, we drastically reduced its amyloidogenicity.
Thus, minor structural differences in an intermediate can shape the
folding landscape decisively to favor either folding or misfolding.
Effect of Thioxopeptide Bonds on r-Helix Structure and Stability
31.05.2008
Journal of the American Chemical Society,
2008,
130,
8079-84
published on 31.05.2008
JACS, online article
JACS, online article
Thioxoamide (thioamide) bonds are nearly isosteric substitutions for amides but have altered hydrogen-bonding and photophysical properties. They are thus well-suited backbone modifications for
physicochemical studies on peptides and proteins. The effect of thioxoamides on protein structure and stability has not been subject to detailed experimental investigations up to date. We used alanine-based model peptides to test the influence of single thioxoamide bonds on R-helix structure and stability. The
results from circular dichroism measurements show that thioxoamides are strongly helix-destabilizing. The effect of an oxo-to-thioxoamide backbone substitution is of similar magnitude as an alanine-to-glycine
substitution resulting in a helix destabilization of about 7 kJ/mol. NMR characterization of a helical peptide with a thioxopeptide bond near the N-terminus indicates that the thioxopeptide moiety is tolerated in helical structures. The thioxoamide group is engaged in an i, i+4 hydrogen bond, arguing against the formation of a 310-helical structure as suggested for the N-termini of R-helices in general and for thioxopeptides in particular.
Active remodelling of the TIM23 complex during translocation of preproteins into mitochondria
21.05.2008
The TIM23 (translocase of the mitochondrial inner membrane) complex mediates translocation of preproteins across and their insertion into the mitochondrial inner membrane. How the translocase mediates sorting of preproteins into the two different subcompartments is poorly understood. In particular, it is not clear whether association of two operationally defined parts of the translocase, the membrane-integrated part and the import
motor, depends on the activity state of the translocase. We established conditions to in vivo trap the TIM23 complex
in different translocation modes. Membraneintegrated
part of the complex and import motor were always found in one complex irrespective of whether an arrested preprotein was present or not. Instead, we detected different conformations of the complex in
response to the presence and, importantly, the type of preprotein being translocated. Two non-essential subunits of the complex, Tim21 and Pam17, modulate its activity in an antagonistic manner. Our data demonstrate that the TIM23 complex acts as a single structural and functional entity that is actively remodelled to sort preproteins into different mitochondrial subcompartments.
Contribution of Trimeric Autotransporter C-Terminal Domains of Oligomeric Coiled-Coil Adhesin (Oca) Family Members YadA, UspA1, EibA, and Hia to Translocation of the YadA Passenger Domain and Virulence of Yersinia enterocolitica
07.05.2008
Journal of Bacteriology,
2008,
190, 14,
5031-43
published on 07.05.2008
Journal of Bacteriology, online article
Journal of Bacteriology, online article
The Oca family is a novel class of autotransporter-adhesins with highest structural similarity in their C-terminal transmembrane region, which supposedly builds a beta-barrel pore in the outer membrane (OM). The prototype of the Oca family is YadA, an adhesin of Yersinia enterocolitica and Yersinia pseudotuberculosis. YadA forms a homotrimeric lollipop-like structure on the bacterial surface. The C-terminal regions of three
YadA monomers form a barrel in the OM and translocate the trimeric N-terminal passenger domain, consisting of stalk, neck, and head region to the exterior. To elucidate the structural and functional role of the C-terminal translocator domain (TLD) and to assess its promiscuous capability with respect to transport of
related passenger domains, we constructed chimeric YadA proteins, which consist of the N-terminal YadA passenger domain and C-terminal TLDs of Oca family members UspA1 (Moraxella catarrhalis), EibA (Escherichia coli), and Hia (Haemophilus influenzae). These constructs were expressed in Y. enterocolitica and compared for OM localization, surface exposure, oligomerization, adhesion properties, serum resistance, and mouse virulence. We demonstrate that all chimeric YadA proteins translocated the YadA passenger domain across the OM. Y. enterocolitica strains producing YadA chimeras or wild-type YadA showed comparable binding to collagen and epithelial cells. However, strains producing YadA chimeras were attenuated in serum resistance and mouse virulence. These results demonstrate for the first time that TLDs of Oca proteins of
different origin are efficient translocators of the YadA passenger domain and that the cognate TLD of YadA is essential for bacterial survival in human serum and mouse virulence.
Actin: its cumbersome pilgrimage through cellular compartments
26.04.2008
Histochemistry and Cell Biology,
2008,
DOI 10.1007/s00418-008-0430-y,
published on 26.04.2008
Histochemistry; online article
Histochemistry; online article
In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days’ knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin.
Energetics of protein translocation into mitochondria
12.04.2008
Biochimica et Biophysica Acta,
2008,
doi:10.1016/j.bbabio.2008.04.009,
published on 12.04.2008
Biochimica et Biophysica Acta, online article
Biochimica et Biophysica Acta, online article
Biogenesis of mitochondria depends on the coordinated action of at least six protein translocases present in both mitochondrial membranes. They use different energy sources to drive unidirectional transport of proteins across and into mitochondrial membranes. Here we present an overview on the energetic
requirements of different mitochondrial import pathways.
Unusual, Virulence Plasmid-Dependent Growth Behavior of Yersinia enterocolitica in Three-Dimensional Collagen Gels
04.04.2008
Journal of Bacteriology,
2008,
190, 12,
4111-20
published on 04.04.2008
Journal of Bacteriology, online article
Journal of Bacteriology, online article
As a first approach to establishing a three-dimensional culture infection model, we studied the growth behavior of the extracellular pathogen Yersinia enterocolitica in three-dimensional collagen gels (3D-CoG). Surprisingly, we observed that plasmidless Y. enterocolitica was motile in the 3D-CoG in contrast to its growth in traditional motility agar at 37°C. Motility at 37°C was abrogated in the presence of the virulence plasmid
pYV or the exclusive expression of the pYV-located Yersinia adhesion gene yadA. YadA-producing yersiniae formed densely packed (dp) microcolonies, whereas pYVyadA-carrying yersiniae formed loosely packedmicrocolonies at 37°C in 3D-CoG. Furthermore, we demonstrated that the packing density of the microcolonies was dependent on the head domain of YadA. Moreover, dp microcolony formation did not depend on the
capacity of YadA to bind to collagen fibers, as demonstrated by the use of yersiniae producing collagen nonbinding YadA. By using a yopE-gfp reporter, we demonstrated Ca2-dependent expression of this pYVlocalized virulence gene by yersiniae in 3D-CoG. In conclusion, this study revealed unique plasmid-dependent
growth behavior of yersiniae in a three-dimensional matrix environment that resembles the behavior of yersiniae (e.g., formation of microcolonies) in infected mouse tissue. Thus, this 3D-CoG model may be a first step to a more complex level of in vitro infection models that mimic living tissue, enabling us to study the dynamics of pathogen-host cell interactions.
Monitoring Protein Conformation along the Pathway of Chaperonin-Assisted Folding
04.04.2008
The GroEL/GroES chaperonin system mediates protein folding in the bacterial cytosol. Newly synthesized proteins reach GroEL via transfer from upstream chaperones such as DnaK/DnaJ (Hsp70).Here we employed single molecule and ensemble FRET to monitor the conformational transitions of amodel substrate as
it proceeds along this chaperone pathway. We find that DnaK/DnaJ stabilizes the protein in collapsed states that fold exceedingly slowly. Transfer to GroEL results in unfolding,with a fraction ofmolecules reaching locally highly expanded conformations. ATP-induced domain movements in GroEL cause transient further unfolding andrapidmobilizationofprotein segments with moderate hydrophobicity, allowing partial compaction on the GroEL surface. The more hydrophobic regions are released upon subsequent protein encapsulation in the central GroEL cavity by GroES,
completing compaction and allowing rapid folding. Segmental chain release and compaction may be important in avoiding misfolding by proteins that fail to fold efficiently through spontaneous hydrophobic
collapse.
TIC62 Redox-regulated Translocon Composition and Dynamics
14.03.2008
The Jounal of Biological Chemistry,
2008,
283,
6656-67
published on 14.03.2008
www.jbc.org, online article
www.jbc.org, online article
The preprotein translocon at the inner envelope of chloroplasts (Tic complex) facilitates the import of nuclear-encoded preproteins into the organelle. Seven distinct subunits have been identified so far. For each of those, specific functions have been proposed based on structural prediction or experimental evidence. Three of those subunits possess modules that could act as redox-active regulatory components in the import process. To date, however, the mode of redox regulation of the import process remains enigmatic. To investigate how the chloroplast redox state influences translocon behavior and composition, we studied the Tic component and the putative redox sensor Tic62 in more detail. The experimental results provide evidence that Tic62 can act as a bona fide dehydrogenase in vitro, and that it changes its localization in the chloroplast dependent on the NADP+/NADPH ratio in the stroma. Moreover, the redox state influences the interactions of Tic62 with the translocon and the flavoenzyme ferredoxin-NADP+ oxidoreductase. Additionally, we give initial experimental insights into the Tic62 structure using circular dichroism measurements and demonstrate that the protein consists of two structurally different domains. Our results indicate that Tic62 possesses redox-dependent properties that would allow it to fulfill a role as redox sensor protein in the chloroplast.
Structural Dynamics of Archaeal Small Heat Shock Proteins
12.02.2008
Small heat shock proteins (sHsps) are a widespread and diverse class of molecular chaperones. In vivo, sHsps contribute to thermotolerance. Recent evidence suggests that their function in the cellular chaperone network is to maintain protein omeostasis by complexing a variety of non-native proteins. One of the most characteristic features of sHsps is their organization into large, sphere-like structures commonly consisting of 12 or 24 subunits.
Here, we investigated the functional and structural properties of Hsp20.2, an sHsp from Archaeoglobus fulgidus, in comparison to its relative, Hsp16.5 from Methanocaldococcus jannaschii. sp20.2 is active in suppressing the aggregation of different model substrates at physiological and heat-stress temperatures. Electron microscopy showed that Hsp20.2 forms two distinct types of octahedral oligomers of slightly different sizes, indicating certain structural flexibility of the oligomeric assembly. By three-dimensional analysis of electron microscopic images of negatively stained specimens, we were able to reconstitute 3D models of the assemblies at a resolution of 19 Å. Under conditions of heat stress, the distribution of the structurally different Hsp20.2 assemblies changed, and this change was correlated with an increased chaperone activity. In analogy to Hsp20.2, Hsp16.5 oligomers displayed structural dynamics and exhibited increased chaperone activity under conditions of heat stress. Thus, temperature-induced conformational regulation of the activity of sHsps may be a general phenomenon in hermophilic archaea.
Activation of the Chaperone Hsp26 is Controlled by the Rearrangement of Its Thermosensor Domain
01.02.2008
Cells respond to a sudden increase in temperature with the transcription of a special set of genes, a phenomenon known as the heat shock response. In the yeast S. cerevisiae, the molecular chaperone Hsp26 is one component of the heat shock response.
Hsp26 has the remarkable ability to sense increases in temperature directly and can switch from an inactive to a chaperone-active state. The underlying principle of this temperature regulation has remained
enigmatic. Hsp26 variants with altered spectroscopic properties allowed us to identify structural elements controlling this activation process. We show that temperature sensing by Hsp26 is a feature of its middle domain that changes its conformation within a narrow temperature range. This structural rearrangement allows Hsp26 to respond autonomously and directly to heat stress by reversibly unleashing its chaperone activity. Thus, the Hsp26 middle domain
is a thermosensor and intrinsic regulator of chaperone activity.
