Refine
Document Type
- Article (6) (remove)
Language
- English (6)
Has Fulltext
- yes (6)
Is part of the Bibliography
- no (6) (remove)
Keywords
- Proteins (6) (remove)
Institute
The endoplasmic reticulum–mitochondria encounter structure (ERMES) connects the mitochondrial outer membrane with the ER. Multiple functions have been linked to ERMES, including maintenance of mitochondrial morphology, protein assembly and phospholipid homeostasis. Since the mitochondrial distribution and morphology protein Mdm10 is present in both ERMES and the mitochondrial sorting and assembly machinery (SAM), it is unknown how the ERMES functions are connected on a molecular level. Here we report that conserved surface areas on opposite sides of the Mdm10 β-barrel interact with SAM and ERMES, respectively. We generated point mutants to separate protein assembly (SAM) from morphology and phospholipid homeostasis (ERMES). Our study reveals that the β-barrel channel of Mdm10 serves different functions. Mdm10 promotes the biogenesis of α-helical and β-barrel proteins at SAM and functions as integral membrane anchor of ERMES, demonstrating that SAM-mediated protein assembly is distinct from ER-mitochondria contact sites.
The prediction of protein–ligand interactions and their corresponding binding free energy is a challenging task in structure-based drug design and related applications. Docking and scoring is broadly used to propose the binding mode and underlying interactions as well as to provide a measure for ligand affinity or differentiate between active and inactive ligands. Various studies have revealed that most docking software packages reliably predict the binding mode, although scoring remains a challenge. Here, a diverse benchmark data set of 99 matched molecular pairs (3D-MMPs) with experimentally determined X-ray structures and corresponding binding affinities is introduced. This data set was used to study the predictive power of 13 commonly used scoring functions to demonstrate the applicability of the 3D-MMP data set as a valuable tool for benchmarking scoring functions.
COMP and TSP-4 interact specifically with the novel GXKGHR motif only found in fibrillar collagens
(2018)
COMP (cartilage oligomeric matrix protein) is a member of the thrombospondin family and forms homopentamers as well as mixed heterooligomers with its closely related family member TSP-4. COMP is long known to bind to collagens and to influence collagen fibril formation. Recent work indicates that already intracellular interaction with collagen is important for collagen secretion. However, the exact binding site of COMP on the collagen triple helix has not been described up to now. In this study we have identified a GXKGHR motif on the collagen II helix to bind to COMP, using a recombinantly expressed collagen II peptide library. This binding sequence is conserved throughout evolution and we demonstrate that TSP-4 binds to the same sequence. The identified binding motif overlaps with the recognition sites of many other collagen-binding partners (e.g. PEDF, Heparin) and also spans the lysine residues, which form collagen cross-links. COMP might thereby protect collagen helices from premature modification and cross-linking. Interestingly, this motif is only found in classical fibrillar collagens, although COMP is known to also bind other types. This might indicate that COMP has a unique interface for fibrillar collagens, thus making it an interesting target for the development of antifibrotic drugs.
The access to information on the dynamic behaviour of large proteins is usually hindered as spectroscopic methods require the site-specific attachment of biophysical probes. A powerful emerging tool to tackle this issue is amber codon suppression. Till date, its application on large and complex multidomain proteins of MDa size has not been reported. Herein, we systematically investigate the feasibility to introduce different non-canonical amino acids into a 540 kDa homodimeric fatty acid synthase type I by genetic code expansion with subsequent fluorescent labelling. Our approach relies on a microplate-based reporter assay of low complexity using a GFP fusion protein to quickly screen for sufficient suppression conditions. Once identified, these findings were successfully utilized to upscale both the expression scale and the protein size to full-length constructs. These fluorescently labelled samples of fatty acid synthase were subjected to initial biophysical experiments, including HPLC analysis, activity assays and fluorescence spectroscopy. Successful introduction of such probes into a molecular machine such as fatty acid synthases may pave the way to understand the conformational variability, which is a primary intrinsic property required for efficient interplay of all catalytic functionalities, and to engineer them.
In bioengineering, scaffold proteins have been increasingly used to recruit molecules to parts of a cell, or to enhance the efficacy of biosynthetic or signalling pathways. For example, scaffolds can be used to make weak or non-immunogenic small molecules immunogenic by attaching them to the scaffold, in this role called carrier. Here, we present the dodecin from Mycobacterium tuberculosis (mtDod) as a new scaffold protein. MtDod is a homododecameric complex of spherical shape, high stability and robust assembly, which allows the attachment of cargo at its surface. We show that mtDod, either directly loaded with cargo or equipped with domains for non-covalent and covalent loading of cargo, can be produced recombinantly in high quantity and quality in Escherichia coli. Fusions of mtDod with proteins of up to four times the size of mtDod, e.g. with monomeric superfolder green fluorescent protein creating a 437 kDa large dodecamer, were successfully purified, showing mtDod’s ability to function as recruitment hub. Further, mtDod equipped with SYNZIP and SpyCatcher domains for post-translational recruitment of cargo was prepared of which the mtDod/SpyCatcher system proved to be particularly useful. In a case study, we finally show that mtDod-peptide fusions allow producing antibodies against human heat shock proteins and the C-terminus of heat shock cognate 70 interacting protein (CHIP).
Vibrational energy transfer (VET) is essential for protein function. It is responsible for efficient energy dissipation in reaction sites, and has been linked to pathways of allosteric communication. While it is understood that VET occurs via backbone as well as via non-covalent contacts, little is known about the competition of these two transport channels, which determines the VET pathways. To tackle this problem, we equipped the β-hairpin fold of a tryptophan zipper with pairs of non-canonical amino acids, one serving as a VET injector and one as a VET sensor in a femtosecond pump probe experiment. Accompanying extensive non-equilibrium molecular dynamics simulations combined with a master equation analysis unravel the VET pathways. Our joint experimental/computational endeavor reveals the efficiency of backbone vs. contact transport, showing that even if cutting short backbone stretches of only 3 to 4 amino acids in a protein, hydrogen bonds are the dominant VET pathway.