The mature part of proNGF induces the structure of its pro-peptide

AbstractHuman nerve growth factor (NGF) belongs to the structural family of cystine knot proteins, characterized by a disulfide pattern in which one disulfide bond threads through a ring formed by a pair of two other disulfides connecting two adjacent β-strands. Oxidative folding of NGF revealed that the pro-peptide of NGF stimulates in vitro structure formation. In order to learn more about this folding assisting protein fragment, a biophysical analysis of the pro-peptide structure has been performed. While proNGF is a non-covalent homodimer, the isolated pro-peptide is monomeric. No tertiary contacts stabilize the pro-peptide in its isolated form. In contrast, the pro-peptide appears to be structured when bound to the mature part. The results presented here demonstrate that the mature part stabilizes the structure in the pro-peptide region. This is the first report that provides a biophysical analysis of a pro-peptide of the cystine knot protein family

Capacitation induces changes in metabolic pathways supporting motility of epididymal and ejaculated sperm

Mammalian sperm require sufficient energy to support motility and capacitation for successful fertilization. Previous studies cataloging the changes to metabolism in sperm explored ejaculated human sperm or dormant mouse sperm surgically extracted from the cauda epididymis. Due to the differences in methods of collection, it remains unclear whether any observed differences between mouse and human sperm represent species differences or reflect the distinct maturation states of the sperm under study. Here we compare the metabolic changes during capacitation of epididymal versus ejaculated mouse sperm and relate these changes to ejaculated human sperm. Using extracellular flux analysis and targeted metabolic profiling, we show that capacitation-induced changes lead to increased flux through both glycolysis and oxidative phosphorylation in mouse and human sperm. Ejaculation leads to greater flexibility in the ability to use different carbon sources. While epididymal sperm are dependent upon glucose, ejaculated mouse and human sperm gain the ability to also leverage non-glycolytic energy sources such as pyruvate and citrate

Mouse sperm energy restriction and recovery (SER) revealed novel metabolic pathways

Mammalian sperm must undergo capacitation to become fertilization-competent. While working on mice, we recently developed a new methodology for treating sperm in vitro, which results in higher rates of fertilization and embryo development after in vitro fertilization. Sperm incubated in media devoid of nutrients lose motility, although they remain viable. Upon re-adding energy substrates, sperm resume motility and become capacitated with improved functionality. Here, we explore how sperm energy restriction and recovery (SER) treatment affects sperm metabolism and capacitation-associated signaling. Using extracellular flux analysis and metabolite profiling and tracing via nuclear magnetic resonance (NMR) and mass spectrometry (MS), we found that the levels of many metabolites were altered during the starvation phase of SER. Of particular interest, two metabolites, AMP and L-carnitine, were significantly increased in energy-restricted sperm. Upon re-addition of glucose and initiation of capacitation, most metabolite levels recovered and closely mimic the levels observed in capacitating sperm that have not undergone starvation. In both control and SER-treated sperm, incubation under capacitating conditions upregulated glycolysis and oxidative phosphorylation. However, ATP levels were diminished, presumably reflecting the increased energy consumption during capacitation. Flux data following the fate of 13C glucose indicate that, similar to other cells with high glucose consumption rates, pyruvate is converted into 13C-lactate and, with lower efficiency, into 13C-acetate, which are then released into the incubation media. Furthermore, our metabolic flux data show that exogenously supplied glucose is converted into citrate, providing evidence that in sperm cells, as in somatic cells, glycolytic products can be converted into Krebs cycle metabolites

Recognition of T-rich single-stranded DNA by the cold shock protein Bs-CspB in solution

Cold shock proteins (CSP) belong to the family of single-stranded nucleic acid binding proteins with OB-fold. CSP are believed to function as ‘RNA chaperones’ and during anti-termination. We determined the solution structure of Bs-CspB bound to the single-stranded DNA (ssDNA) fragment heptathymidine (dT(7)) by NMR spectroscopy. Bs-CspB reveals an almost invariant conformation when bound to dT(7) with only minor reorientations in loop β1–β2 and β3–β4 and of few aromatic side chains involved in base stacking. Binding studies of protein variants and mutated ssDNA demonstrated that Bs-CspB associates with ssDNA at almost diffusion controlled rates and low sequence specificity consistent with its biological function. A variation of the ssDNA affinity is accomplished solely by changes of the dissociation rate. (15)N NMR relaxation and H/D exchange experiments revealed that binding of dT(7) increases the stability of Bs-CspB and reduces the sub-nanosecond dynamics of the entire protein and especially of loop β3–β4

New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein

A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1∶3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins – designed ankyrin repeat proteins – without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies

Folding and Stability of Ankyrin Repeats Control Biological Protein Function

Ankyrin repeat proteins are found in all three kingdoms of life. Fundamentally, these proteins are involved in protein-protein interaction in order to activate or suppress biological processes. The basic architecture of these proteins comprises repeating modules forming elongated structures. Due to the lack of long-range interactions, a graded stability among the repeats is the generic properties of this protein family determining both protein folding and biological function. Protein folding intermediates were frequently found to be key for the biological functions of repeat proteins. In this review, we discuss most recent findings addressing this close relation for ankyrin repeat proteins including DARPins, Notch receptor ankyrin repeat domain, IκBα inhibitor of NFκB, and CDK inhibitor p19INK4d. The role of local folding and unfolding and gradual stability of individual repeats will be discussed during protein folding, protein-protein interactions, and post-translational modifications. The conformational changes of these repeats function as molecular switches for biological regulation, a versatile property for modern drug discovery

Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain.

The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state