We have used a mass spectrometry-based proteomic approach to compile an atlas of the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans and covering melting temperatures of 30–90 °C. Protein sequence, composition and size affect thermal stability in prokaryotes and eukaryotic proteins show a nonlinear relationship between the degree of disordered protein structure and thermal stability. The data indicate that evolutionary conservation of protein complexes is reflected by similar thermal stability of their proteins, and we show examples in which genomic alterations can affect thermal stability. Proteins of the respiratory chain were found to be very stable in many organisms, and human mitochondria showed close to normal respiration at 46 °C. We also noted cell-type-specific effects that can affect protein stability or the efficacy of drugs. This meltome atlas broadly defines the proteome amenable to thermal profiling in biology and drug discovery and can be explored online at http://meltomeatlas.proteomics.wzw.tum.de:5003/ and http://www.proteomicsdb.org .

The discovery of novel and robust enzymes for the breakdown of plant biomass bears tremendous potential for the development of sustainable production processes in the rapidly evolving new bioeconomy. By functional screening of a metagenomic library from a volcano soil sample a novel thermostable endo-β-glucanase (EngU) which is unusual with regard to its module architecture and cleavage specificity was identified. Various recombinant EngU variants were characterized. Assignment of EngU to an existing glycoside hydrolase (GH) family was not possible. Two regions of EngU showed weak sequence similarity to proteins of the GH clan GH-A, and acidic residues crucial for catalytic activity of EngU were identified by mutation. Unusual, a carbohydrate-binding module (CBM4) which displayed binding affinity for β-glucan, lichenin and carboxymethyl-cellulose was found as an insertion between these two regions. EngU hydrolyzed β-1,4 linkages in carboxymethyl-cellulose, but displayed its highest activity with mixed linkage (β-1,3-/β-1,4-) glucans such as barley β-glucan and lichenin, where in contrast to characterized lichenases cleavage occurred predominantly at the β-1,3 linkages of C4-substituted glucose residues. EngU and numerous related enzymes with previously unknown function represent a new GH family of biomass-degrading enzymes within the GH-A clan. The name assigned to the new GH family is GH148.

Background: Clostridium thermocellum is a paradigm for efficient cellulose degradation and a promising organism for the production of second generation biofuels. It owes its high degradation rate on cellulosic substrates to the presence of supra-molecular cellulase complexes, cellulosomes, which comprise over 70 different single enzymes assembled on protein-backbone molecules of the scaffold protein CipA.Results: Although all 24 single-cellulosomal cellulases were described previously, we present the first comparative catalogue of all these enzymes together with a comprehensive analysis under identical experimental conditions, including enzyme activity, binding characteristics, substrate specificity, and product analysis. In the course of our study, we encountered four types of distinct enzymatic hydrolysis modes denoted by substrate specificity and hydrolysis product formation: (i) exo-mode cellobiohydrolases (CBH), (ii) endo-mode cellulases with no specific hydrolysis pattern, endoglucanases (EG), (iii) processive endoglucanases with cellotetraose as intermediate product (pEG4), and (iv) processive endoglucanases with cellobiose as the main product (pEG2). These modes are shown on amorphous cellulose and on model cello-oligosaccharides (with degree of polymerization DP 3 to 6). Artificial mini-cellulosomes carrying combinations of cellulases showed their highest activity when all four endoglucanase-groups were incorporated into a single complex. Such a modeled nonavalent complex (n = 9 enzymes bound to the recombinant scaffolding protein CipA) reached half of the activity of the native cellulosome. Comparative analysis of the protein architecture and structure revealed characteristics that play a role in product formation and enzyme processivity.Conclusions: The identification of a new endoglucanase type expands the list of known cellulase functions present in the cellulosome. Our study shows that the variety of processivities in the enzyme complex is a key enabler of its high cellulolytic efficiency. The observed synergistic effect may pave the way for a better understanding of the enzymatic interactions and the design of more active lignocellulose-degrading cellulase cocktails in the future.

We analyze the dynamics of helical peptides that were designed de novo to mimic the transmembrane domains (TMDs) of natural fusogenic proteins. Their fusogenicity and the deuterium/proton exchange rates of the deuterated backbone amides increase with the content of helix-destabilizing residues (Val, Gly, Pro) corroborating the link between backbone dynamics and lipid mixing (Poschner et al., 2009. J. Mol. Biol. 386:733-741). Recent experiments indicate that these peptides also promote lipid flip. The efficiency by which they flip reporter lipids with different head-groups critically depends on the core sequence, the charge of the polar flanking residues, and the composition of the host membrane. In order to examine the complex peptide/lipid interactions we carried out all-atom MD simulations of these peptides in isotropic membrane-mimetic solvent and in a mixeds bilayer (DOPC:DOPE:DOPS 3:1:1). The simulations suggest that the local backbone dynamics of the TMDs is comparable less pronounced in the membrane than in isotropic solvent while the rank order of sequences is maintained. Efficient snorkeling of flanking lysine residues minimizes hydrophobic mismatch. The ordering of lipids around the TMDs shows a considerable dependence not only on the charge state of helix termini and head-groups, but also on the flexibility of the cores. Local lipids respond differently to the presence of the TMDs. The extents to which head-groups are pulled in and lipid tail order is disturbed will be discussed in relation to the extent by which these model TMDs promote fusion and induce lipid flip. Email: [email protected]