An iterative computational design approach to increase the thermal endurance of a mesophilic enzyme.

Background: Strategies for maximizing the microbial production of bio-based chemicals and fuels include eliminating branched points to streamline metabolic pathways. While this is often achieved by removing key enzymes, the introduction of nonnative enzymes can provide metabolic shortcuts, bypassing branched points to decrease the production of undesired side-products. Pyruvate decarboxylase (PDC) can provide such a shortcut in industrially promising thermophilic organisms; yet to date, this enzyme has not been found in any thermophilic organism.

Natural diversity of glycoside hydrolase family 48 exoglucanases: insights from structure.

Glycoside hydrolase (GH) family 48 is an understudied and increasingly important exoglucanase family found in the majority of bacterial cellulase systems. Moreover, many thermophilic enzyme systems contain GH48 enzymes. Deletion of GH48 enzymes in these microorganisms results in drastic reduction in biomass deconstruction.

Development of an Optical Zn Probe Based on a Single Fluorescent Protein.

Various fluorescent probes have been developed to reveal the biological functions of intracellular labile Zn. Here, we present Green Zinc Probe (GZnP), a novel genetically encoded Zn sensor design based on a single fluorescent protein (single-FP). The GZnP sensor is generated by attaching two zinc fingers (ZF) of the transcription factor Zap1 (ZF1 and ZF2) to the two ends of a circularly permuted green fluorescent protein (cpGFP).

Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein.

Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue toward energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption.

Comparing Residue Clusters from Thermophilic and Mesophilic Enzymes Reveals Adaptive Mechanisms.

Understanding how proteins adapt to function at high temperatures is important for deciphering the energetics that dictate protein stability and folding. While multiple principles important for thermostability have been identified, we lack a unified understanding of how internal protein structural and chemical environment determine qualitative or quantitative impact of evolutionary mutations. In this work we compare equivalent clusters of spatially neighboring residues between paired thermophilic and mesophilic homologues to evaluate adaptations under the selective pressure of high temperature.

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover.

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall.

The catalytic mechanism and unique low pH optimum of Caldicellulosiruptor bescii family 3 pectate lyase.

The unique active site of the Caldicellulosiruptor bescii family 3 pectate lyase (PL3) enzyme has been thoroughly characterized using a series of point mutations, X-ray crystallography, pK(a) calculations and biochemical assays. The X-ray structures of seven PL3 active-site mutants, five of them in complex with intact trigalacturonic acid, were solved and characterized structurally, biochemically and computationally. The results confirmed that Lys108 is the catalytic base, but there is no clear candidate for the catalytic acid.

Predicting enzyme adsorption to lignin films by calculating enzyme surface hydrophobicity.

The inhibitory action of lignin on cellulase cocktails is a major challenge to the biological saccharification of plant cell wall polysaccharides. Although the mechanism remains unclear, hydrophobic interactions between enzymes and lignin are hypothesized to drive adsorption. Here we evaluate the role of hydrophobic interactions in enzyme-lignin binding.

Computationally designed peptide inhibitors of the ubiquitin E3 ligase SCF(Fbx4).

A structure-based computational approach was used to rationally design peptide inhibitors that can target an E3 ligase (SCF(Fbx4) )-substrate (TRF1) interface and subsequent ubiquitylation. Characterization of the inhibitors demonstrates that our sequence-optimization protocol results in an increase in peptide-TRF1 affinity without compromising peptide-protein specificity.

Cellulase linkers are optimized based on domain type and function: insights from sequence analysis, biophysical measurements, and molecular simulation.

Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs) to carbohydrate-binding modules (CBMs). Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function.

Transmembrane peptides used to investigate the homo-oligomeric interface and binding hotspot of latent membrane protein 1.

Epstein-Barr virus (EBV), a human γ-herpesvirus, establishes lifelong infection by targeting the adaptive immune system of the host through memory B cells. Although normally benign, EBV contributes to lymphoid malignancies and lymphoproliferative syndromes in immunocompromised individuals. The viral oncoprotein latent membrane protein 1 (LMP-1) is essential for B lymphocyte immortalization by EBV.

Computational design of the sequence and structure of a protein-binding peptide.

The de novo design of protein-binding peptides is challenging because it requires the identification of both a sequence and a backbone conformation favorable for binding. We used a computational strategy that iterates between structure and sequence optimization to redesign the C-terminal portion of the RGS14 GoLoco motif peptide so that it adopts a new conformation when bound to Gα(i1). An X-ray crystal structure of the redesigned complex closely matches the computational model, with a backbone root-mean-square deviation of 1.

Structural determinants of affinity enhancement between GoLoco motifs and G-protein alpha subunit mutants.

GoLoco motif proteins bind to the inhibitory G(i) subclass of G-protein α subunits and slow the release of bound GDP; this interaction is considered critical to asymmetric cell division and neuro-epithelium and epithelial progenitor differentiation. To provide protein tools for interrogating the precise cellular role(s) of GoLoco motif/Gα(i) complexes, we have employed structure-based protein design strategies to predict gain-of-function mutations that increase GoLoco motif binding affinity. Here, we describe fluorescence polarization and isothermal titration calorimetry measurements showing three predicted Gα(i1) point mutations, E116L, Q147L, and E245L; each increases affinity for multiple GoLoco motifs.

Computational design of second-site suppressor mutations at protein-protein interfaces.

The importance of a protein-protein interaction to a signaling pathway can be established by showing that amino acid mutations that weaken the interaction disrupt signaling, and that additional mutations that rescue the interaction recover signaling. Identifying rescue mutations, often referred to as second-site suppressor mutations, controls against scenarios in which the initial deleterious mutation inactivates the protein or disrupts alternative protein-protein interactions. Here, we test a structure-based protocol for identifying second-site suppressor mutations that is based on a strategy previously described by Kortemme and Baker.

A peptide antagonist of the TLR4-MD2 interaction.

Toll-like receptors are an integral part of innate immunity in the central nervous system (CNS); they orchestrate a robust defense in response to both exogenous and endogenous danger signals. Recently, toll-like receptor 4 (TLR4) has emerged as a therapeutic target for the treatment of CNS-related diseases such as sepsis and chronic pain. We herein report a chemical biology approach by using a rationally designed peptide inhibitor to disrupt the TLR4-MD2 association, thereby blocking TLR4 signaling.

Structure-based protocol for identifying mutations that enhance protein-protein binding affinities.

The ability to manipulate protein binding affinities is important for the development of proteins as biosensors, industrial reagents, and therapeutics. We have developed a structure-based method to rationally predict single mutations at protein-protein interfaces that enhance binding affinities. The protocol is based on the premise that increasing buried hydrophobic surface area and/or reducing buried hydrophilic surface area will generally lead to enhanced affinity if large steric clashes are not introduced and buried polar groups are not left without a hydrogen bond partner.

Normal metabolism but different physical properties of myelin from mice deficient in proteolipid protein.

Proteolipid protein (PLP) is the primary protein component of CNS myelin, yet myelin from the PLP(null) mouse has only minor ultrastructural abnormalities. Might compensation for a potentially unstable structure involve increased myelin synthesis and turnover? This was not the case; neither accumulation nor in vivo synthesis rates for the myelin-specific lipid cerebroside was altered in PLP(null) mice relative to wild-type (wt) animals. However, the yield of myelin from PLP(null) mice, assayed as levels of cerebroside, was only about 55% of wt control levels.

Alterations in metabolism and gene expression in brain regions during cuprizone-induced demyelination and remyelination.

Exposure of mice to the copper chelator, cuprizone, results in CNS demyelination. There is remyelination after removal of the metabolic insult. We present brain regional studies identifying corpus callosum as particularly severely affected; 65% of cerebroside is lost after 6 weeks of exposure.