Timothy A. Whitehead

Computational design of proteins targeting the conserved stem region of influenza hemagglutinin.

Proteins can be designed that bind to specific patches on target proteins to alter their subsequent interactions. We describe a general computational method for designing proteins that bind a surface patch of interest on a target macromolecule. Favorable interactions between disembodied amino acid residues and the target surface are identified and used to anchor de novo designed interfaces. The method was used to design proteins that bind a conserved surface patch on the stem of the influenza hemagglutinin (HA) from the 1918 H1N1 pandemic virus. After affinity maturation, two of the designed proteins, HB36 and HB80, bind H1 and H5 HAs with low nanomolar affinity. Further, HB80 inhibits the HA fusogenic conformational changes induced at low pH. The crystal structure of HB36 in complex with 1918/H1 HA revealed that the actual binding interface is nearly identical to that in the computational design model. Such designed binding proteins may be useful for both diagnostics and therapeutics.

Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing

We show that comprehensive sequence-function maps obtained by deep sequencing can be used to reprogram interaction specificity and to leapfrog over bottlenecks in affinity maturation by combining many individually small contributions not detectable in conventional approaches. We use this approach to optimize two computationally designed inhibitors against H1N1 influenza hemagglutinin and, in both cases, obtain variants with subnanomolar binding affinity. The most potent of these, a 51-residue protein, is broadly cross-reactive against all influenza group 1 hemagglutinins, including human H2, and neutralizes H1N1 viruses with a potency that rivals that of several human monoclonal antibodies, demonstrating that computational design followed by comprehensive energy landscape mapping can generate proteins with potential therapeutic utility.

Community-wide assessment of protein-interface modeling suggests improvements to design methodology

The CAPRI (Critical Assessment of Predicted Interactions) and CASP (Critical Assessment of protein Structure Prediction) experiments have demonstrated the power of community-wide tests of methodology in assessing the current state of the art and spurring progress in the very challenging areas of protein docking and structure prediction. We sought to bring the power of community-wide experiments to bear on a very challenging protein design problem that provides a complementary but equally fundamental test of current understanding of protein-binding thermodynamics. We have generated a number of designed protein-protein interfaces with very favorable computed binding energies but which do not appear to be formed in experiments, suggesting that there may be important physical chemistry missing in the energy calculations. A total of 28 research groups took up the challenge of determining what is missing: we provided structures of 87 designed complexes and 120 naturally occurring complexes and asked participants to identify energetic contributions and/or structural features that distinguish between the two sets. The community found that electrostatics and solvation terms partially distinguish the designs from the natural complexes, largely due to the nonpolar character of the designed interactions. Beyond this polarity difference, the community found that the designed binding surfaces were, on average, structurally less embedded in the designed monomers, suggesting that backbone conformational rigidity at the designed surface is important for realization of the designed function. These results can be used to improve computational design strategies, but there is still much to be learned; for example, one designed complex, which does form in experiments, was classified by all metrics as a nonbinder.

Mechanical nanosensor based on fret within a thermosome: Damage-reporting polymeric materials

Unter Spannung: Andert sich in einem Protein-Polymer-Hybridmaterial die mechanische Spannung der Polymermatrix, so lost dies eine Konformationsanderung des Proteinkomplexes aus: Das Material meldet eine strukturelle Schadigung (siehe Bild). Die Reporterkomponente ist ein Chaperonin, das ein Paar fluoreszierender Proteine kovalent bindet. Wird das Chaperonin deformiert, andert sich der Abstand zwischen den Fluorophoren und folglich auch das FRET-Signal. Under stress: Changes in stress of the polymer matrix in a protein-polymer hybrid material result in changes of conformation of the protein complex, thus resulting in a damage-reporting material (see picture). The reporter is an engineered chaperonin that covalently entraps a pair of fluorescent proteins. Deformation of the chaperonin leads to a change in fluorophore distance and a change in the fluorescene resonance energy transfer (FRET) signal.

Minimal protein-folding systems in hyperthermophilic archaea

Although many archaeal species thrive in extreme environments, including hydrothermal vents, geothermal springs, acid seeps or hypersaline pools, there are also numerous species that are mesophilic. Mesophilic archaeal genomes encode complex protein-folding systems, which include combinations of bacterial and eukaryotic heat-shock proteins. Hyperthermophilic archaea, however, typically have reduced genomes that encode simplified heat-shock systems, with chaperones that are homologous to eukaryotic chaperones, and are reviewed here.

Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification.

BackgroundNon-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification.ResultsIn this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansiona (AFEX™-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin.ConclusionsOur findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.

Hotspot-Centric De Novo Design of Protein Binders

Protein-protein interactions play critical roles in biology, and computational design of interactions could be useful in a range of applications. We describe in detail a general approach to de novo design of protein interactions based on computed, energetically optimized interaction hotspots, which was recently used to produce high-affinity binders of influenza hemagglutinin. We present several alternative approaches to identify and build the key hotspot interactions within both core secondary structural elements and variable loop regions and evaluate the methods performance in natural-interface recapitulation. We show that the method generates binding surfaces that are more conformationally restricted than previous design methods, reducing opportunities for off-target interactions.

The interrelationship between promoter strength, gene expression, and growth rate

In exponentially growing bacteria, expression of heterologous protein impedes cellular growth rates. Quantitative understanding of the relationship between expression and growth rate will advance our ability to forward engineer bacteria, important for metabolic engineering and synthetic biology applications. Recently, a work described a scaling model based on optimal allocation of ribosomes for protein translation. This model quantitatively predicts a linear relationship between microbial growth rate and heterologous protein expression with no free parameters. With the aim of validating this model, we have rigorously quantified the fitness cost of gene expression by using a library of synthetic constitutive promoters to drive expression of two separate proteins (eGFP and amiE) in E. coli in different strains and growth media. In all cases, we demonstrate that the fitness cost is consistent with the previous findings. We expand upon the previous theory by introducing a simple promoter activity model to quantitatively predict how basal promoter strength relates to growth rate and protein expression. We then estimate the amount of protein expression needed to support high flux through a heterologous metabolic pathway and predict the sizable fitness cost associated with enzyme production. This work has broad implications across applied biological sciences because it allows for prediction of the interplay between promoter strength, protein expression, and the resulting cost to microbial growth rates.

Biotemplated Metal Nanowires Using Hyperthermophilic Protein Filaments

The fabrication of multifunctional nanostructures with complex geometries is necessary for advancement in the field of nanoelectronics and nanosensors. This is evident by the limited ability of present systems to handle optimum current loads, meet smaller device configurations, and provide integration. Electron transport in nanostructures has received a lot of attention for these reasons. Fortunately, nature offers a diverse assortment of self-assembling biomolecular templates for the construction of circuits, electrodes, and wires. These includeDNA,viruses, proteins, andpeptides that self-assemble into supramolecular structures, possess unique architectures (rods, spheres, filaments), and serve as scaffolds for nanoparticle synthesis andassembly. For example,DNAstrands have been used to template metal nanowires or two-dimensional (2D) nanogrids; the tobacco mosaic virus has been decorated with small gold nanoparticles and peptide nanotubes or protein functionalized carbon nanotubes have been mineralized with copper, gold, and silver. These biotemplated structures highlight the unique structures obtainable through the use of biomolecular templates. Moreover, biotemplating enables one to acquire nano–bio hybrids exhibiting electrical properties ranging from insulator to conductor with the conductivity close to bulkmetals, mainly due to the fact that electron transport through the metal nanowires formed fromvariousbiotemplates is governedby the metal domain-to-domain interface and/or grain boundaries. In the case of crystalline nanowires, the absence of a grain boundary or interface results in high conductivity and failure current density.

Tying up the loose ends: circular permutation decreases the proteolytic susceptibility of recombinant proteins

Recombinant proteins often suffer from poor expression because of proteolysis. Existing genetic engineering or fermentation strategies work for only a subset of cases where higher recombinant protein expression is needed. In this paper, we describe the use of circular permutation, wherein the original termini of a protein are concatenated and new termini are generated elsewhere with the sequence, as a general protein engineering strategy to produce full-length, active recombinant protein. We show that a circularly permuted variant of the thermosome (Group II chaperonin) from Methanocaldococcus jannaschii exhibited reduced proteolysis and increased expression in three different strains of Escherichia coli. Circular permutation of a different protein, TEM-1 beta-lactamase, by a similar method increased the expression lifetime of the protein in the periplasm of E. coli. Both circularly permuted proteins maintained activity near their wild-type counterparts and design criteria for selecting the sites for circular permutation are discussed. It is expected that this method will find broad utility for enhanced expression of recombinant proteins when proteolysis is a factor.