James A. Stapleton

The khmer software package: enabling efficient nucleotide sequence analysis

The khmer package is a freely available software library for working efficiently with fixed length DNA words, or k-mers. khmer provides implementations of a probabilistic k-mer counting data structure, a compressible De Bruijn graph representation, De Bruijn graph partitioning, and digital normalization. khmer is implemented in C++ and Python, and is freely available under the BSD license at https://github.com/dib-lab/khmer/.

High-Resolution Sequence-Function Mapping of Full-Length Proteins

Comprehensive sequence-function mapping involves detailing the fitness contribution of every possible single mutation to a gene by comparing the abundance of each library variant before and after selection for the phenotype of interest. Deep sequencing of library DNA allows frequency reconstruction for tens of thousands of variants in a single experiment, yet short read lengths of current sequencers makes it challenging to probe genes encoding full-length proteins. Here we extend the scope of sequence-function maps to entire protein sequences with a modular, universal sequence tiling method. We demonstrate the approach with both growth-based selections and FACS screening, offer parameters and best practices that simplify design of experiments, and present analytical solutions to normalize data across independent selections. Using this protocol, sequence-function maps covering full sequences can be obtained in four to six weeks. Best practices introduced in this manuscript are fully compatible with, and complementary to, other recently published sequence-function mapping protocols.

Plasmid-based one-pot saturation mutagenesis

Deep mutational scanning is a foundational tool for addressing the functional consequences of large numbers of mutants, but a more efficient and accessible method for construction of user-defined mutagenesis libraries is needed. Here we present nicking mutagenesis, a robust, single-day, one-pot saturation mutagenesis method performed on routinely prepped plasmid dsDNA. The method can be used to produce comprehensive or single- or multi-site saturation mutagenesis libraries.

Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Significance The ability to construct novel proteins from basic principles of molecular structure is the fundamental goal of protein design. This is particularly challenging in the case of the β-barrel outer membrane proteins, where our understanding of the rules governing structure and function lags behind that of other classes of proteins. Here, we took a step toward understanding β-barrel membrane protein architecture by focusing on the outward-facing amino acid positions that contact the cell membrane. We replaced the membrane-facing surface of OmpA with new surfaces designed to resemble natural β-barrel surfaces. We were able to design versions of OmpA with mutations at about two-thirds of all surface positions, indicating that β-barrel membrane protein surface design is achievable. Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface of Escherichia coli OmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion.

Haplotype-Phased Synthetic Long Reads from Short-Read Sequencing.

Next-generation DNA sequencing has revolutionized the study of biology. However, the short read lengths of the dominant instruments complicate assembly of complex genomes and haplotype phasing of mixtures of similar sequences. Here we demonstrate a method to reconstruct the sequences of individual nucleic acid molecules up to 11.6 kilobases in length from short (150-bp) reads. We show that our method can construct 99.97%-accurate synthetic reads from bacterial, plant, and animal genomic samples, full-length mRNA sequences from human cancer cell lines, and individual HIV env gene variants from a mixture. The preparation of multiple samples can be multiplexed into a single tube, further reducing effort and cost relative to competing approaches. Our approach generates sequencing libraries in three days from less than one microgram of DNA in a single-tube format without custom equipment or specialized expertise.

Fine Epitope Mapping of Two Antibodies Neutralizing the Bordetella Adenylate Cyclase Toxin

Adenylate cyclase toxin (ACT) is an important Bordetella pertussis virulence factor that is not included in current acellular pertussis vaccines. We previously demonstrated that immunization with the repeat-in-toxin (RTX) domain of ACT elicits neutralizing antibodies in mice and discovered the first two antibodies to neutralize ACT activities by occluding the receptor-binding site. Here, we fully characterize these antibodies and their epitopes. Both antibodies bind ACT with low nanomolar affinity and cross-react with ACT homologues produced by B. parapertussis and B. bronchiseptica. Antibody M1H5 binds B. pertussis RTX751 ∼100-fold tighter than RTX751 from the other two species, while antibody M2B10 has similar affinity for all three variants. To initially map the antibody epitopes, we generated a series of ACT chimeras and truncation variants, which implicated the repeat blocks II-III. To identify individual epitope residues, we displayed randomly mutated RTX751 libraries on yeast and isolated clones with decreased antibody binding by flow cytometry. Next-generation sequencing identified candidate epitope residues on the basis of enrichment of clones with mutations at specific positions. These epitopes form two adjacent surface patches on a predicted structural model of the RTX751 domain, one for each antibody. Notably, the cellular receptor also binds within blocks II-III and shares at least one residue with the M1H5 epitope. The RTX751 model supports the notion that the antibody and receptor epitopes overlap. These data provide insight into mechanisms of ACT neutralization and guidance for engineering more stable RTX variants that may be more appropriate vaccine antigens.

Electrostatics in Protein Engineering and Design

The electrostatic interactions between charged atoms in natural proteins play a central role in specifying protein topology, modulating stability of the molecule, and allowing for the impressive catalytic properties of enzymes. In this chapter, we discuss how protein engineers use the principles of electrostatics and computational protein modeling to develop new proteins for biomedical and biotechnological applications. First, a general introduction is given to familiarize the reader with the important factors to consider in protein electrostatics, and the nature of these electrostatic forces. The next section describes various levels of theory used for modeling electrostatics in proteins. The last sections focus on specific applications in two conceptual classes: the engineering of ionic interactions (1) on protein surfaces, and (2) within the hydrophobic protein core. In both cases, the aim is to promote stability or to control molecular recognition.