Leveraging nature's biomolecular designs in next-generation protein sequencing reagent development.

Next-generation approaches for protein sequencing are now emerging that could have the potential to revolutionize the field in proteomics. One such sequencing method involves fluorescence-based imaging of immobilized peptides in which the N-terminal amino acid of a polypeptide is readout sequentially by a series of fluorescently labeled biomolecules. When selectively bound to a specific N-terminal amino acid, the NAAB (N-terminal amino acid binder) affinity reagent identifies the amino acid through its associated fluorescence tag. A key technical challenge in implementing this fluoro-sequencing approach is the need to develop NAAB affinity reagents with the high affinity and selectivity for specific N-terminal amino acids required for this biotechnology application. One approach to develop such a NAAB affinity reagent is to leverage naturally occurring biomolecules that bind amino acids and/or peptides. Here, we describe several candidate biomolecules that could be considered for this purpose and discuss the potential for developability of each. Key points • Next-generation sequencing methods are emerging that could revolutionize proteomics. • Sequential readout of N-terminal amino acids by fluorescent-tagged affinity reagents. • Native peptide/amino acid binders can be engineered into affinity reagents. • Protein size and structure contribute to feasibility of reagent developability.

A single amino acid substitution alters ClpS2 binding specificity.

ClpS2 is a small protein under development as a probe for selectively recognizing N-terminal amino acids of N-degron peptide fragments. To understand the structural basis of ClpS2 specificity for an N-terminal amino acid, all atom molecular dynamics (MD) simulations were conducted using the sequence of a bench-stable mutant of ClpS2, called PROSS. We predicted that a single amino acid leucine to asparagine substitution would switch the specificity of PROSS ClpS2 to an N-terminal tyrosine over the preferred phenylalanine. Experimental validation of the mutant using a fluorescent yeast-display assay showed an increase in tyrosine binding over phenylalanine, in support of the proposed hypothesis.

Strategies for Development of a Next-Generation Protein Sequencing Platform.

Proteomic analysis can be a critical bottleneck in cellular characterization. The current paradigm relies primarily on mass spectrometry of peptides and affinity reagents (i.e., antibodies), both of which require a priori knowledge of the sample. An unbiased protein sequencing method, with a dynamic range that covers the full range of protein concentrations in proteomes, would revolutionize the field of proteomics, allowing a more facile characterization of novel gene products and subcellular complexes. To this end, several new platforms based on single-molecule protein-sequencing approaches have been proposed. This review summarizes four of these approaches, highlighting advantages, limitations, and challenges for each method towards advancing as a core technology for next-generation protein sequencing.

Engineering ClpS for selective and enhanced N-terminal amino acid binding.

One of the central challenges in the development of single-molecule protein sequencing technologies is achieving high-fidelity sequential recognition and detection of specific amino acids that comprise the peptide sequence. An approach towards achieving this goal is to leverage naturally occurring proteins that function through recognition of amino (N)-terminal amino acids (NAAs). One such protein, the N-end rule pathway adaptor protein ClpS, natively recognizes NAAs on a peptide chain. The native ClpS protein has a high specificity albeit modest affinity for the amino acid Phe at the N-terminus but also recognizes the residues Trp, Tyr, and Leu at the N-terminal position. Here, we employed directed evolution methods to select for ClpS variants with enhanced affinity and selectivity for two NAAs (Phe and Trp). Using this approach, we identified two promising variants of the Agrobacterium tumefaciens ClpS protein with native residues 34-36 ProArgGlu mutated to ProMetSer and CysProSer. In vitro surface binding assays indicate that the ProMetSer variant has enhanced affinity for Phe at the N-terminus with sevenfold tighter binding relative to wild-type ClpS, and that the CysProSer variant binds selectively to Trp over Phe at the N-terminus while having a greater affinity for both Trp and Phe. Taken together, this work demonstrates the utility of engineering ClpS to make it more effective for potential use in peptide sequencing applications.

A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination.

BACKGROUND: Saccharification of lignocellulosic material by xylanases and other glycoside hydrolases is generally conducted at high concentrations of the final reaction products, which frequently inhibit the enzymes used in the saccharification process. Using a random nonhomologous recombination strategy, we have fused the GH11 xylanase from Bacillus subtilis (XynA) with the xylose binding protein from Escherichia coli (XBP) to produce an enzyme that is allosterically stimulated by xylose. RESULTS: The pT7T3GFP_XBP plasmid containing the XBP coding sequence was randomly linearized with DNase I, and ligated with the XynA coding sequence to create a random XynA-XBP insertion library, which was used to transform E. coli strain JW3538-1 lacking the XBP gene. Screening for active XBP was based on the expression of GFP from the pT7T3GFP_XBP plasmid under the control of a xylose inducible promoter. In the presence of xylose, cells harboring a functional XBP domain in the fusion protein (XBP+) showed increased GFP fluorescence and were selected using FACS. The XBP+ cells were further screened for xylanase activity by halo formation around xylanase producing colonies (XynA+) on LB-agar-xylan media after staining with Congo red. The xylanase activity ratio with xylose/without xylose in supernatants from the XBP+/XynA+ clones was measured against remazol brilliant blue xylan. A clone showing an activity ratio higher than 1.3 was selected where the XynA was inserted after the asparagine 271 in the XBP, and this chimera was denominated as XynA-XBP271. The XynA-XBP271 was more stable than XynA at 55 °C, and in the presence of xylose the catalytic efficiency was ~3-fold greater than the parental xylanase. Molecular dynamics simulations predicted the formation of an extended protein-protein interface with coupled movements between the XynA and XBP domains. In the XynA-XBP271 with xylose bound to the XBP domain, the mobility of a ß-loop in the XynA domain results in an increased access to the active site, and may explain the observed allosteric activation. CONCLUSIONS: The approach presented here provides an important advance for the engineering enzymes that are stimulated by the final product.

Enzymatic protein switches built from paralogous input domains.

Protein switches have a variety of potential applications in biotechnology and medicine that motivate efforts to accelerate their development. Switches can be built by the proper fusion of two proteins with the prerequisite input and output functions. However, the exact fusion geometry for switch creation, which typically involves insertion of one protein domain into the other, is difficult to predict. Based on our previous work developing protein switches using periplasmic binding proteins as input domains, we wondered whether there are "hot spots" for insertion of output domains and successful switch creation within this class of proteins. Here we describe directed evolution experiments that identified switches in which TEM-1 beta-lactamase (BLA) is inserted into the class I periplasmic binding proteins ribose binding protein (RBP), glucose binding protein (GBP), and xylose binding protein (XBP). Although some overlap in sites for successful switch insertion could be found among the paralogs, successful switches at these sites required different linkers between the domains and different circular permutations of the BLA protein. Our data suggests that sites for successful switch creation are not easily transferable between paralogs. Furthermore, by comparison to a previous study in which switches were created by inserting a xylanase into XBP, we find no correlation between insertion sites when using two different output domains. We conclude that the switch property likely depends on the precise molecular details of the fusions and cannot be easily predicted from some overall general structural property of the fusion topology.

Insertion of a xylanase in xylose binding protein results in a xylose-stimulated xylanase.

BACKGROUND: Product inhibition can reduce catalytic performance of enzymes used for biofuel production. Different mechanisms can cause this inhibition and, in most cases, the use of classical enzymology approach is not sufficient to overcome this problem. Here we have used a semi-rational protein fusion strategy to create a product-stimulated enzyme. RESULTS: A semi-rational protein fusion strategy was used to create a protein fusion library where the Bacillus subtilis GH11 xylanase A (XynA) was inserted at 144 surface positions of the Escherichia coli xylose binding protein (XBP). Two XynA insertions at XBP positions 209 ([209]XBP-Xyn-XBP) and 262 ([262]XBP-Xyn-XBP) showed a 20% increased xylanolytic activity in the presence of xylose, conditions where native XynA is inhibited. Random linkers of 1-4 Gly/Ala residues were inserted at the XynA N- and C-termini in the [209]XBP and [262]XBP, and the chimeras 2091A and 2621B were isolated, showing a twofold increased xylanolytic activity in the presence of xylose and k cat values of 200 and 240 s(-1) in the 2091A and 2621B, respectively, as compared to 70 s(-1) in the native XynA. The xylose affinity of the XBP was unchanged in the chimeras, showing that the ~3- to 3.5-fold stimulation of catalytic efficiency by xylose was the result of allosteric coupling between the XBP and XynA domains. Molecular dynamics simulations of the chimeras suggested conformation alterations in the XynA on xylose binding to the XBP resulted in exposure of the catalytic cavity and increased mobility of catalytic site residues as compared to the native XynA. CONCLUSIONS: These results are the first report of engineered glycosyl hydrolase showing allosteric product stimulation and suggest that the strategy may be more widely employed to overcome enzyme product inhibition and to improve catalytic performance. Graphical abstractProtein fusion of a GH11 xylanase (in red) and a xylose binding protein (XBP, in blue) results in a xylanase-XBP chimera that presents allosteric activation of the xylanase activity by xylose (shown as a space-filled molecule bound to the xylanase-XBP chimera).

Recovery of an HMWP/hmwBP (pUL48/pUL47) complex from virions of human cytomegalovirus: subunit interactions, oligomer composition, and deubiquitylase activity.

UNLABELLED: We report that the human cytomegalovirus (HCMV) high-molecular-weight tegument protein (HMWP, pUL48; 253 kDa) and the HMWP-binding protein (hmwBP, pUL47; 110 kDa) can be recovered as a complex from virions disrupted by treatment with 50 mM Tris (pH 7.5), 0.5 M NaCl, 0.5% NP-40, and 10 mM dithiothreitol [DTT]. The subunit ratio of the complex approximates 1:1, with a shape and structure consistent with an elongated heterodimer. The HMWP/hmwBP complex was corroborated by reciprocal coimmunoprecipitation experiments using antipeptide antibodies and lysates from both infected cells and disrupted virus particles. An interaction of the amino end of pUL48 (amino acids [aa] 322 to 754) with the carboxyl end of pUL47 (aa 693 to 982) was identified by fragment coimmunoprecipitation experiments, and a head-to-tail self-interaction of hmwBP was also observed. The deubiquitylating activity of pUL48 is retained in the isolated complex, which cleaves K11, K48, and K63 ubiquitin isopeptide linkages. IMPORTANCE: Human cytomegalovirus (HCMV, or human herpesvirus 5 [HHV-5]) is a large DNA-containing virus that belongs to the betaherpesvirus subfamily and is a clinically important pathogen. Defining the constituent elements of its mature form, their organization within the particle, and the assembly process by which it is produced are fundamental to understanding the mechanisms of herpesvirus infection and developing drugs and vaccines against them. In this study, we report isolating a complex of two large proteins encoded by HCMV open reading frames (ORFs) UL47 and UL48 and identifying the binding domains responsible for their interaction with each other and of pUL47 with itself. Our calculations indicate that the complex is a rod-shaped heterodimer. Additionally, we determined that the ubiquitin-specific protease activity of the ORF UL48 protein was functional in the complex, cleaving K11-, K48-, and K63-linked ubiquitin dimers. This information builds on and extends our understanding of the HCMV tegument protein network that is required to interface the HCMV envelope and capsid.

Total synthesis of a functional designer eukaryotic chromosome.

Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871-base pair designer eukaryotic chromosome, synIII, which is based on the 316,617-base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATα allele on synIII. The complete design and synthesis of synIII establishes S. cerevisiae as the basis for designer eukaryotic genome biology.

Protein switch engineering by domain insertion.

The switch-like regulation of protein activity by molecular signals is abundant in native proteins. The ability to engineer proteins with novel regulation has applications in biosensors, selective protein therapeutics, and basic research. One approach to building proteins with novel switch properties is creating combinatorial libraries of gene fusions between genes encoding proteins that have the prerequisite input and output functions of the desired switch. These libraries are then subjected to selections and/or screens to identify those rare gene fusions that encode functional switches. Combinatorial libraries in which an insert gene is inserted randomly into an acceptor gene have been useful for creating switches, particularly when combined with circular permutation of the insert gene. Methods for creating random domain insertion libraries are described. Three methods for creating a diverse set of insertion sites in the acceptor gene are presented and compared: DNase I digestion, S1 nuclease digestion, and multiplex inverse PCR. A PCR-based method for creating a library of circular permutations of the insert gene is also presented.

Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA.

We demonstrate that S1 nuclease converts supercoiled plasmid DNA to unit-length, linear dsDNA through the creation of a single, double-stranded break in a plasmid molecule. These double-stranded breaks occur not only in the origin of replication near inverted repeats but also at a wide variety of locations throughout the plasmid. S1 nuclease exhibits this activity under conditions typically employed for the nuclease's single-stranded nuclease activity. Thus, S1 nuclease digestion of plasmid DNA, unlike analogous digestion with DNaseI, effectively halts after the first double-stranded break. This property makes easier the construction of large domain insertion libraries in which the goal is to insert linear DNA at a variety of locations throughout a plasmid. We used this property to create a library in which a circularly permuted TEM1 ß-lactamase gene was inserted throughout a plasmid containing the gene encoding Escherichia coli ribose binding protein. Gene fusions that encode allosteric switch proteins in which ribose modulates ß-lactamase catalytic activity were isolated from this library using a combination of a genetic selection and a screen.