Combinatorial protein design

Combinatorial protein libraries permit the examination of a wide range of sequences. Such methods are being used for denovo design and to investigate the determinants of protein folding. The exponentially large number of possible sequences, however, necessitates restrictions on the diversity of sequences in a combinatorial library. Recently, progress has been made in developing theoretical tools to bias and characterize the ensemble of sequences that fold into a given structure - tools that can be applied to the design and interpretation of combinatorial experiments.     

Using changes in hydrostatic and osmotic pressure to manipulate metabolic function in chondrocytes

Articular cartilage has distinct histological depth zones. In each zone, chondrocytes are subject to different hydrostatic (HP) and osmotic pressure (OP) due to weight-bearing and joint-loading. Previous in vitro studies of regeneration and pathophysiology in cartilage have failed to consider the characteristics of histological heterogeneity and the effects of combinations of changes in HP and OP. Thus, we have constructed molecular, biochemical, and histological profiles of anabolic and catabolic molecules produced by chondrocytes from each depth zone isolated from bovine articular cartilage in response to changes in HP and OP. We cultured the chondrocytes with combinations of loading or off-loading of HP at 0-0.5 MPa, 0.5 Hz, and changes in OP of 300-450 mosM over 1 wk, and evaluated mRNA expression and immunohistology of both anabolic and catabolic molecules and amounts of accumulated sulfated glycosaminoglycan. Any changes in HP and OP upregulated mRNA of anabolic and catabolic molecules in surface-, middle-, and deep-zone cells, in descending order of magnitude. Off-loading HP maintained the anabolic and reduced the catabolic mRNA; high OP retained upregulation of catabolic mRNA. These molecular profiles were consistent with immunohistological and biochemical findings. Changes in HP and OP are essential for simulating chondrocyte physiology and useful for manipulating phenotypes.     

Combinatorial approaches to protein stability and structure.

Why do proteins adopt the conformations that they do, and what determines their stabilities? While we have come to some understanding of the forces that underlie protein architecture, a precise, predictive, physicochemical explanation is still elusive. Two obstacles to addressing these questions are the unfathomable vastness of protein sequence space, and the difficulty in making direct physical measurements on large numbers of protein variants. Here, we review combinatorial methods that have been applied to problems in protein biophysics over the last 15 years. The effects of hydrophobic core composition, the most important determinant of structure and stability, are still poorly understood. Particular attention is given to core composition as addressed by library methods. Increasingly useful screens and selections, in combination with modern high-throughput approaches borrowed from genomics and proteomics efforts, are making the empirical, statistical correlation between sequence and structure a tractable problem for the coming years.     

Inactivation of taste receptor cell function by two cationic protein modification reagents

Two cationic protein modification reagents, 1-cyclohexyl-3-(2-morpholinylethyl) carbodiimide (CMCD) and dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNB-dmS), inhibit taste receptor cell stimulation by NaCl, sucrose, and HCl. Modified inactive derivatives of the reagents under the same conditions are ineffective. Inactivation by HNB-dmS is essentially irreversible. The effects of inactivation by CMCD are reversible after about 10–15 minutes of a water rinse, however, when applied in the presence of glycine methyl ester, the inhibited response is stabilized and only recovers after about 1.5–3 hours. Glycine methyl ester alone has no inhibitory properties. The kinetics of inactivation by both HNB-dmS and CMCD are consistent with a second-order reaction with rate constants of 0.041 ± 0.001 M^?1 sec^?1 and 0.121 ± 0.012 M^?1 sec^?1, respectively. The rate of inactivation by both compounds is independent of NaCl concentration as well as degree of receptor stimulation. This, together with the observation that the response to stimulation by all effectors examined is altered, suggests the inactivation occurs at an event which is common to the transduction of the response from all three stimuli. The ether:water partition coefficients, as well as previous results from inactivation by N–substituted maleimides, indicate that hydrophilic reagents do not cross the cell membrane in significant concentrations within the time period of application. This suggests the site of modification by the cationic protein modification reagents is at the surface of the cell membrane. Significant residual NaCl, sucrose, and HCl activity remains after total inactivation. To account for this, a two-state membrane receptor system is postulated.     

Affinity-capture reagents for protein arrays

The simultaneous identification and quantitative measurement of the production levels of thousands of different proteins in a biological specimen remains an unachieved goal of modern proteomic research. Advances in the development of microarray-based platforms for highly parallel detection of proteins have therefore received a considerable impulse during the last few years. Here, we review the existing reagents for affinity capture of protein targets, as well as the techniques used for their immobilization on solid supports and methods for the detection of binding events, underlining the problems and the opportunities in this continuously evolving research field.     

libcov: A C++ bioinformatic library to manipulate protein structures,sequence alignments and phylogeny

Background  

An increasing number of bioinformatics methods are considering the phylogenetic relationships between biological sequences. Implementing new methodologies using the maximum likelihood phylogenetic framework can be a time consuming task.     

Immunomodulation of jasmonate to manipulate the wound response

Jasmonates are signals in plant stress responses and development. The exact mode of their action is still controversial. To modulate jasmonate levels intracellularly as well as compartment-specifically, transgenic Nicotiana tabacum plants expressing single-chain antibodies selected against the naturally occurring (3R,7R)-enantiomer of jasmonic acid (JA) were created in the cytosol and the endoplasmic reticulum. Consequently, the expression of anti-JA antibodies in planta caused JA-deficient phenotypes such as insensitivity of germinating transgenic seedlings towards methyl jasmonate and the loss of wound-induced gene expression. Results presented here suggest an essential role for cytosolic JA in the wound response of tobacco plants. The findings support the view that substrate availability takes part in regulating JA biosynthesis upon wounding. Moreover, high JA levels observed in immunomodulated plants in response to wounding suggest that tobacco plants are able to perceive a reduced level of physiologically active JA and attempt to compensate for this by increased JA accumulation.     

Combinatorial control of protein phosphatase-1.

The catalytic subunit of the type 1 Ser/Thr protein phosphatases (PP1) can interact with many different regulatory (R) subunits. These R subunits function as activity-modulators, targeting subunits and/or substrates. The specificity of the R subunits can be accounted for by their interaction with specific subsets of binding pockets on the catalytic subunit and by the presence of subcellular targeting sequences. Hormones, growth factors and metabolites control the function of PP1 holoenzymes mainly by modulating the interaction of the subunits.     

Combinatorial approaches to probe the sequence determinants of protein aggregation and amyloidogenicity

Elucidation of the molecular determinants that drive proteins to aggregate is important both to advance our fundamental understanding of protein folding and misfolding, and as a step towards successful intervention in human disease. Combinatorial strategies enable unbiased and model-free approaches to probe sequence/structure relationships. Through the use of combinatorial methods, it is possible (i) to probe the sequence determinants of natural amyloid proteins by screening libraries of amino acid substitutions (mutations) to identify those that prevent amyloid formation; and (ii) to test new hypotheses about the mechanism of formation of amyloid fibrils by using these hypotheses to guide the design of combinatorial libraries of de novo amyloid-like proteins. Here, we review how these two approaches have been used to study the molecular determinants of protein aggregation and amyloidogenicity.     

Salmonella enterica serotype Typhimurium usurps the scaffold protein IQGAP1 to manipulate Rac1 and MAPK signalling

Salmonella enterica serotype Typhimurium invades eukaryotic cells by re-arranging the host-cell cytoskeleton. However, the precise mechanisms by which Salmonella induces cytoskeletal changes remain undefined. IQGAP1 (IQ motif-containing GTPase-activating protein 1) is a scaffold protein that binds multiple proteins including actin, the Rho GTPases Rac1 and Cdc42 (cell division cycle 42), and components of the MAPK (mitogen-activated protein kinase) pathway. We have shown previously that optimal invasion of Salmonella into HeLa cells requires IQGAP1. In the present paper, we use IQGAP1-null MEFs (mouse embryonic fibroblasts) and selected well-characterized IQGAP1 mutant constructs to dissect the molecular determinants of Salmonella invasion. Knockout of IQGAP1 expression reduced Salmonella invasion into MEFs by 75%. Reconstituting IQGAP1-null MEFs with wild-type IQGAP1 completely rescued invasion. By contrast, reconstituting IQGAP1-null cells with mutant IQGAP1 constructs that specifically lack binding to either Cdc42 and Rac1 (termed IQGAP1ΔMK24), actin, MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase] or ERK only partially restored Salmonella entry. Cell-permeant inhibitors of Rac1 activation or MAPK signalling reduced Salmonella invasion into control cells by 50%, but had no effect on bacterial entry into IQGAP1-null MEFs. Importantly, the ability of IQGAP1ΔMK24 to promote Salmonella invasion into IQGAP1-null cells was abrogated by chemical inhibition of MAPK signalling. Collectively, these results imply that the scaffolding function of IQGAP1, which integrates Rac1 and MAPK signalling, is usurped by Salmonella to invade fibroblasts and suggest that IQGAP1 may be a potential therapeutic target for Salmonella pathogenesis.     

ARROGANT: an application to manipulate large gene collections

ARROGANT (ARRay OrGANizing Tool) is a software tool developed to facilitate the identification, annotation and comparison of large collections of genes or clones. The objective is to enable users to compile gene/clone collections from different databases, allowing them to design experiments and analyze the collections as well as associated experimental data efficiently. ARROGANT can relate different sequence identifiers to their common reference sequence using the UniGene database, allowing for the comparison of data from two different microarray experiments. ARROGANT has been successfully used to analyze microarray expression data for colon cancer, to compile genes potentially related to cardiac diseases for subsequent resequencing (to identify single nucleotide polymorphisms, SNPs), to design a new comprehensive human cDNA microarray for cancer, to combine and compare expression data generated by different microarrays and to provide annotation for genes on custom and Affymetrix chips.     

Combinatorial triple-selective labeling as a tool to assist membrane protein backbone resonance assignment

Obtaining NMR assignments for slowly tumbling molecules such as detergent-solubilized membrane proteins is often compromised by low sensitivity as well as spectral overlap. Both problems can be addressed by amino-acid specific isotope labeling in conjunction with ¹⁵N–¹H correlation experiments. In this work an extended combinatorial selective in vitro labeling scheme is proposed that seeks to reduce the number of samples required for assignment. Including three different species of amino acids in each sample, ¹⁵N, 1-¹³C, and fully ¹³C/¹⁵N labeled, permits identification of more amino acid types and sequential pairs than would be possible with previously published combinatorial methods. The new protocol involves recording of up to five 2D triple-resonance experiments to distinguish the various isotopomeric dipeptide species. The pattern of backbone NH cross peaks in this series of spectra adds a new dimension to the combinatorial grid, which otherwise mostly relies on comparison of [¹⁵N, ¹H]–HSQC and possibly 2D HN(CO) spectra of samples with different labeled amino acid compositions. Application to two α-helical membrane proteins shows that using no more than three samples information can be accumulated such that backbone assignments can be completed solely based on 3D HNCA/HN(CO)CA experiments. Alternatively, in the case of severe signal overlap in certain regions of the standard suite of triple-resonance spectra acquired on uniformly labeled protein, or missing signals due to a lack of efficiency of 3D experiments, the remaining gaps can be filled.     

Prebiotics: tools to manipulate the gut microbiome and metabolome

Journal of Industrial Microbiology & Biotechnology - The human gut is an ecosystem comprising trillions of microbes interacting with the host. The composition of the microbiota and their...     

The kinetics of protein precipitation by different reagents

The kinetics of precipitation of soya protein has been examined in a tubular flow reactor with the precipitants, ammonium sulfate, ethanol, divalent calcium, and sulfuric acid. The precipitate growth profiles obtained in all cases showed the rapid formation of a precipitate phase and the attainment of a final size within 16 s. The final mean particle diameter d(m) varied with precipitating agent used in the order: sulfuric acid (12.5 mum) > ethanol (7.5 mum) approximately calcium ion (7.2 mum) > ammonium sulfate (3.1 mum). In the case of ethanol precipitation, changes in the design of the contacting section of the reactor led to differences in the precipitate growth curve. Protein solubility curves are also presented, and with the reactor data, they provide a convenient method for assessing the effect of precipitating agent on the design of protein precipitation reactors.     

Combinatorial control of the specificity of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs), the enzymes that dephosphorylate tyrosyl phosphoproteins, were initially believed to be few in number and serve a 'housekeeping' role in signal transduction. Recent work indicates that this is totally incorrect. Instead, PTPs comprise a large superfamily whose members play critical roles in a wide variety of cellular processes. Moreover, PTPs exhibit exquisite substrate specificity in vivo. Recent evidence has led us to propose that members of the PTP family achieve selectivity through different combinations of specific targeting strategies and intrinsic catalytic domain specificity.     

Using regulatory information to manipulate glycerol metabolism in Saccharomyces cerevisiae

Metabolic engineering has emerged as an attractive alternative to random mutagenesis and screening to design cell factories for industrial fermentation processes. The design of metabolic networks has been realized by gene deletions or strong overexpression of heterologous genes. There is an increasing body of evidence that indicates complete inactivation of native genes and high-level activity of heterologous enzymes may be deleterious to the cell. To moderately implement their expression, genes of interest are expressed under the control of promoters with different strengths. Constructing a promoter library is labor-intensive and requires precise quantification of the promoter strength. However, when the mechanisms of pathway regulation are known, it is possible to exploit this information to effect genetic changes efficiently. We report the implementation of this concept to reducing glycerol production during aerobic growth of Saccharomyces cerevisiae. Glycerol is produced to dispose excess cytosolic reduced nicotinamide adenine dinucleotide (NADH), and the regulating step in the pathway is mediated by glycerol 3-phosphate dehydrogenase (encoded by GPD1 and GPD2 genes). We expressed NADH oxidase in S. cerevisiae under the control of the GPD2 promoter to modulate the decrease in cytosolic NADH to the right level where the heterologous enzyme does not compete with oxidative phosphorylation while at the same time, decreasing glycerol production. This metabolic design resulted in substantially decreasing glycerol production and indeed, the excess carbon was redirected to biomass, resulting in a 14% increase in the specific growth rate. We believe that such strategies are more efficient than conventional methods and will find applications in bioprocesses.     

Programmable technologies to manipulate gene expression at the RNA level

RNA has long been an enticing therapeutic target, but is now garnering increased attention, largely driven by clinical successes of RNA interference–based drugs. While gene knockdown by well-established RNA interference– and other oligonucleotide-based strategies continues to advance in the clinic, the repertoire of targetable effectors capable of altering gene expression at the RNA level is also rapidly expanding. In this review, we focus on several recently developed bifunctional molecular technologies that both interact with and act upon a target RNA. These new approaches for programmable RNA knockdown, editing, splicing, translation, and chemical modifications stand to provide impactful new modalities for therapeutic development in the coming decades.