The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
23 Related JoVE Articles!
Interview: Protein Folding and Studies of Neurodegenerative Diseases
Institutions: MIT - Massachusetts Institute of Technology.
In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.
Neuroscience, issue 17, protein folding, brain, neuron, prion, neurodegenerative disease, yeast, screen, Translational Research
Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking
Institutions: National Institutes of Health.
This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo
. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1
. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli
together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii
. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 Å. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.
Immunology, Issue 82, Autotransporters, Bam complex, Molecular chaperones, protein-protein interactions, Site-specific photocrosslinking
The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin
Institutions: European Institute of Oncology.
Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics
), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP
in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.
Biochemistry, Issue 86, chromatin, histone post-translational modifications (hPTMs), epigenetics, mass spectrometry, proteomics, SILAC, chromatin immunoprecipitation , histone variants, chromatome, hPTMs cross-talks
Magnetic Tweezers for the Measurement of Twist and Torque
Institutions: Delft University of Technology.
Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a “conventional” magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the “conventional” magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.
Bioengineering, Issue 87, magnetic tweezers, magnetic torque tweezers, freely-orbiting magnetic tweezers, twist, torque, DNA, single-molecule techniques
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis
Institutions: Institut Nanosciences et Cryogénie, CEA-Grenoble, Université Paris-Sud, Institut de Biologie Structurale.
In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.
Bioengineering, Issue 91, electronic tongue, combinatorial cross-reactive receptor, surface plasmon resonance imaging, pattern recognition, continuous evolution profiles, continuous evolution landscapes, protein analysis
In Situ Neutron Powder Diffraction Using Custom-made Lithium-ion Batteries
Institutions: University of Sydney, University of Wollongong, Australian Synchrotron, Australian Nuclear Science and Technology Organisation, University of Wollongong, University of New South Wales.
Li-ion batteries are widely used in portable electronic devices and are considered as promising candidates for higher-energy applications such as electric vehicles.1,2
However, many challenges, such as energy density and battery lifetimes, need to be overcome before this particular battery technology can be widely implemented in such applications.3
This research is challenging, and we outline a method to address these challenges using in situ
NPD to probe the crystal structure of electrodes undergoing electrochemical cycling (charge/discharge) in a battery. NPD data help determine the underlying structural mechanism responsible for a range of electrode properties, and this information can direct the development of better electrodes and batteries.
We briefly review six types of battery designs custom-made for NPD experiments and detail the method to construct the ‘roll-over’ cell that we have successfully used on the high-intensity NPD instrument, WOMBAT, at the Australian Nuclear Science and Technology Organisation (ANSTO). The design considerations and materials used for cell construction are discussed in conjunction with aspects of the actual in situ
NPD experiment and initial directions are presented on how to analyze such complex in situ
Physics, Issue 93, In operando, structure-property relationships, electrochemical cycling, electrochemical cells, crystallography, battery performance
Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders
Institutions: Purdue University.
Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g.
, FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast
) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.
Chemistry, Issue 98, Amide hydrogen/deuterium exchange, photolytic labeling, mass spectrometry, lyophilized formulations, photo-leucine, solid-state, protein structure, protein conformation, protein dynamics, secondary structure, protein stability, excipients
A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay
Institutions: Wright-Patterson Air Force Base, The Henry M. Jackson Foundation, UES, Inc..
Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.
Molecular Biology, Issue 96, Aptamer, structure-switching, SELEX, small molecule, cortisol, next generation sequencing, gold nanoparticle, assay
Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins
Institutions: San Diego State University, San Diego State University, San Diego State University, San Diego State University, San Diego State University, Argonne National Laboratory, Broad Institute.
Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.
Immunology, Issue 100, phenomics, phage, viral metagenome, Multi-phenotype Assay Plates (MAPs), continuous culture, metabolomics
Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients
Institutions: Hadassah Hebrew-University Medical Center.
In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.
Medicine, Issue 86, Optic neuritis, visual impairment, dynamic visual functions, motion perception, stereopsis, demyelination, remyelination
Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction
Institutions: Rutgers University, Koç University, New York University, Fairfield University.
We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be controlled by one computer.
Behavior, Issue 84, genetics, cognitive mechanisms, behavioral screening, learning, memory, timing
Designing Silk-silk Protein Alloy Materials for Biomedical Applications
Institutions: Rowan University, Rowan University, Cooper Medical School of Rowan University, Rowan University.
Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi
) and domestic mulberry silk (Bombyx mori
) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.
Bioengineering, Issue 90, protein alloys, biomaterials, biomedical, silk blends, computational simulation, implantable electronic devices
Molecular Evolution of the Tre Recombinase
Institutions: Max Plank Institute for Molecular Cell Biology and Genetics, Dresden.
Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells.
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.
Cell Biology, Issue 15, HIV-1, Tre recombinase, Site-specific recombination, molecular evolution
Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding
Institutions: School of Medicine, New York University.
The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' surface envelope glycoprotein. We previously proposed that the crown of the V3 loop, although dynamic and sequence variable, is constrained throughout the population of HIV-1 viruses to an immunologically relevant β-hairpin tertiary structure. Importantly, there are thousands of different V3 loop crown sequences in circulating HIV-1 viruses, making 3D structural characterization of trends across the diversity of viruses difficult or impossible by crystallography or NMR. Our previous successful studies with folding of the V3 crown1, 2
used the ab initio
accessible in the ICM-Pro molecular modeling software package (Molsoft LLC, La Jolla, CA) and suggested that the crown of the V3 loop, specifically from positions 10 to 22, benefits sufficiently from the flexibility and length of its flanking stems to behave to a large degree as if it were an unconstrained peptide freely folding in solution. As such, rapid ab initio
folding of just this portion of the V3 loop of any individual strain of the 60,000+ circulating HIV-1 strains can be informative. Here, we folded the V3 loop of the R2 strain to gain insight into the structural basis of its unique properties. R2 bears a rare V3 loop sequence thought to be responsible for the exquisite sensitivity of this strain to neutralization by patient sera and monoclonal antibodies4, 5
. The strain mediates CD4-independent infection and appears to elicit broadly neutralizing antibodies. We demonstrate how evaluation of the results of the folding can be informative for associating observed structures in the folding with the immunological activities observed for R2.
Infection, Issue 43, HIV-1, structure-activity relationships, ab initio simulations, antibody-mediated neutralization, vaccine design
A Protocol for Computer-Based Protein Structure and Function Prediction
Institutions: University of Michigan , University of Kansas.
Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently >20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.
Biochemistry, Issue 57, On-line server, I-TASSER, protein structure prediction, function prediction
Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
Institutions: Royal Institute of Technology.
Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1
. To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2
. The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3
. The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4
, were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5
, a small, bispecific molecule with affinity for both HSA and the novel target was identified6
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli
with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7
Molecular Biology, Issue 59, Affinity chromatography, albumin-binding domain, human serum albumin, Z-domain
Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids
Institutions: Washington University, Washington University, Washington University.
Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13
C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13
C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes.
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2
, we grow cells with 13
C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4
, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third
, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2
. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1
. In this protocol, we will use Cyanothece
51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.
Molecular Biology, Issue 59, GC-MS, novel pathway, metabolism, labeling, phototrophic microorganism
Perceptual and Category Processing of the Uncanny Valley Hypothesis' Dimension of Human Likeness: Some Methodological Issues
Institutions: University of Zurich.
Mori's Uncanny Valley Hypothesis1,2
proposes that the perception of humanlike characters such as robots and, by extension, avatars (computer-generated characters) can evoke negative or positive affect (valence) depending on the object's degree of visual and behavioral realism along a dimension of human likeness
) (Figure 1
). But studies of affective valence of subjective responses to variously realistic non-human characters have produced inconsistent findings 3, 4, 5, 6
. One of a number of reasons for this is that human likeness is not perceived as the hypothesis assumes. While the DHL can be defined following Mori's description as a smooth linear change in the degree of physical humanlike similarity, subjective perception of objects along the DHL can be understood in terms of the psychological effects of categorical perception (CP) 7
. Further behavioral and neuroimaging investigations of category processing and CP along the DHL and of the potential influence of the dimension's underlying category structure on affective experience are needed. This protocol therefore focuses on the DHL and allows examination of CP. Based on the protocol presented in the video as an example, issues surrounding the methodology in the protocol and the use in "uncanny" research of stimuli drawn from morph continua to represent the DHL are discussed in the article that accompanies the video. The use of neuroimaging and morph stimuli to represent the DHL in order to disentangle brain regions neurally responsive to physical human-like similarity from those responsive to category change and category processing is briefly illustrated.
Behavior, Issue 76, Neuroscience, Neurobiology, Molecular Biology, Psychology, Neuropsychology, uncanny valley, functional magnetic resonance imaging, fMRI, categorical perception, virtual reality, avatar, human likeness, Mori, uncanny valley hypothesis, perception, magnetic resonance imaging, MRI, imaging, clinical techniques
Setting Limits on Supersymmetry Using Simplified Models
Institutions: University College London, CERN, Lawrence Berkeley National Laboratories.
Experimental limits on supersymmetry and similar theories are difficult to set because of the enormous available parameter space and difficult to generalize because of the complexity of single points. Therefore, more phenomenological, simplified models are becoming popular for setting experimental limits, as they have clearer physical interpretations. The use of these simplified model limits to set a real limit on a concrete theory has not, however, been demonstrated. This paper recasts simplified model limits into limits on a specific and complete supersymmetry model, minimal supergravity. Limits obtained under various physical assumptions are comparable to those produced by directed searches. A prescription is provided for calculating conservative and aggressive limits on additional theories. Using acceptance and efficiency tables along with the expected and observed numbers of events in various signal regions, LHC experimental results can be recast in this manner into almost any theoretical framework, including nonsupersymmetric theories with supersymmetry-like signatures.
Physics, Issue 81, high energy physics, particle physics, Supersymmetry, LHC, ATLAS, CMS, New Physics Limits, Simplified Models
Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology
Institutions: California Institute of Technology, California Institute of Technology, Massachusetts Institute of Technology, University of Minnesota.
Ideal cell-free expression systems can theoretically emulate an in vivo
cellular environment in a controlled in vitro
This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3
To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo
cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli
based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5
The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli
, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6
Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7
The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8
Accompanying mathematical models are available.9,10
The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."
Cellular Biology, Issue 79, Bioengineering, Synthetic Biology, Chemistry Techniques, Synthetic, Molecular Biology, control theory, TX-TL, cell-free expression, in vitro, transcription-translation, cell-free protein synthesis, synthetic biology, systems biology, Escherichia coli cell extract, biological circuits, biomolecular breadboard
Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation
Institutions: University of Rochester, University of Rochester, University of Rochester Medical Center.
One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young’s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.
primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;
H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.
Chemistry, Issue 80, Poly(ethylene glycol), peptides, polymerization, polymers, methacrylation, peptide functionalization, 1H-NMR, MALDI-ToF, hydrogels, macromer synthesis
Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues
Institutions: The Research Institute at Nationwide Children's Hospital.
Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g.,
domains). Traditional consensus-building models fail to account for interpositional dependencies – functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple “majority rule” or even HMM-based consensus models, and the result can be a biologically invalid “consensus” which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.
Chemistry, Issue 101, protein engineering, covariation, codependent residues, visualization