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.
20 Related JoVE Articles!
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
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 (http://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.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Microfluidic Mixers for Studying Protein Folding
Institutions: Michigan State University, Hong Kong University of Science and Technology, University of California, Davis .
The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1
. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 μs2
. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4
Laminar flow is achieved for Reynolds numbers Re
≤100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 μm wide and 10 μm deep (See Figure 1
). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 μm in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re
~10, but the protein consumption is only ~0.5 nL/s or 1.8 μL/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 μM (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2
). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 μm in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5
Bioengineering, Issue 62, microfluidic mixing, laminar flow, protein folding, fluorescence, FRET
Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy
Institutions: University of California San Diego - UCSD.
Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, ΔG°H2O
, for a representative integral membrane protein from E. coli
. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.
Bioengineering, Issue 50, tryptophan, peptides, Gibbs free energy, protein stability, vesicles
Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin
Institutions: NYU Hospital for Joint Diseases, New York University School of Medicine.
Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1
, inflammation 2, 3
, and host defense 4
. PGRN also functions as a neurotrophic factor 5
, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7
. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11
. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12
, extracellular matrix protein 10, 13
, and growth factor14
. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15
Molecular Biology, Issue 59, Modified yeast two-hybrid screen, PGRN, TNFR2, inflammation, autoimmune diseases
4D Imaging of Protein Aggregation in Live Cells
Institutions: Hebrew University of Jerusalem .
One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1
. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2
, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1
. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4
. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1
, target them for degradation by the ubiquitin-proteasome system5
, or direct them to protective aggregation inclusions6-9
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10
: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1
- model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12
) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts
is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 °C, or when the cell faces misfolding stress, Ubc9ts
misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.
Cellular Biology, Issue 74, Molecular Biology, Genetics, Proteins, Aggregation quality control, protein folding quality control, GFP, JUNQ (juxtanuclear quality control compartment), IPOD (insoluble protein deposit), proteostasis sensor, 4D live cell imaging, live cells, laser, cell biology, protein folding, Ubc9ts, yeast, assay, cell, imaging
Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism
Institutions: Ben-Gurion University of the Negev.
The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro
, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans
. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo
, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.
Biochemistry, Issue 82, aging, Caenorhabditis elegans, heat shock response, neurodegenerative diseases, protein folding homeostasis, proteostasis, stress, temperature-sensitive
Aip1p Dynamics Are Altered by the R256H Mutation in Actin
Institutions: University of Iowa, University of Iowa.
Mutations in actin cause a range of human diseases due to specific molecular changes that often alter cytoskeletal function. In this study, imaging of fluorescently tagged proteins using total internal fluorescence (TIRF) microscopy is used to visualize and quantify changes in cytoskeletal dynamics. TIRF microscopy and the use of fluorescent tags also allows for quantification of the changes in cytoskeletal dynamics caused by mutations in actin. Using this technique, quantification of cytoskeletal function in live cells valuably complements in vitro
studies of protein function. As an example, missense mutations affecting the actin residue R256 have been identified in three human actin isoforms suggesting this amino acid plays an important role in regulatory interactions. The effects of the actin mutation R256H on cytoskeletal movements were studied using the yeast model. The protein, Aip1, which is known to assist cofilin in actin depolymerization, was tagged with green fluorescent protein (GFP) at the N-terminus and tracked in vivo
using TIRF microscopy. The rate of Aip1p movement in both wild type and mutant strains was quantified. In cells expressing R256H mutant actin, Aip1p motion is restricted and the rate of movement is nearly half the speed measured in wild type cells (0.88 ± 0.30 μm/sec in R256H cells compared to 1.60 ± 0.42 μm/sec in wild type cells, p < 0.005).
Developmental Biology, Issue 89, green fluorescent protein, actin, Aip1p, total internal fluorescence microscopy, yeast, cloning
Oscillation and Reaction Board Techniques for Estimating Inertial Properties of a Below-knee Prosthesis
Institutions: University of Northern Colorado, Arizona State University, Iowa State University.
The purpose of this study was two-fold: 1) demonstrate a technique that can be used to directly estimate the inertial properties of a below-knee prosthesis, and 2) contrast the effects of the proposed technique and that of using intact limb inertial properties on joint kinetic estimates during walking in unilateral, transtibial amputees. An oscillation and reaction board system was validated and shown to be reliable when measuring inertial properties of known geometrical solids. When direct measurements of inertial properties of the prosthesis were used in inverse dynamics modeling of the lower extremity compared with inertial estimates based on an intact shank and foot, joint kinetics at the hip and knee were significantly lower during the swing phase of walking. Differences in joint kinetics during stance, however, were smaller than those observed during swing. Therefore, researchers focusing on the swing phase of walking should consider the impact of prosthesis inertia property estimates on study outcomes. For stance, either one of the two inertial models investigated in our study would likely lead to similar outcomes with an inverse dynamics assessment.
Bioengineering, Issue 87, prosthesis inertia, amputee locomotion, below-knee prosthesis, transtibial amputee
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
Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
Institutions: University of Wuerzburg, Max Planck Institute of Neurobiology, Martinsried, Ludwig-Maximilians University of Munich.
Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+
indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+
indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro
. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+
indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+
indicator and a hydrophilic fluorescent dye/Ca2+
complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0
Cellular Biology, Issue 75, Neurobiology, Neuroscience, Molecular Biology, Biochemistry, Biomedical Engineering, Bioengineering, Virology, Medicine, Anatomy, Physiology, Surgery, Endoplasmic Reticulum, ER, Calcium Signaling, calcium store, calcium imaging, calcium indicator, metabotropic signaling, Ca2+, neurons, cells, mouse, animal model, cell culture, targeted esterase induced dye loading, imaging
Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR
Institutions: The Hebrew University of Jerusalem, The Hebrew University of Jerusalem.
Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.
Chemistry, Issue 82, solution structure determination, NMR, peptide models, copper-binding proteins, copper complexes
Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry
Institutions: University of Heidelberg.
All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1
H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g.
crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex.
Chemistry, Issue 81, Molecular Chaperones, mass spectrometers, Amino Acids, Peptides, Proteins, Enzymes, Coenzymes, Protein dynamics, conformational changes, allostery, protein folding, secondary structure, mass spectrometry
Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro
using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro
. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo
. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo
tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro
and in vivo
and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
Nanomanipulation of Single RNA Molecules by Optical Tweezers
Institutions: University at Albany, State University of New York, University at Albany, State University of New York, University at Albany, State University of New York, University at Albany, State University of New York, University at Albany, State University of New York.
A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.
Bioengineering, Issue 90, RNA folding, single-molecule, optical tweezers, nanomanipulation, RNA secondary structure, RNA tertiary structure
In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae
Institutions: VU University Amsterdam.
In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo
, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871
, who showed that it was possible to reconstitute these complexes in vitro
starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro
reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g.,
pigment binding sites) or protein domain (e.g.,
protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro
currently used in our laboratory,
and examples describing applications of the method are provided.
Biochemistry, Issue 92, Reconstitution, Photosynthesis, Chlorophyll, Carotenoids, Light Harvesting Protein, Chlamydomonas reinhardtii, Arabidopsis thaliana
Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies
Institutions: University of Texas Southwestern Medical Center at Dallas.
To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.
Molecular Biology, Issue 77, Biochemistry, Genetics, Cellular Biology, Structural Biology, Biophysics, Membrane Lipids, Phospholipids, Carrier Proteins, Membrane Proteins, Micelles, Molecular Motor Proteins, life sciences, biochemistry, Amino Acids, Peptides, and Proteins, lipid-protein interaction, channel reconstitution, lipid-dependent gating, voltage-gated ion channel, conformation-specific ligands, lipids
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
Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer
Institutions: Scientific Instruments.
Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF · (OD260-OD320)· NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1
Protein concentration results can be expressed as μg/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.
Molecular Biology, Issue 48, Nucleic acid quantitation, protein quantitation, micro-volume analysis, label quantitation
Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System
Institutions: National Institute on Aging, NIH.
Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN
) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN
through its catalytic activities and protein interactions. The human WRN
gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN
, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication  at www.impactaging.com. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.
Microbiology, Issue 37, Werner syndrome, helicase, topoisomerase, RecQ, Bloom's syndrome, Sgs1, genomic instability, genetics, DNA repair, yeast
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