Research Tools : Protein interactions & function

Research Tools Portfolios


Monomeric Fluorescent Protein-Ligand Complexes with Strong Fluorescence in the Far-Red Region

Research from the University of Wisconsin-Washington County in collaboration with the Institute for Stem Cell Biology and Regenerative Medicine in India, has resulted in the development of monomeric variants of the naturally occurring Sandercyanin Fluorescent Protein (SFP) using site-directed mutagenesis. This work has stemmed from earlier research focused on development of the tetrameric form of SFP, a biliverdin-binding lipocalin protein originally isolated from the mucus of the blue walleye fish, Sander vitreus. Monomeric variants of SFP (mSFPs) have been found to possess the same non-covalent, bili-binding characteristics of the tetramer but are one-quarter the size (~18.6kDa) and do not oligomerize. They are therefore anticipated to be more useful in a host of biotechnology applications. Like the tetrameric form, the mSFPs have a large stokes shift (375nm/675nm) and fluoresce in the far-red or near infrared region, which is advantageous for a wide range of applications including investigation of protein-protein interactions, spatial and temporal gene expression, assessing cell biology distribution and mobility, studying protein activity and protein interactions in vivo, as well as cancer research, immunology, and stem cell research and sub-cellular localization. In addition, the newly developed mSFP’s far-red fluorescence is particularly advantageous for in vivo, deep-tissue imaging.

Reagents for Bioreversible Protein Esterification

UW–Madison researchers have developed an optimized diazo compound, derived from phenylglycine amide, for converting carboxylate groups into an ester in high yield in buffered water. The ensuing esters are labile to esterase enzymes such as reside in all human cells, making the modification bioreversible. The novel compound is small, avoids deleterious side reactions and has a modularity that enables broad utility.

Enhancing Cell Penetration to Improve Drug Delivery

UW–Madison researchers have developed a method for enhancing cellular uptake of a cargo molecule by covalently bonding fluorenyl groups to it. The fluorenylated molecule is then contacted with the cell or tissue. Cellular uptake may be in vivo or in vitro and includes at least partial penetration into the cytosol.

New Amphiphiles for Manipulating Membrane Proteins

UW–Madison researchers have developed improved amphiphiles for solubilizing, isolating and characterizing membrane proteins. They can be prepared from cholic acid, deoxycholic acid and lithocholic acid, which are steroids found in bile.

The new amphiphiles, called CAO, DCAO and LCAO, are effective in challenging biochemical systems, such as extraction of delicate photosynthetic superassemblies from native lipid bilayers.

Improved Ref Nuclease for Site-Specific DNA Cleavage

The researchers have now developed a truncated variant of Ref nuclease that is twice as efficient as the original. It can be used along with RecA protein for site-specific cleavage of double-stranded DNA.

The new Ref variant was derived from a related phage called ϕW39, in which several dozen amino acid residues were deleted.

New Disulfide-Bond Reducing Agent

UW–Madison researchers have developed a fast-working pyrazine dithiol that can be prepared from inexpensive starting material. The new reagent, 2,3-bis(mercaptomethyl)pyrazine (BMMP), is synthesized in three simple steps from the commonplace aromatic chemical 2,3-dimethylpyrazine.

Fluorescent Ligand Complexes with Strong Fluorescence in the Far-Red Region for Tissue and Cell Labeling

UW – Washington County researchers have developed a fluorescent biomarker with strong fluorescence in the far-red region. These biomarkers can be used to localize and monitor proteins of interest or detect gene expression in cells, tissues, and whole organisms. The fluorescent molecules were discovered in a wild walleye fish species and their properties enhanced by targeted mutation of particular amino acid residues. The fluorescent molecules have several advantages over currently used reporters such as GFP, including small size, large stoke shift, far-red fluorescence, and long quenching time.

New Amphiphiles for Manipulating Integral Membrane Proteins

UW–Madison researchers have developed new amphiphiles that better aid in the solubilization, isolation, purification, stabilization, crystallization and structural determination of integral membrane proteins.

The new molecules use a multifused ring system as a lipophilic group. They are synthesized from commercially available steroidal precursors (cholesterol, in most cases) and fall into two types – GLC and GCT amphiphiles.

Membrane preparations containing some protein of interest can be treated with the novel amphiphiles to achieve protein extraction and solubilization. The amphiphiles are able to stabilize membrane proteins for several weeks.

Liquid Crystal Device for Identifying and Validating Cleaning Processes for Biofouling

UW–Madison researchers have developed a model liquid crystal-based system for studying the behavior and conformation of proteins at a surface or other interface. The system can be used to rapidly assess cleaning compositions for removing proteins or biofilms, agents for preventing adsorption of proteins and other molecules to surfaces and potential protein stabilizing agents.

The system includes one or more protein molecules disposed at a liquid crystal-aqueous interface. As proteins are removed from the interface, the liquid crystal surprisingly undergoes a continuous orientational ordering transition, which is correlated to the extent and speed of protein removal. By measuring this ordering transition, the model system can be used to rapidly assay the effectiveness of a given cleaning composition in removing proteins or biofilms from a surface.

In the model system, proteins that have aged or deformed are more difficult to remove from the interface and proteins that are crowded on the interface (i.e., less likely to be deformed) are easier to remove. Accordingly, the system also can be used to assay the state or conformation of proteins.

Improved Disulfide-Bond Reducing Agents

UW–Madison researchers have developed dithioamine reducing agents that can be prepared from inexpensive starting materials. The new reagent, dithiobutylamine (DTBA), is synthesized from the common amino acid, aspartic acid, by a short and simple route.

Solubilizing and Characterizing Membrane Proteins Using Tandem Facial Amphiphiles

UW–Madison researchers have developed tandem facial amphiphiles (TFAs) that can aid the solubilization, isolation, purification, stabilization, crystallization and structural determination of membrane proteins.

A membrane preparation containing the protein of interest is treated with TFA to achieve protein extraction and solubilization. The TFAs can contain a pair of maltose-functionalized deoxycholate units that are long enough to match the width of a lipid bilayer and form a sheath around the protein’s nonpolar surfaces. The TFAs can stabilize intrinsic membrane proteins in native-like conformations.

New Antimicrobials for Treating Bacterial Infection and Contamination

UW–Madison researchers have developed a lead compound and synthetic analogs that represent a new class of antimicrobial weapons.

The researchers identified a new family of small molecules from a high-throughput screen that are inhibitors of bacterial cell division. These compounds are toxic to a range of Gram-negative bacteria, including Escherichia coli, Caulobacter crescentus, Vibrio cholera, Shigella boydii and Acinetobacter baumannii. Compound treatment blocks the assembly and maturation of the divisome in bacteria and leads to the incomplete constriction of the cell division plane. The division process resumes once the compound is washed away.

New Tools for Solubilizing, Isolating and Characterizing Membrane Proteins

Researchers from UW–Madison and Stanford have now developed a family of synthetic carbohydrate-based amphiphiles, which are useful as tools for the manipulation of membrane proteins.  The new amphiphiles have novel chemical structures and are relatively easy to synthesize.  In addition, they exhibit favorable solubilization and stabilization properties in challenging biochemical systems, such as lipid bilayers, photosynthetic superassemblies and G protein-coupled receptors.

Controlling and Predicting the Stability of a Protein Against Degradation by Proteases

UW–Madison researchers have developed methods for predicting and controlling the stability of expressed polypeptides in prokaryotes, particularly mycobacteria like M. smegmatis.  They previously showed that DesA3 expressed in M. smegmatis with a modified C-terminal sequence had higher catalytic activity and stability than with the natural C-terminal sequence.  The researchers found that the identity of the last two or three amino acid residues of the C-terminus is a predictor and determinant of protein stability and resistance to proteolytic degradation. 

Specifically, altering one or more of the last three amino acid residues at the C-termini of polypeptides can make the proteins more stable during heterologous expression in mycobacterial hosts.  Identifying the last three residues also can be used to predict the relatively stability of proteins against degradation by proteases.

New Tools for Solubilizing, Isolating and Characterizing Membrane Proteins

UW-Madison researchers have developed new tools for solubilizing, isolating and characterizing membrane proteins.  Specifically, they developed synthetic amphiphiles that exhibit favorable solubilization and stabilization properties in biological systems, including lipid bilayers, photosynthetic superassemblies and G protein-coupled receptors.  The amphiphiles can feature carbohydrate-derived hydrophilic groups and branchpoints in the hydrophilic moiety or in a lipophilic moiety.  The invention also includes methods of using these amphiphiles to solubilize or stabilize a membrane protein.

Novel Antimicrobial Compounds

UW-Madison researchers have developed a method for identifying potential antibiotic compounds that block the association of bacterial SSB with a target protein. The inventors determined—for the first time—the high-resolution structure of the E. coli SSB segment bound to Exonuclease I, a target protein. They used this structure to develop a rapid fluorescence polarization method for measuring SSB-Exonuclease I binding in solution. This method was then used to identify small molecules that inhibit the interaction between bacterial SSB and its target proteins.

Because of the importance of protein interactions with SSB for bacterial viability and the high conservation of the SSB protein binding sequence across bacterial species, these molecules have potent broad spectrum antibacterial properties. Because the SSB peptide sequence is not found in human and other eukaryotic SSBs, these small molecules are also likely to be non-toxic to human cell lines. Together, these features make these compounds excellent candidates for novel, broad spectrum antibiotics.

Multidimensional Spectrometer

UW-Madison researchers have developed a spectrometer capable of measuring multidimensional spectra in a straightforward manner. The spectrometer utilizes a pulse shaper and a simple collinear geometry so that alignment is easy and data collection can be automated. Researchers can simply insert their samples and select one of several options for collecting 2D IR spectra. With this invention, along with some engineering and computer programming, the powerful new optical techniques are ready to be commercialized in a user-friendly apparatus that will have broad appeal among researchers.

Methods and Reagents for Appending Functional Groups to Proteins

UW-Madison researchers have developed a bifunctional molecule for the site-selective functionalization of peptides and proteins. This molecule can be used to covalently link a peptide or protein to a biological or chemical entity, such as a particular functional group, a reporter molecule, a biological molecule, a ligand that in turn binds a receptor, a small molecule like an antibiotic, or a biological/substrate surface. It has the formula H2N-NH-CH2-M-L-FG, where M is a single bond or a chemical group carrying a non-bonding electron pair, L is a linker, and FG is a functional group, such as an azido group (-N3), which has different reactivity than the hydrazino group (H2NHN-). The hydrazino group reacts with a thioester group at the C-terminus of the target peptide or protein to link the protein to the bifunctional molecule, while the azido group links to a functionalized surface or other chemical species.

Methods for Determining Protein Binding Specificity Using Peptide Libraries

UW-Madison researchers have developed a novel, high throughput method for determining deacetylase substrate specificity. The method uses a one-bead, one-compound (OBOC) acetyl-peptide library with a quantum dot tagging strategy and automated bead sorting. The library is screened using a deacetylase, such as a SIRT protein, to identify the most efficiently deacetylated sequences. Each bead is then labeled with a streptavidin-coated quantum dot. After fluorescent bead sorting, the brightest and most deacetylated beads can be sequenced via mass spectrometry.

Defined Surfaces of Self-Assembled Monolayers and Stem Cells

UW-Madison researchers have developed a method of identifying what peptides can be used to support a culture of hES cells. Self-assembled monolayer chemistry is used to create an array of alkane thiols on which peptide ligands attach in selected areas. Then cells, such as hES cells, are cultured with the monolayer as a substrate and the ligands that promote growth and self-renewal of cells are identifed.

Device That Rapidly Detects and Characterizes Interactions Between a Drug and Potential Protein Target

UW-Madison researchers have developed a device that detects and characterizes interactions between two molecules, such as a drug and a potential protein target, under conditions similar to in vivo conditions. This device can quickly detect transient interactions and requires little sample.

The device uses two laser beams, each with a frequency that can be tuned to the drug or the target biomolecule. If the drug binds to the protein target, it changes the signal beam. The resulting signal can then be measured to determine if the two molecules interact and to assess the strength of binding.

Protein-Acrylamide Copolymer Hydrogels for Measuring Protein Concentration and Activity

UW-Madison researchers have developed a method of specifically attaching proteins to glass surfaces via copolymerization with a polyacrylamide hydrogel. They also have developed techniques for using the arrays formed using this process to detect proteins and measure their concentrations, binding affinities and kinetics.

Their method uses a surface functionalized with an acrylic acid- or acrylamide-based hydrogel. Proteins are labeled with an acrylic moiety and then attached to the functionalized surface through copolymerization with the hydrogel. The attached protein is then available for use in a variety of assays.

Microfluidics Platform and Method That Mimic the Cellular Environment

UW-Madison researchers have developed a microfluidics-based platform for mimicking the environment within a cell. This model environment is simpler than a cell, yet captures the basic characteristics of the cellular nano-environment, including charge, crowding, water content and structure. It has many advantages over current systems, including the fact it uses much less protein, can detect weaker interactions and requires less time for experiments.

The platform includes a microfluidic device that contains a chamber. At least one hydrogel post is positioned within the chamber. Each post may contain a different density of polymers or a different cross-linker to simulate various crowding or caging effects. A solution containing proteins of interest is introduced into the chamber and the proteins diffuse into the hydrogel posts. The interactions between the proteins are then observed inside the posts.

Fluorescence Polarization Assay to Detect Protease Cleavage

UW-Madison researchers have developed a fluorescence polarization technology to measure protease activity in real time. Fluorescent polarization can be used to estimate the size of a protein to which a fluorescent complex is attached. In the method, an uncharacterized protein is conjugated to a fluorescence tag. The fluorescent tag exhibits little fluorescence until it binds to a specific fluorescent ligand to create a highly fluorescent complex. The complex comprising the protein, fluorescent tag and fluorescent ligand is then placed in contact with a protease. When the protease cleaves the protein into two or more fragments, the fluorescence polarization of the complex decreases. The rate of change in fluorescence polarization can be measured in real time, and is equivalent to the rate of protease cleavage.

Heterogeneous Protein Foldamers Containing Alpha, Beta and Gamma Amino Acids

UW-Madison researchers have developed polypeptide foldamers containing alpha-amino acids along with cyclically constrained beta-amino acids and gamma-amino acids. These unnatural compounds contain rotationally constrained amino acid residues that are not amenable to enzymatic degradation, making them useful to probe protein-protein and other large molecule interactions. Because the backbone is heterogeneous, a portion of the residues, such as the alpha-amino acids, can provide functional diversity, while the cyclically constrained residues confer conformational specificity and stability.

Method and Device for Measuring Electrical Conductance of Membranes with a Radio Frequency Probe

A UW-Madison researcher has developed a method for measuring the conductance of single channels from biochemical membranes such as supported bilayers and cell membranes. The method uses a probe that creates a highly localized radio frequency field that interacts with the membrane. The sharp-tipped probe, which may have an inner core tip surrounded by a coaxial shield, is positioned adjacent to the exposed surface of the biochemical membrane. Radio frequency power is supplied to the probe to apply the radio frequency field to the membrane. Channel protein activities, such as transport and binding, are detected by changes in the electromagnetic field transmitted through the membrane.

Microwave Dielectric Spectroscopy Method and Device

UW-Madison researchers have developed a method of using dielectric spectroscopy to detect protein conformational changes. This alternative method relies on the fact that proteins in solution are surrounded by one or more shells of “bound” water. In response to changes in a protein’s conformation, bound water is released or rearranged, causing a change in the solution’s permittivity that can be easily measured by using this invention.

Previous dielectric spectroscopy methods have not been widely implemented because they involve complicated analysis. In this invention, data analysis is as simple as in conventional optical spectroscopy. In addition, no labeling of receptor or ligand is required for detection.

Modified Strains PJ69-7A and PJ69-7B for the Yeast Two-Hybrid System

A UW-Madison researcher has developed two strains, known as PJ69-7A and PJ69-7B, that eliminate many of the problems associated with false positives. In addition, the strains include both mating types, allowing high throughput screening.

Predicting Protein Hot Spot Residues

UW–Madison researchers have created the most accurate program ever developed to predict hot spot residues in proteins and model the effects of alanine substitution on each of the amino acids. The program uses a modified Knowledge-based FADE and Contacts (or KFC2) approach.

Strain and Vector for Use with the Three Hybrid System

UW-Madison researchers have corrected this problem by the addition of the screening marker ade2 onto the plasmids containing the hybrid RNA and the ura3 marker.  They created a yeast cell line containing the sequence Lex A-CP-CP (where CP is coat protein) in its genome, and plasmids that encode a hybrid RNA molecule and ura3 as a selectable marker.

The researchers have utilized this three hybrid system to detect RNA-protein interactions as measured by reporter function activity, such as transcription of a his3 reporter gene. The most common application is one in which the RNA is known and interacting proteins unknown. In this case, a library of cDNAs is screened to detect a protein of interest.