Site-specific immobilization of proteins in a microarray using intein-mediated protein splicing

One of the critical issues in the generation of a protein microarray lies in the choice of immobilization strategies, which ensure proteins are adhered to the glass surface while properly retaining their native biological activities. Herein, we report a bacterium-based, intein-mediated strategy to generate N-terminal cysteine-containing proteins which are then chemoselectively immobilized to a thioester-functionalized glass slide to generate the corresponding protein microarray. We also showed preliminary data of the strategy in a yeast host system.     

Site-specific pegylation of G-CSF by reversible denaturation

A new strategy has been developed for extending the possibility of poly(ethylene glycol) (PEG) modification to accessible thiol groups of biologically active proteins. In particular, thiol-reactive PEGs have been coupled to the cysteine 17 of granulocyte colony stimulating factor (G-CSF), which is known to be partially buried in a hydrophobic protein pocket. The PEG linking was accomplished by partial protein denaturation with 3 M guanidine.HCl in the absence of any reducing agent in order to preserve the native protein's disulfide bridges. PEG coupling occurred also, but at a lower degree, by using a 3 M solution of urea as the denaturing agent. Following the PEGylation, which was carried out in the unfolded state, the conjugated protein was refolded using dialysis or gel filtration chromatography to eliminate the denaturant. Different thiol-reactive PEGs and polymer molecular weights (5, 10, or 20 kDa) were investigated for G-CSF conjugation under denaturation. The secondary structure of the protein in the G-CSF-PEG conjugates, evaluated using circular dichroism and biological activity assay in cell culture, was maintained with respect to the native protein. Unexpectedly, conjugation enhanced the G-CSF tendency to aggregate, a problem that was overcome by a proper formulation.     

Site-specific incorporation of arginine analogs into proteins using arginyl-tRNA synthetase

Arginine analogs were incorporated site-specifically into proteins using an in vitro translation system. In this system, mRNAs containing a CGGG codon were translated by an aminoacyl-tRNA_CCCG, which was charged with arginine analogs using yeast arginyl-tRNA synthetase. N^G-monomethyl-l-arginine, l-citrulline and l-homoarginine were incorporated successfully into proteins using this method. The influence of arginine monomethylation in histone H3 on the acetylation of lysine residues by histone acetyltransferase hGCN5 was investigated, and the results demonstrated that K9 acetylation was suppressed by the methylation of R8 and R17 but not by R26 methylation. K18 acetylation was not affected by the methylation of R8, R17 and R26. This site-specific modification strategy provides a way to explore the roles of post-translational modifications in the absence of heterogeneity due to other modifications.     

Tollip and Tom1 form a complex and recruit ubiquitin-conjugated proteins onto early endosomes

Tom1 (target of Myb1) is a protein of unknown function. Tom1 and its relative Tom1L1 have an N-terminal VHS (Vps27p/Hrs/Stam) domain followed by a GAT (GGA and Tom1) domain, both of which are also found in the GGA (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor-binding protein) family of proteins. Although the VHS and GAT domains of GGA proteins bind to transmembrane cargo proteins and the small GTPase ADP-ribosylation factor, respectively, the VHS and GAT domains of Tom1 are unable to interact with these proteins. In this study, we show that the GAT domains of Tom1 and Tom1L1 interact with ubiquitin and Tollip (Toll-interacting protein). Ubiquitin bound the GAT domains of Tom1, Tom1L1, and GGA proteins, whereas Tollip interacted specifically with Tom1 and Tom1L1. Ubiquitin and Tollip bound to an overlapping region of the Tom1-GAT domain in a mutually exclusive manner. Tom1 was predominantly cytosolic when expressed in cells. On the other hand, Tollip was localized on early endosomes and recruited Tom1 and ubiquitinated proteins. These observations suggest that Tollip and Tom1 form a complex and regulate endosomal trafficking of ubiquitinated proteins.     

Traceless and Site-specific Attachment of Proteins onto Solid Supports

Many experimental approaches in biology and biophysics, as well as applications in diagnosis and drug discovery, require proteins to be immobilized on solid supports. Protein microarrays, for example, provide a high-throughput format to study biomolecular interactions. The technique employed for protein immobilization is a key to the success of these applications. Recent biochemical developments are allowing, for the first time, the selective and traceless immobilization of proteins generated by cell-free systems without the need for purification and/or reconcentration prior to the immobilization step.     

Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex

Microtubule assembly is initiated by the gamma-tubulin ring complex (gamma-TuRC). In yeast, the microtubule is nucleated from gamma-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors gamma-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with gamma-tubulin through binding with gamma-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and gamma-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with gamma-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring gamma-TuRC.     

Identification of an heterologous histone complex using reversible crosslinking

Using formaldehyde as a reversible crosslinker, it has been shown that an heterologous dimer is the principal components in a mixture of calf thymus H2A and sea urchin H2B₁. Comparison of the crosslinking ability of formaldehyde with dimethyl suberimidate demonstrates that suberimidate gives a better representation of the free-solution equilibrium of the protein mixture.     

Site-specific covalent labeling of proteins inside live cells using small molecule probes

The study of dynamic movement and interactions of proteins inside living cells in real time is critical for a better understanding of cellular mechanisms and functions in molecular detail. Genetically encoded fusions to fluorescent protein(s) (FP) have been widely used for this purpose [Annu. Rev. Biochem. 1998, 67, 509-544]. To obviate some of the drawbacks associated with the use of FPs [Curr. Opin. Biotechnol. 2005, 16, 1-6; Nat. Methods2006, 3, 591-596], we report a small molecule-based approach that exploits the unique reactivity between the cysteine residue at the N-terminus of a target protein and cell-permeable, thioester-based small molecule probes resulting in site-specific, covalent tagging of proteins. This approach has been demonstrated by the in vivo labeling of proteins in both bacterial and mammalian systems thereby making it potentially useful for future bioimaging applications.     

Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase

We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction. Because ketones are absent from native cell surfaces, AP-fused recombinant cell surface proteins can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage protein labeling methodology on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor (EGFR) expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe are incorporated, with total labeling times as short as 20 min.     

Site-specific polymer modification of therapeutic proteins

Recent advances in chemoselective ligation technology have made possible the modification of proteins with polymers in a site-specific and controlled manner. These approaches rely on the incorporation of chemoselective anchors into the protein backbone by either chemical or recombinant means, and subsequent modification with a polymer carrying a complementary linker. As a result, the assembly process and the covalent structure of the resulting protein-polymer conjugate are completely controlled, enabling the rational optimization of drug properties, in particular efficacy and pharmacokinetic properties. Application of chemoselective ligation technologies to cytokines and chemokines has led to the generation of new lead proteins for use as erythropoietic agents and HIV fusion inhibitors.     

Structure of a cellulose II-hydrazine complex

The structure of a crystalline cellulose II-hydrazine complex has been determined by x-ray diffraction methods as part of an investigation of cellulose-solvent interaction. The complex studied was that formed when Fortisan fibers were swollen in hydrazine and then vacuumdried. The unit cell is monoclinic with dimensions a = 9.37 Å, b = 19.88 Å, c = 10.39 Å, and γ = 120.0° and contains disaccharide segments of four chains, with one hydrazine per glucose residue. In view of the limited x-ray intensity data, the structure has been determined based on an approximate unit cell containing two chain segments, with a = 4.69 Å, using the linked-atom least-squares refinement procedures. The refined model contains antiparallel cellulose chains that are linked by both intermolecular hydrogen bonds and hydrogen-bonded hydrazine molecules. The parallel chains in the 020 planes are packed in register, leading to stacks of chains analogous to those in chitin. All the hydroxyl groups are satisfactorily hydrogen-bonded, and each hydrazine forms four donor and two acceptor hydrogen bonds, including an N? H…N bond between hydrazines. From this work it can be seen that the interaction of cellulose II with hydrazine involves scission of the intermolecular hydrogen bonds followed by disruption of the stacks of quarter-staggered chains. The latter effect is probably necessary for hydrazine to act as a cellulose solvent.     

Labeling of specific cysteines in proteins using reversible metal protection

Fluorescence spectroscopy is an indispensible tool for studying the structure and conformational dynamics of protein molecules both in isolation and in their cellular context. The ideal probes for monitoring intramolecular protein motions are small, cysteine-reactive fluorophores. However, it can be difficult to obtain specific labeling of a desired cysteine in proteins with multiple cysteines, in a mixture of proteins, or in a protein's native environment, in which many cysteine-containing proteins are present. To obtain specific labeling, we developed a method we call cysteine metal protection and labeling (CyMPL). With this method, a desired cysteine can be reversibly protected by binding group 12 metal ions (e.g., Cd²⁺ and Zn²⁺) while background cysteines are blocked with nonfluorescent covalent modifiers. We increased the metal affinity for specific cysteines by incorporating them into minimal binding sites in existing secondary structural motifs (i.e., α-helix or β-strand). After the metal ions were removed, the deprotected cysteines were then available to specifically react with a fluorophore.     

Site-specific, covalent attachment of proteins to a solid surface

Immobilized and site-specifically labeled proteins are becoming invaluable tools in proteomics. Here, we describe a strategy to attach a desired protein to a solid surface in a covalent, site-specific manner. This approach employs an enzymatic posttranslational modification method to site-specifically label a target protein with an azide; an alternative substrate for protein farnesyl transferase containing an azide group was developed for this purpose. A bio-orthogonal Cu(I)-catalyzed cycloaddition reaction is then used to covalently attach the protein to agarose beads bearing an alkyne functional group. We demonstrate that both the azide incorporation and the capture steps can be performed on either a purified protein target or on a protein present within a complex mixture. This approach involves the use of a four-residue tag which is significantly smaller than most other tags reported to date and results in covalent immobilization of the target protein. Hence it should have significant applicability in protein science.     

Site-specific incorporation of PEGylated amino acids into proteins using nonnatural amino acid mutagenesis

Site-directed incorporation of PEGylated nonnatural amino acids with 4, 8, and 12 repeated ethylene glycol units was examined in a cell-free translation system. PEGylated aminophenylalanine derivatives were successfully incorporated into proteins, whereas PEGylated lysines were not. The incorporation efficiency of the PEGylated amino acids decreased with an increase in PEG chain length. The present method will be useful for preparation of proteins which are PEGylated in a site-specific and quantitative manner.     

Free energy of charges in solvated proteins: microscopic calculations using a reversible charging process

Evaluation of the free energy of ionization of acidic groups in proteins may be used as a powerful and general test case for determining the reliability of calculations of electrostatic energies in macromolecules. This work attacks this test case by using an adiabatic charging process that evaluates the changes in free energies associated with ionizing the acidic groups Asp-3 and Glu-7 in bovine pancreatic trypsin inhibitor and aspartic acid in solution. The results of these free energy calculations are very encouraging; the error range is about 1 kcal/mol for these free energy changes of about-70 kcal/mol. This indicates that we are finally approaching the stage of obtaining quantitative results in modeling the energetics of solvated proteins.     

Structure and thermal behavior of a cellulose I-ethylenediamine complex

We prepared highly crystalline samples of a cellulose I-ethylenediamine (EDA) complex by immersing oriented films of algal (Cladophora) cellulose microcrystals in EDA at room temperature for a few days. The unit-cell parameters were determined to be a = 0.455, b = 1.133, and c = 1.037 nm (fiber repeat) and gamma = 94.02 degrees. The space group was P2(1). On the basis of unit cell, density, and thermogravimetry analyses, the asymmetric unit is composed of one anhydrous glucose residue and one EDA molecule. The chemical and thermal stabilities of the cellulose I-EDA complex were also investigated by the use of X-ray diffraction. When the cellulose I-EDA complex was immersed in methanol or water at room temperature, cellulose III I or I beta was obtained, respectively. However, immersion in a nonpolar solvent such as toluene did not affect the crystal structure of the complex. The cellulose I-EDA complex was stable up to a temperature of approximately 130 degrees C, whereas the boiling point of EDA is 117 degrees C. This thermal stability of the complex is probably caused by intermolecular hydrogen bonds between EDA molecules and cellulose. When heated above 150 degrees C, the cellulose I-EDA complex decomposed into cellulose I beta.     

Reagents for reversible coupling of proteins to the active centres of trypsin-like serine proteinases.

In order to extend the scope for the application of acyl-enzymes as fibrinolytic agents, p-amidinophenyl ester enzyme substrates were prepared that gave stabilized acyl-enzymes capable of being coupled to other proteins. Coupling was achieved by reaction of protein thiol functions with a 2-pyridyldithio moiety within the acyl group. Acyl-enzymes derived from such substrates were stable enough to permit isolation of reversible conjugates between proteins and the active centres of plasminogen activators. Hydrolytic release of active enzyme from a conjugate of human immunoglobulin G and urokinase could be demonstrated.