A comprehensive system for protein purification and biochemical analysis based on antibodies to c-myc peptide.

The genomics revolution has created a need for increased speed and generality for recombinant protein production systems as well as general methods for conducting biochemical assays with the purified protein products. 9E10 is a well-known high-affinity antibody that has found use in a wide variety of biochemical assays. Here we present a standardized system for purifying proteins with a simple epitope tag based on c-myc peptide using an antibody affinity column. Antibodies with binding parameters suitable for protein purification have been generated and characterized. To purify these antibodies from serum-containing medium without carrying through contaminating immunoglobulin G, a peptide-based purification process was developed. A fluorescence polarization binding assay was developed to characterize the antigen-antibody interaction. Protein purification protocols were optimized using a fluorescein-labeled peptide as a surrogate "protein." Binding and elution parameters were evaluated and optimized and basic operating conditions were defined. Several examples using this procedure for the purification of recombinant proteins are presented demonstrating the generality of the system. In all cases tested, highly pure final products are obtained in good yields. The combination of the antibodies described here and 9E10 allow for almost any biochemical application to be utilized with a single simple peptide tag.

Structural consequences of an amino acid deletion in the B1 domain of protein G.

We describe the NMR structure of a deletion mutant of the B1 IgG-binding domain from Group G Streptococcus. The deletion occurs within the last beta-strand of the protein, where it may potentially have a deleterious effect on the stability of the protein if the protein were not able to conformationally adjust to the perturbation. In particular, the deletion changes the registry of the final three residues in the sheet, forcing a polar Thr to be buried in the interior of the protein and exposing a hydrophobic Val to solvent. The deletion could also potentially create a large cavity in the beta-sheet and force the alpha- and gamma-carboxylates of the C-terminal Glu residue into a partially buried region of the sheet. The structure of the mutant illustrates how the conformation of the protein adjusts to the deletion, thereby mitigating some of the potentially deleterious consequences. Although the elements of secondary structure are retained between the mutant and the wt domain, there are multiple small adjustments in the segments connecting secondary structure elements. In particular, a hydrogen bond between the Glu57 carboxylates and two main chain amides is introduced that alters the conformation in the loop connecting the helix to strand 3. In addition, to minimize hydrophobic surface exposure, the turn connecting strands 1 and 2 folds toward the core so that the molecular volume is decreased.

Aspartyl beta -hydroxylase (Asph) and an evolutionarily conserved isoform of Asph missing the catalytic domain share exons with junctin.

The mouse aspartyl beta-hydroxylase gene (Asph, BAH) has been cloned and characterized. The mouse BAH gene spans 200 kilobase pairs of genomic DNA and contains 24 exons. Of three major BAH-related transcripts, the two largest (6,629 and 4,419 base pairs) encode full-length protein and differ only in the use of alternative polyadenylation signals. The smallest BAH-related transcript (2,789 base pairs) uses an alternative 3' terminal exon, resulting in a protein lacking a catalytic domain. Evolutionary conservation of this noncatalytic isoform of BAH (humbug) is demonstrated in mouse, man, and Drosophila. Monoclonal antibody reagents were generated, epitope-mapped, and used to definitively correlate RNA bands on Northern blots with protein species on Western blots. The gene for mouse junctin, a calsequestrin-binding protein, was cloned and characterized and shown to be encoded from the same locus. When expressed in heart tissue, BAH/humbug preferably use the first exon and often the fourth exon of junctin while preserving the reading frame. Thus, three individual genes share common exons and open reading frames and use separate promoters to achieve differential expression, splicing, and function in a variety of tissues. This unusual form of exon sharing suggests that the functions of junctin, BAH, and humbug may be linked.

An early intermediate in the folding reaction of the B1 domain of protein G contains a native-like core.

The folding kinetics of a 57-residue IgG binding domain of streptococcal protein G has been studied under varying solvent conditions, using stopped-flow fluorescence methods. Although GB1 has been cited as an example of a protein that obeys a two-state folding mechanism, the following kinetic observations suggest the presence of an early folding intermediate. Under stabilizing conditions (low denaturant concentrations, especially in the presence of sodium sulfate), the kinetics of folding shows evidence of a major unresolved fluorescence change during the 1.5 ms dead time of the stopped-flow experiment (burst phase). Together with some curvature in the rate profile for the single observable folding phase, this provides clear evidence of the rapid formation of compact states with native-like fluorescence for the single tryptophan at position 43. In refolding experiments at increasing denaturant concentrations, the amplitude of the sub-millisecond phase decreases sharply and the corresponding slope (m value) is only about 30% lower than that of the equilibrium unfolding curve indicative of a pre-equilibrium transition involving cooperative unfolding of an ensemble of compact intermediates. The dependence on guanidine hydrochloride concentration of both rates and amplitudes (including the equilibrium transition) is described quantitatively by a sequential three-state mechanism, U [symbol: see text] I [symbol: see text] N, where an intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-limiting formation of the native state (N). A 66-residue fragment of GB1 with an N-terminal extension containing five apolar side chains exhibits three-state kinetic behavior virtually identical to that of the 57-residue fragment. This is consistent with the presence of a well-shielded native-like core excluding the N-terminal tail in the early folding intermediate and argues against a mechanism involving random hydrophobic collapse, which would predict a correlation between overall hydrophobicity and stability of compact states.

Expression of de novo designed alpha-helical bundles.

The successful design of proteins requires careful consideration of the multiplicity of forces that stabilize their three-dimensional structures including hydrophobic interactions, hydrogen-bonding, electrostatics and weakly polar interactions. Early attempts to design proteins relied too heavily on hydrophobic interactions to provide stability, resulting in structures with dynamic properties. Addition of more specific interactions to these initial designs gives rise to proteins with more native-like properties. This manuscript describes the design of native-like three- and four-helix bundles, and their cloning and expression of these proteins.

Protein design: a hierarchic approach.

The de novo design of peptides and proteins has recently emerged as an approach for investigating protein structure and function. Designed, helical peptides provide model systems for dissecting and quantifying the multiple interactions that stabilize secondary structure formation. De novo design is also useful for exploring the features that specify the stoichiometry and stability of alpha-helical coiled coils and for defining the requirements for folding into structures that resemble native, functional proteins. The design process often occurs in a series of discrete steps. Such steps reflect the hierarchy of forces required for stabilizing tertiary structures, beginning with hydrophobic forces and adding more specific interactions as required to achieve a unique, functional protein.

Phage display: protein engineering by directed evolution.

Phage display of proteins has become an important tool for protein engineering. Over the past year, the versatility of the technology has expanded to include the development of DNA-binding proteins with novel specificities, energetics of protein folding and directed evolution of antibodies. In addition, display of expressed cDNA libraries opens an exciting opportunity for studying protein-protein interactions.

Thermodynamic genetics of the folding of the B1 immunoglobulin-binding domain from streptococcal protein G.

A method has been developed to select proteins that are thermodynamically destabilized yet still folded and functional. The DNA encoding the B1 IgG-binding domain from Group G Streptococcus (Strp G) has been fused to gene III of bacteriophage M13. The resulting fusion protein is displayed on the surface of the phage thus enabling the phage to bind to IgG molecules. In addition, these phage exhibit a small plaque phenotype that is reversed by mutations that destabilize the Strp G domain. By selecting phage with large plaque morphology that retain their IgG-binding function, it is possible to identify mutants that are folded but destabilized compared with wild-type Strp G. Such mutants can be divided into three general categories: 1) those that disrupt packing of hydrophobic side chains in the protein interior; 2) those that destabilize secondary structure; and 3) those that alter specific hydrogen bonds involving amino acid side chains. A number of the mutants have been physically characterized by circular dichroism and nuclear magnetic resonance and have been shown to have structures similar to wild-type Strp G but stabilities that were decreased by 2-5 kcal/mol.

Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library.

Methods have recently been developed to present vast libraries of random peptides on the surface of filamentous phage. To introduce a degree of conformational constraint into random peptides, a library of hexapeptides flanked by cysteine residues (capable of forming cyclic disulfides) was constructed. This library was screened using the platelet glycoprotein, IIb/IIIa, which mediates the aggregation of platelets through binding of fibrinogen. A variety of peptides containing the sequence Arg-Gly-Asp or Lys-Gly-Asp were discovered and synthesized. The cyclic, disulfide-bonded forms of the peptides bound IIb/IIIa with dissociation constants in the nanomolar range, while reduced forms or an analogue in which Ser replaced the Cys residues bound considerably less tightly. These results demonstrate the feasibility for introducing conformational constraints into random peptide libraries and also demonstrates the potential for using phage peptide libraries to discover pharmacologically active lead compounds.

DNA-induced increase in the alpha-helical content of C/EBP and GCN4.

Leucine zipper proteins comprise a recently identified class of DNA binding proteins that contain a bipartite structural motif consisting of a "leucine zipper" dimerization domain and a segment rich in basic residues responsible for DNA interaction. Protein fragments encompassing the zipper plus basic region domains (bZip) have previously been used to determine the conformational and dynamic properties of this motif. In the absence of DNA, the coiled-coil portion is alpha-helical and dimeric, whereas the basic region is flexible and partially disordered. Addition of DNA containing a specific recognition sequence induces a fully helical conformation in the basic regions of these fragments. However, the question remained whether the same conformational change would be observed in native bZip proteins where the basic regions might be stabilized in an alpha-helical conformation even in the absence of DNA, through interactions with portions of the protein not included in the bZip motif. We have now examined the DNA-induced conformational transition for an intact bZip protein, GCN4, and for the bZip fragment of C/EBP with two enhancers that are differentially symmetric. Our results are consistent with the induced helical fork model wherein the basic regions are largely flexible in the absence of DNA and become fully helical in the presence of the specific DNA recognition sequence.

A thermodynamic scale for the helix-forming tendencies of the commonly occurring amino acids.

Amino acids have distinct conformational preferences that influence the stabilities of protein secondary and tertiary structures. The relative thermodynamic stabilities of each of the 20 commonly occurring amino acids in the alpha-helical versus random coil states have been determined through the design of a peptide that forms a noncovalent alpha-helical dimer, which is in equilibrium with a randomly coiled monomeric state. The alpha helices in the dimer contain a single solvent-exposed site that is surrounded by small, neutral amino acid side chains. Each of the commonly occurring amino acids was substituted into this guest site, and the resulting equilibrium constants for the monomer-dimer equilibrium were determined to provide a list of free energy difference (delta delta G degree) values.

Design of DNA-binding peptides based on the leucine zipper motif.

A class of transcriptional regulator proteins bind to DNA at dyad-symmetric sites through a motif consisting of (i) a "leucine zipper" sequence that associates into noncovalent, parallel, alpha-helical dimers and (ii) a covalently connected basic region necessary for binding DNA. The basic regions are predicted to be disordered in the absence of DNA and to form alpha helices when bound to DNA. These helices bind in the major groove forming multiple hydrogen-bonded and van der Waals contacts with the nucleotide bases. To test this model, two peptides were designed that were identical to natural leucine zipper proteins only at positions hypothesized to be critical for dimerization and DNA recognition. The peptides form dimers that bind specifically to DNA with their basic regions in alpha-helical conformations.

How calmodulin binds its targets: sequence independent recognition of amphiphilic alpha-helices.

Calmodulin (CaM) is a protein capable of recognizing positively charged, amphiphilic alpha-helical peptides independent of their precise amino acid sequences; this structural feature has also been found in many CaM-binding proteins. Recent work involving crystallography and site-directed mutagenesis of CaM along with studies of photoreactive and fluorescent CaM-binding peptides have helped define how calmodulin interacts with amphiphilic helices.

Photolabeling of calmodulin with basic, amphiphilic alpha-helical peptides containing p-benzoylphenylalanine.

A novel photoreactive amino acid has been incorporated synthetically into two model peptides and the calmodulin-binding domain from myosin light chain kinase. Cross-linked photoadducts of each peptide with calmodulin have been prepared and digested by chemical and/or enzymatic methods to determine the site of label attachment. Depending on the position of the photoprobe in the peptide sequence, either Met-144 or Met-71 is photolabeled. These results are discussed in relation to the three-dimensional structure of calmodulin obtained crystallographically and the known solution properties of calmodulin.

The interaction of calmodulin with fluorescent and photoreactive model peptides: evidence for a short interdomain separation.

Calmodulin is known to bind target enzymes and basic, amphiphilic peptides in a Ca2(+)-dependent manner. Recently, we introduced a photoaffinity label, p-benzoylphenylalanine (Bpa), into the sequence of a model, alpha-helical, calmodulin-binding peptide. When the Bpa residue was introduced at the third position of the peptide, Met-144 on the C-terminal domain of calmodulin was labeled, whereas when the photolabel was placed at the thirteenth position, Met-71 on the N-terminal domain was labeled. Assuming that both peptides bind in similar orientations, these results are not consistent with the crystal structure of calmodulin, in which the domains are held at a significant distance from one another by a long alpha-helical segment. To test the assumption that both peptides bind in similar orientations, we have synthesized a calmodulin-binding peptide with the photolabel in both the third and the thirteenth positions. Upon photolysis, this peptide forms a cross-link between Met-71 and Met-124 on the N- and C-terminal domains, respectively. Furthermore, a peptide with a Bpa in the thirteenth position and a Trp residue in the third position was also synthesized. After photocross-linking the Bpa residue of this peptide to Met-71 of calmodulin, it could be shown that the fluorescence properties of the Trp residue were consistent with its side chain being buried in a hydrophobic pocket on the C-terminal domain of calmodulin. These data indicate that, when complexed with basic, amphiphilic peptides, calmodulin can adopt a conformation in which its two domains are significantly closer than in the crystal structure of the uncomplexed protein.

Fluorescence properties of calmodulin-binding peptides reflect alpha-helical periodicity.

A basic amphiphilic alpha-helix is a structural feature common to many calmodulin-binding peptides and proteins. A set of fluorescent analogues of a very tight binding inhibitor (dissociation constant of 200 picomolar) of calmodulin has been synthesized. The fluorescent amino acid tryptophan has been systematically moved throughout the sequence of this peptide. The fluorescence properties for the peptides repeat every three to four residues and are consistent with the periodicity observed for an alpha-helix.

Predicted calmodulin-binding sequence in the gamma subunit of phosphorylase b kinase.

A basic, amphiphilic alpha helix is a structural feature common to a variety of inhibitors of calmodulin and to the calmodulin-binding domains of myosin light chain kinases. To aid in recognizing this structural feature in sequences of peptides and proteins we have developed a computer algorithm which searches for sequences of appropriate length, hydrophobicity, helical hydrophobic moment, and charge to be considered as potential calmodulin-binding sequences. Such sequences occurred infrequently in proteins of known crystal structure. This algorithm was used to find the most likely site in the catalytic (gamma) subunit of phosphorylase b kinase for interaction with calmodulin (the delta subunit). A peptide corresponding to this site (residues 341-361 of the gamma subunit) was synthesized and found to bind calmodulin with approximately an 11 nM dissociation constant. A variant of this peptide in which an aspartic acid at position 7 in its sequence (347 of the gamma subunit) was replaced with an asparagine was found to bind calmodulin with approximately a 3 nM dissociation constant.

A predicted structure of calmodulin suggests an electrostatic basis for its function.

By using interactive computer graphics, two models for calmodulin have been constructed based on the structures of two functionally and structurally related proteins, intestinal calcium-binding protein and carp parvalbumin. The two models have been compared and contrasted to the parent proteins with respect to proportion of solvent-exposed hydrophobic residues, solvent-accessible surface area, and side-chain packing. Electrostatic potential surfaces generated for the models suggest a probable binding site for basic amphiphilic alpha-helical peptides located between the last E and F helices in the second domain of calmodulin. Both electrostatic and hydrophobic complementarity can contribute to stabilization of a peptide-protein complex in this region.