Chimeric genes containing the coding sequence for bacterial chloramphenicol acetyl transferase (CAT) have been introduced by electroporation into maize protoplasts (Black Mexican Sweet) and transient expression monitored by enzyme assays. Levels of CAT expression were enhanced 12-fold and 20-fold respectively by the inclusion of maize alcohol dehydrogenase-1 introns 2 and 6 in the chimeric construct. This enhancement was seen when the intron was placed within the 5′ translated region but not when it was located upstream of the promoter or within the 3′ untranslated region. Deletion of exon sequences adjacent to intron 2 abolished its ability to mediate enhancement of CAT gene expression. Northern analysis of protoplasts electroporated with intron constructs revealed elevated levels of CAT mRNA. However, this elevation was insufficient to account for the increased enzyme activity. One explanation of these results is that splicing affects both the quantity of mRNA.

Intron-mediated enhancement of heterologous gene expression in maize.

The level of intracellular calcium is strictly regulated in all cells. In a resting cell, the [Ca2+] is < or = 10(-7) M and during activation it rises to approximately 10(-6) M. Calmodulin (CaM) is the secondary messenger protein that has to translate this modest rise in intracellular calcium into a physiological response in all eukaryotic cells. CaM can activate almost 30 different target systems, including smooth muscle contraction, protein kinases and phosphatases, nitric oxide synthases, and calcium-extruding pumps. It is an acidic protein of 148 amino acids with four helix-loop-helix calcium-binding domains and it has a characteristic dumbbell shape in the crystal structure. In this review I discuss which features of CaM allow it to be such a universal and versatile calcium regulator. First of all, the positive cooperative calcium binding to all four binding sites of CaM in the presence of a target protein allows the protein to act effectively during a calcium transient. Secondly, the high Met content of two hydrophobic surface patches on the two domains of CaM creates a flexible and pliable, yet sticky, interaction surface that does not place high demands on the specificity of the interaction. Consequently, calcium-CaM can bind effectively to the CaM-binding domains of all its target proteins, despite their lack of amino acid sequence homology; their only common feature is that they are hydrophobic basic peptides that have a propensity to form an alpha-helix. CaM's capacity to recognize its CaM-binding domains is further enhanced by its third crucial feature, the intrinsic flexibility of the central linker region; this allows the two domains of CaM to slide over the surface of the alpha-helical bound peptide, to find their most favourable binding orientation. In this review I have also presented selected examples of a variety of experimental techniques that have contributed to our understanding of this unique multitasking protein. These include studies with well-established techniques such as site-directed mutagenesis, chemical modification, limited proteolysis, circular dichroism, and two-dimensional nuclear magnetic resonance (NMR), as well as novel or less common approaches involving the use of unnatural amino acids, metal-ion NMR, lysine pKa determinations, and isotope-edited Fourier transform infrared spectroscopy. In combination with available structural information, these studies have provided considerable detail in our understanding of this versatile calcium regulatory protein.

Calmodulin: a versatile calcium mediator protein

Here we show that, as a consequence of binding the drug trifluoperazine, a major conformational movement occurs in Ca(2+)-calmodulin (CaM). The tertiary structure changes from an elongated dumb-bell, with exposed hydrophobic surfaces, to a compact globular form which can no longer interact with its target enzymes. It is likely that inactivation of Ca(2+)-CaM by trifluoperazine is due to this major tertiary-structural alteration in Ca(2+)-CaM, which is initiated and stabilized by drug binding. This conformational change is similar to that which occurs on the binding of Ca(2+)-CaM to target peptides. Two hydrophobic binding pockets, created by amino acid residues adjacent to Ca(2+)-coordinating residues, form the key recognition sites on Ca(2+)-CaM for both inhibitors and target enzymes.

Trifluoperazine-induced conformational change in Ca(2+)-calmodulin

We present a rapid, sensitive enzymatic assay for chloramphenicol acetyltransferase (CAT) that does not require chromatography, HPLC, or autoradiography. The assay is based on the use of an inexpensive substrate, tritiated acetate, instead of [14C]chloramphenicol. The method is adapted from one originally used by de Crombrugghe et al. (1973) and by Shaw (1975), but with simplifications appropriate for routine use. In our hands, the method is as sensitive as the customary thin-layer chromatography assay and is far more efficient for the performance of many assays, both in terms of labor and expense.

A rapid, sensitive, and inexpensive assay for chloramphenicol acetyltransferase.

The development of materials that undergo shape changes occupies a central theme in materials science and has proven important in several applications. Several classes of hydrogels, which are cross-linked water-soluble polymers, can change their properties (such as, volume, cross-link density) in response to temperature, pH value, or ionic strength. These dynamic hydrogels can also be modified with biochemical moieties to give materials that change their properties in response to proteins and ligands. 6] For example, hydrogels that undergo volume changes because of antigen–antibody and lectin–carbohydrate interactions have been used as biosensors. The underlying principle of operation of dynamic hydrogel materials relates to a change in their physical or chemical cross-linking density in response to environmental cues. An unexplored alternative to these approaches relies on the use of a natural protein that undergoes a conformational change as a mechanism to alter the characteristics of a material. The functional importance of protein motions in biological systems, together with the wide range of protein motions that can be harnessed, offers a flexible approach to the preparation of dynamic materials. We describe herein an example of a protein-based dynamic material, the functional nature of which is derived from the conformational properties of the protein calmodulin (CaM). Calmodulin is a 16.5-kDa protein with two distinct conformational states (Figure 1). In the presence of calcium ions, CaM has an extended, dumbbell-shaped conformation (herein termed “extended CaM”). This calciumbound CaM undergoes a transition from an extended dumbbell to a collapsed conformation (herein termed “collapsed CaM”) upon the binding of ligands, which include certain antipsychotic drugs (such as, trifluoperazine (TFP)), 13,16] peptides, and a variety of proteins . Recently, Daunert and co-workers described a class of hydrogels that incorporate CaM and a small-molecule ligand as pendant moieties within the network. The binding of the CaM units and ligands resulted in an increased cross-linking density and a decreased swelling of the network. This approach is analogous to the development of dynamic hydrogels based on antigen–antibody and lectin–carbohydrate interactions, but differs from our approach in that the dynamic response of the gel is not due primarily to the conformational properties of CaM. We prepared an engineered version of CaM in which cysteine residues are included in place of tyrosine residues at the ends of the dumbbell-shaped protein (CaM Y34C, Y110C). The distance that separates the two cysteine residues is approximately 50 : in the extended conformation, but is decreased to approximately 15 : in the collapsed conformation. We incorporated the CaM building blocks into a poly(ethylene glycol)-based hydrogel by using a four-armed poly(ethylene glycol) (PEG) molecule terminated at each end with an acrylate group. The acrylate groups react selectively with the sulfhydryl groups on the engineered CaM and therefore serve to cross-link the CaM proteins into watersoluble conjugates. The Ellman test, which measures the amount of free sulfhydryl groups, was performed after a period of 5 minutes and showed that the reaction of the PEG tetraacrylate with the engineered CaM was complete (Figure 2a). Furthermore, MALDI-TOF mass spectrometry confirmed the conversion of the free CaM protein into crosslinked products (Figure 2b). The MALDI spectra, for example, showed a diffuse peak at 27 kDa, which represents a conjugate of a single PEG tetraacrylate chain ( 10.2 kDa) and a single CaM molecule (16.5 kDa). This peak is not observed when the PEG acrylate is present in a high concentration, and these data, along with the loss of the sulfhydryl groups, demonstrate the formation of higher-order conjugates by partial cross-linking. To complete the formation of the hydrogel, solutions containing conjugates of the CaM Figure 1. The two conformational states of calmodulin: an extended, dumbbell-shaped conformation in the presence of calcium ions (left), and a collapsed conformation upon binding to a ligand (right). Dark red: hinge region; blue: ends of dumbbell-shaped protein; light-red spheres: acrylate–thiol linkage; black ribbons, : poly(ethylene glycol) moiety.

Dynamic Hydrogels: Translating a Protein Conformational Change into Macroscopic Motion

Effects of the binding of myosin light chain kinase on the reactivities of calmodulin lysines.

An assay based on high-performance liquid chromotography (HPLC) has been developed for determining the expression of the acetyl coenzyme A: chloramphenicol 3-O-acetyltransferase gene in tr...

Detection of chloramphenicol acetyl transferase activity in transfected cells: a rapid and sensitive HPLC-based method.

Binding of trifluoperazine and fluorene-containing compounds to calmodulin and adducts.

Effects of interaction with calcineurin on the reactivities of calmodulin lysines.

A method is described for rapidly surveying the effects of modifying individual amino acid residues of a protein on its ability to interact specifically with another macromolecule. The procedure has been used to examine the individual roles of the seven lysyl residues of calmodulin in its ability to bind to smooth muscle myosin light chain kinase; previous studies by Jackson et al. (J. Biol. Chem. 261:1226-12232, 1986) have suggested that certain lysines may be located close to the interaction site. Trace [3H]-acetylated calmodulin, consisting predominantly of molecules acetylated at single sites together with unmodified protein, was incubated in excess (five- to 20-fold) with smooth muscle MLC kinase to allow the modified and unmodified molecules to compete for binding to the enzyme. Subsequently, the calmodulin-enzyme complex was separated from unbound calmodulin, and the level of acetylation of each of the seven lysines of the bound fraction of calmodulin was determined and compared to that of each corresponding group of the starting preparation. Significant changes were found at only two of the lysines, 21 and 75, where the extent of acetylation in the bound fraction was three- and fivefold lower, respectively, than that in the original preparation. These results were reproducible in three separate selection experiments employing both chicken and turkey gizzard MLC kinase. It is concluded that acetylation of calmodulin at either lysine 21 or 75 markedly reduces its affinity for MLC kinase, but acetylation at any of the other lysines (13, 30, 77, 94, or 148) has only minor effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthur E. Jackson

Papers

Effects of the binding of myosin light chain kinase on the reactivities of calmodulin lysines.

Detection of chloramphenicol acetyl transferase activity in transfected cells: a rapid and sensitive HPLC-based method.

Binding of trifluoperazine and fluorene-containing compounds to calmodulin and adducts.

Effects of interaction with calcineurin on the reactivities of calmodulin lysines.

Association of calmodulin and smooth muscle myosin light chain kinase: application of a label selection technique with trace acetylated calmodulin