Jeff Stock

Predicting coiled coils from protein sequences

The probability that a residue in a protein is part of a coiled-coil structure was assessed by comparison of its flanking sequences with sequences of known coiled-coil proteins. This method was used to delineate coiled-coil domains in otherwise globular proteins, such as the leucine zipper domains in transcriptional regulators, and to predict regions of discontinuity within coiled-coil structures, such as the hinge region in myosin. More than 200 proteins that probably have coiled-coil domains were identified in GenBank, including alpha- and beta-tubulins, flagellins, G protein beta subunits, some bacterial transfer RNA synthetases, and members of the heat shock protein (Hsp70) family.

Photoactivation turns green fluorescent protein red

In the few years since its gene was first cloned, the Aequorea victoria green fluorescent protein (GFP) has become a powerful tool in cell biology, functioning as a marker for gene expression, protein localization and protein dynamics in living cells. GFP variants with improved fluorescence intensity and altered spectral characteristics have been identified, but additional GFP variants are still desirable for multiple labeling experiments, protein interaction studies and improved visibility in some organisms. In particular, long-wavelength (red) fluorescence has remained elusive. Here we describe a red-emitting, green-absorbing fluorescent state of GFP that is generated by photoactivation with blue light. GFP can be switched to its red-emitting state easily with a laser or fluorescence microscope lamp under conditions of low oxygen concentration. This previously unnoticed ability enables regional, non-invasive marking of proteins in vivo. In particular, we report here the use of GFP photoactivation to make the first direct measurements of protein diffusion in the cytoplasm of living bacteria.

Folding and trimerization of clathrin subunits at the triskelion hub

The triskelion shape of the clathrin molecule enables it to form the polyhedral protein network that covers clathrin-coated pits and vesicles. Domains within the clathrin heavy chain that are responsible for maintaining triskelion shape and function were identified and localized. Sequences that mediate trimerization are distal to the carboxyl terminus and are adjacent to a domain that mediates both light chain binding and clathrin assembly. Structural modeling predicts that within this domain, the region of heavy chain-light chain interaction is a bundle of three or four alpha helices. These studies establish a low resolution model of clathrin subunit folding in the central portion (hub) of the triskelion, thus providing a basis for future mutagenesis experiments.

Sensory transduction in bacterial chemotaxis involves phosphotransfer between CHE proteins

The CheA protein of the Salmonella typhimurium chemotaxis system is phosphorylated by ATP. Phospho-CheA transfers its phosphoryl group to a second chemotaxis protein, CheY. Unlike phospho-CheA, phospho-CheY is relatively unstable, rapidly decaying to phosphate and CheY. We propose that phosphorylation of CheY may play a role in its function as a tumble regulator to control motor behavior in response to attractant and repellent stimuli.

Bacterial chemotaxis: The five sensors of a bacterium

The components of the Escherichia coli chemosensory system have been identified and their activities characterized, but how sensory information is processed to give an integrated response remains an open question.

N-terminal methylation of proteins: Structure, function and specificity

A common site for the posttranslational modification of proteins is at the N‐terminal α‐amino group. Here we consider the enzymatic addition of one or more methyl groups that has been found to occur in several proteins. Although the methylated proteins have different overall functions, they all appear to be involved in large macromolecular structures such as ribosomes, myofibrils, nucleosomes, pilins, or flagella. Structural features at the N‐termini of these methylated proteins suggest that sequences in this region may serve as recognition sites for only a few different types of methylating enzymes. Thus, we propose that three enzymes could account for the N‐methylated species so far identified in bacteria, the hypothetical MAK, QP, and pilin methyltransferases, and a single additional enzyme, the hypothetical PK methyltransferase, could account for all of the α‐amino methylations observed in eukaryotic cells. Finally, we discuss criteria that could be used in conjuction with primary sequence data to predict proteins that might be subject to methylation at their amino termini.

A single activity carboxyl methylates both farnesyl and geranylgeranyl cysteine residues

Members of the Ras superfamily of small GTP‐binding proteins, γ‐subunits of heterotrimeric G proteins and nuclear lamin B are subject to a series of post‐translational modifications that produce prenylcysteine methylester groups at their carboxyl termini. The thioether‐linked polyisoprenoid substituent can be either farnesyl (C15) or geranylgeranyl (C20). Small molecule prenylcysteine derivatives with either the C15 or C20 modification, such as N‐acetyl‐S‐trans,trans‐farnesyl‐L‐cysteine (AFC), S‐trans,trans‐farnesylthiopropionate (FTP), as well as the corresponding geranylgeranyl derivatives (AGGC and GGTP) are substrates for the carboxyl methyltransferase. Saccharomyces cerevisiae ste 14 mutants that lack RAS and a‐factor carboxyl methyltransferase activity are also unable to methylate farnesyl and geranylgeranylcysteine derivatives. Moreover, C20‐substituted cysteine analogs directly compete for carboxyl methylation with the C15‐substituted cysteine analogs and vice versa. Finally, AGGC is even more effective than AFC as an inhibitor of Ras carboxyl methylation, despite the fact that Ras is methylated at a farnesylcysteine rather than a geranylgeranylcysteine residue.

Novel mutations that alter the regulation of sporulation in Bacillus subtilis: Evidence that phosphorylation of regulatory protein spoOA controls the initiation of sporulation

Sporulation in Bacillus subtilis is a complex developmental process that occurs in response to nutrient deprivation. To identify components of the mechanism that allows cells to monitor their nutritional status and to understand how this sensory information is transduced into a signal to activate specific sporulation genes, we have isolated mutants that are able to sporulate efficiently under nutritional conditions that strongly inhibit sporulation in wild-type bacteria, a phenotype we refer to as Coi (control of initiation). Four coi mutations were found to be within the coding sequence of spoOA, a gene in which null mutations prevent the initiation of sporulation and a gene whose product shares a domain of homology with phosphorylation-activated proteins that play signal transduction roles in bacteria. All four of the spoOA mutations were within this conserved domain and in close proximity to the presumptive phosphoacceptor site. The wild-type and one of the mutant SpoOA proteins were purified and shown to be competent to accept phosphoryl groups from a phosphohistidine within a bacterial signal transduction kinase (CheA). The mutant SpoOA protein exhibited enhanced phosphoacceptor activity compared with the wild-type. This property of the mutant protein, together with additional genetic information, supports a model for regulation of sporulation initiation by control of the phosphorylation level of SpoOA.

Signal transduction: Hair brains in bacterial chemotaxis

The conserved cytoplasmic domains of bacterial chemotaxis receptors are a fibrous arrangement of alpha-helical coiled coils that look a lot like hair. Such bundles of alpha-helical filaments mediate sensory-motor responses in all prokaryotic cells. How do they work? Very nearly perfectly is probably as good an answer as any.

Active Site Interference and Asymmetric Activation in the Chemotaxis Protein Histidine Kinase CheA

The histidine protein kinase CheA is a multidomain protein that mediates stimulus-response coupling in bacterial chemotaxis. We have previously shown that the purified protein exhibits an equilibrium between inactive monomer and active dimer (Surette, M., Levit, M., Liu, Y., Lukat, G., Ninfa, E., Ninfa, A., and Stock, J. (1996) J. Biol. Chem.271, 939-945). We report here a study of the kinetics of phosphorylation of the isolated phosphoacceptor domain of CheA catalyzed by the isolated catalytic domain of the protein. The reaction fits Michaelis-Menten kinetics (Km = 0.26 mM for ATP and 0.10 mM for phosphoacceptor domain; kobs = 17 min−1). The catalytic domain exhibits the same equilibrium between inactive monomers and active dimers as the full-length CheA protein. Thus, CheA dimerization is an intrinsic property of this domain, independent of any other portion of the molecule and is required for its catalytic activity. In equimolar mixtures of full-length CheA and catalytic domain, homodimers and heterodimers are formed in equal concentration, indicating that all of the determinants for the dimerization are localized entirely on the catalytic domain. An analysis of the kinetics of phosphorylation catalyzed by CheA-catalytic domain heterodimers indicates half of the sites reactivity. The rate of CheA phosphorylation within this heterodimer is over 5-fold greater than that observed in CheA homodimers. The dramatic increase in activity within this asymmetric dimer raises the possibility that CheA activation by receptors involves a mechanism that directs catalysis to one active site while preventing interference from the other.