A minimal gating model for the cardiac calcium release channel.

A Markovian model of the cardiac Ca release channel, based on experimental single-channel gating data, was constructed to understand the transient nature of Ca release. The rate constants for a minimal gating scheme with one Ca-free resting state, and with two open and three closed states with one bound Ca2+, were optimized to simulate the following experimental findings. In steady state the channel displays three modes of activity: inactivated 1 mode without openings, low-activity L mode with single openings, and high-activity H mode with bursts of openings. At the onset of a Ca2+ step, the channel first activates in H mode and then slowly relaxes to a mixture of all three modes, the distribution of which depends on the new Ca2+. The corresponding ensemble current shows rapid activation, which is followed by a slow partial inactivation. The transient reactivation of the channel (increment detection) in response to successive additions of Ca2+ is then explained by the model as a gradual recruitment of channels from the extant pool of channels in the resting state. For channels in a living cell, the model predicts a high level of peak activation, a high extent of inactivation, and rapid deactivation, which could underlie the observed characteristics of the elementary release events (calcium sparks).     

Biotin binding to avidin. Oligosaccharide side chain not required for ligand association.

A commercially available, purified preparation of avidin was found to comprise two polypeptide bands (Mr 18,000 and Mr 15,500 respectively). Both bands bound biotin as assessed by biotin overlays of protein blots. The Mr 15,500 polypeptide was found to differ from the Mr 18,000 polypeptide only in its sugar content. When the commercial preparation was applied to a concanavalin A affinity column, the glycosylated forms were retarded as expected, and homotypic nonglycosylated avidin tetramers which failed to bind selectively to the column were collected in the effluent. The biotin-binding properties of the nonglycosylated avidin were equivalent to those obtained for the native (glycosylated) avidin molecule, indicating that the oligosaccharide moiety is not essential for the binding activity.     

Modification of voltage-dependent gating of potassium channels by free form of tryptophan side chain.

Indole constitutes a major component of the side chain of the amino acid tryptophan. Application of indole slows activation of voltage-dependent potassium channels and reduces steady-state conductance in a voltage- and concentration-dependent manner. The steep concentration dependence indicates that multiple indole molecules may interact with the channel. Indole does not noticeably change the unitary conductance or the mean open duration, however, it accelerates off-gating currents without altering on-gating currents. These properties of the modification of channel gating induced by indole are consistent with a model in which indole binds independently to every subunit of the channel complex to prevent the final concerted transition to the open state. We suggest that exogenously applied indole and side-chains of the tryptophan residues of the channel protein involved in activation may compete for the same effector position and that indole might be useful as a probe to study functional roles of tryptophan residues.     

A new approach to the rapid determination of protein side chain conformations

Two efficient algorithms have been developed which allow amino acid side chain conformations to be optimized rapidly for a given peptide backbone conformation. Both these approaches are based on the assumption that each side chain can be represented by a small number of rotameric states. These states have been obtained by a dynamic cluster analysis of a large data base of known crystallographic structures. Successful applications of these algorithms to the prediction of known protein conformations are presented.     

Sequence comparison of new prokaryotic and mitochondrial members of the polypeptide chain release factor family predicts a five-domain model for release factor structure.

We have recently reported the cloning and sequencing of the gene for the mitochondrial release factor mRF-1. mRF-1 displays high sequence similarity to the bacterial release factors RF-1 and RF-2. A database search for proteins resembling these three factors revealed high similarities to two amino acid sequences deduced from unassigned genomic reading frames in Escherichia coli and Bacillus subtilis. The amino acid sequence derived from the Bacillus reading frame is 47% identical to E.coli and Salmonella typhimurium RF-2, strongly suggesting that it represents B.subtilis RF-2. Our comparison suggests that the expression of the B.subtilis gene is, like that of the E.coli and S. typhimurium RF-2 genes, autoregulated by a stop codon dependent +1 frameshift. A comparison of prokaryotic and mitochondrial release factor sequences, including the putative B.subtilis RF-2, leads us to propose a five-domain model for release factor structure. Possible functions of the various domains are discussed.     

Role of sgcR3 in positive regulation of enediyne antibiotic C-1027 production of Streptomyces globisporus C-1027

Background  

C-1027, produced by Streptomyces globisporus C-1027, is one of the most potent antitumoral agents. The biosynthetic gene cluster of C-1027, previously cloned and sequenced, contains at least three putative regulatory genes, i.e. sgcR1, sgcR2 and sgcR3. The predicted gene products of these genes share sequence similarities to StrR, regulators of AraC/XylS family and TylR. The purpose of this study was to investigate the role of sgcR3 in C-1027 biosynthesis.     

A new histochemical method for the identification and visualization of both side chain acylated and nonacylated sialic acids.

A new histochemical method is described for the differentiation of mucins that utilizes two different Schiff reagents and allows single section identification of side chain O-acylated, and nonacylated, sialic acids in contrasting colors. In the event of mucins containing only one type of sialic acid, it may allow their specific identification (e.g., C7 or C8 side chain O-acylated). It has been shown to be useful in the identification of some metastases from adenocarcinomas of colon (where the primary is potassium hydroxide/periodic acid-Schiff positive) and should prove of great value in the investigation of diseases of the gastrointestinal tract and particularly those of the colon. It should also be valuable in the general field of epithelial mucin histochemistry, particularly for those mucins of the salivary and parotid glands, etc.     

Role of the phenylalanine B24 side chain in directing insulin interaction with its receptor. Importance of main chain conformation

We have investigated (by use of semisynthetic insulin analogs and isolated canine hepatocytes) the role of invariant residue PheB24 in determining the affinity of insulin-receptor interactions. Our results confirm that replacement of PheB24 by D-Phe is not detrimental to ligand binding to receptor, show that D-Ala is well tolerated at position B24 (whereas Ala is not), and demonstrate that [GlyB24]insulin retains as much as 78% of the receptor binding potency of native insulin. Additional findings show that replacement of PheB24 by D-Pro or by alpha-aminoisobutyric acid results in analogs with severely decreased binding potency, and that the COOH-terminal domain containing residues B26-B30 plays a positive role in determining receptor binding potency in GlyB24-substituted insulin (whereas it plays a negative role in determining the receptor binding potency of its GlyB25-substituted counterpart). We interpret our results as identifying (a) a critical role for the insulin main chain near residue B24 in determining the affinity of receptor for ligand, (b) the importance of main chain flexibility in achieving a high affinity state of receptor-bound hormone, and (c) a potential interaction of the PheB24 side chain with receptor which initiates main chain structural changes in the natural hormone, but which does not itself confer affinity to ligand-receptor interactions.     

Primary structure of peptides and ion channels. Role of amino acid side chains in voltage gating of melittin channels.

Melittin produces a voltage-dependent increase in the conductance of planar lipid bilayers. The conductance increases when the side of the membrane to which melittin has been added (cis-side) is made positive. This paper reports observations on the effect of modifying two positively charged amino acid residues within the NH2-terminal region of the molecule: lysine at position 7 (K7), and the NH2-terminal glycine (G1). We have synthesized melittin analogues in which K7 is replaced by asparagine (K7-N), G1 is blocked by a formyl group (G1-f), and in which both modifications of the parent compound were introduced (G1-f, K7-N). The time required to reach peak conductance during a constant voltage pulse was shorter in membranes exposed to the analogues than in membranes modified by melittin. The apparent number of monomers producing a conducting unit for [K7-N]-melittin and [G1-f]-melittin, eight, was found to be greater than the one for [G1-f], K7-N]-melittin and for melittin itself, four. The apparent gating charge per monomer was less for the analogues, 0.5-0.3 than for melittin, one. Essentially similar results were obtained with melittin analogues in which the charge on K7 or G1 or both was blocked by an uncharged N-linked spin label. These results show that the positive charges in the NH2-terminal region of melittin play a major but not exclusive role in the voltage gating of melittin channels in bilayers.     

A seesaw model for intermolecular gating in the kinesin motor protein

Recent structural observations of kinesin-1, the founding member of the kinesin group of motor proteins, have led to substantial gains in our understanding of this molecular machine. Kinesin-1, similar to many kinesin family members, assembles to form homodimers that use alternating ATPase cycles of the catalytic motor domains, or “heads”, to proceed unidirectionally along its partner filament (the microtubule) via a hand-over-hand mechanism. Cryo-electron microscopy has now revealed 8-Å resolution, 3D reconstructions of kinesin-1?microtubule complexes for all three of this motor’s principal nucleotide-state intermediates (ADP-bound, no-nucleotide, and ATP analog), the first time filament co-complexes of any cytoskeletal motor have been visualized at this level of detail. These reconstructions comprehensively describe nucleotide-dependent changes in a monomeric head domain at the secondary structure level, and this information has been combined with atomic-resolution crystallography data to synthesize an atomic-level "seesaw" mechanism describing how microtubules activate kinesin’s ATP-sensing machinery. The new structural information revises or replaces key details of earlier models of kinesin’s ATPase cycle that were based principally on crystal structures of free kinesin, and demonstrates that high-resolution characterization of the kinesin–microtubule complex is essential for understanding the structural basis of the cycle. I discuss the broader implications of the seesaw mechanism within the cycle of a fully functional kinesin dimer and show how the seesaw can account for two types of "gating" that keep the ATPase cycles of the two heads out of sync during processive movement.     

Forced gating motions by a substituted titratable side chain at the bundle crossing of a potassium channel

Numerous inwardly rectifying potassium (Kir) channels possess an aromatic residue in the helix bundle crossing region, forming the narrowest pore constriction in crystal structures. However, the role of the Kir channel bundle crossing as a functional gate remains uncertain. We report a unique phenotype of Kir6.2 channels mutated to encode glutamate at this position (F168E). Despite a prediction of four glutamates in close proximity, Kir6.2(F168E) channels are predominantly closed at physiological pH, whereas alkalization causes rapid and reversible channel activation. These findings suggest that F168E glutamates are uncharged at physiological pH but become deprotonated at alkaline pH, forcing channel opening due to mutual repulsion of nearby negatively charged side chains. The potassium channel pore scaffold likely brings these glutamates close together, causing a significant pK(a) shift relative to the free side chain (as seen in the KcsA selectivity filter). Alkalization also shifts the apparent ATP sensitivity of the channel, indicating that forced motion of the bundle crossing is coupled to the ATP-binding site and may resemble conformational changes involved in wild-type Kir6.2 gating. The study demonstrates a novel mechanism for engineering extrinsic control of channel gating by pH and shows that conformational changes in the bundle crossing region are involved in ligand-dependent gating of Kir channels.     

A three-state model for connexin37 gating kinetics

The gating behavior of human connexin 37 (hCx37) is unaffected by the nature of the bathing monovalent (for Na, K, Rb). It is modified by [Mg] in the millimolar range. For fitting the kinetics, we propose a simple extension to three states of the canonical 2-state model of the hemichannel. The extra closed state allows for some immobilization of a hemichannel at high transjunctional voltages. The model is reasonably efficient at fitting data at various voltage protocols. Interpreting the fits of the data at different [Mg] is consistent with a binding site for Mg.     

A model of the gating of ion channels

The gating of ion channels in biological membranes has usually been described in terms of Markov transitions between a few discrete open or closed states. Such models predict that the distributions of open and closed durations decay as a sum of exponential terms. Recent experimental data have indicated that certain channels are not easily described by these models. We show that distributions of open and closed times similar to those seen experimentally are predicted by a model that involves only one open and closed state but that assumes the activation energy of the gating process to be stochastic. This model involves only a few parameters and these have direct physical interpretations. Measurements of the correlation between the durations of successive open or closed events is shown to provide an experimental method for distinguishing between this and other models.     

Backbone side chain interactions in peptides. I. Crystal structures of model dipeptides with the Pro-Ser sequence

The preferential occurrence of amino-acid residues having short polar side-chain within beta-folded regions of crystallized proteins suggests the existence of some stabilizing interaction involving the side polar function. Three model dipeptides tBuCO-L-Pro-L-Ser-NHMe 1, tBuCO-L-Pro-D-Ser-NHMe 2 in the pure enantiomeric a and racemic b forms, and iPrCO-L-Pro-D-Ser-OMe 3 have been investigated in the solid state by X-ray crystallography. Homo and heterochiral sequences 1 and 2 are folded in the beta I and beta II types, respectively, whereas 3 obviously accommodates an open conformation. Besides the i + 3 leads to i hydrogen bond typical of beta-bends in 1, 2a, and 2b, the Ser NH group in all four crystal structures is a proton donor to the lone orbitals of the Ser O gamma oxygen atom. The result is that the disposition of the Ser C alpha--C beta bond corresponds to the rotamer III (chi 1 congruent to 60 degrees). As shown by the crystal structure of 3, the intra-Ser NH. . .O gamma hydrogen bonding is not restricted to beta-folded Pro-Ser sequences. Therefore, this interaction is not only a stabilizing factor for beta-turns but it is also probably responsible for the already mentioned stability of rotamer III for the Ser C alpha--C beta bond in peptides and protein.     

Atomic coordinates and calculated intensities for a side by side D.N.A. model

A precise conformation of the side-by-side type is proposed for the B-D.N.A. form. In this model the antiparallel chains are related by a dyadic symmetry and binding between complementary bases is realized using Watson-Crick pairing. The elementary unit, comprising 10 nucleotides for one chain, is composed of one segment in a right handed helix, a second segment rotated to the left and two bends. Cylindrical atomic coordinates are given for atoms in the elementary unit and the square of the Fourier transform is calculated for this conformation and compared to the well known double helix and to experimental data.     

What's new in the antibiotic pipeline.

Many advances have recently been made in the development of chemotherapeutic agents for bacterial infections. As a consequence of problematic antimicrobial-resistant bacteria, research is now directed towards narrow-spectrum agents rather than broad-spectrum agents. Further, orally active agents have always been desirable, but today's cost-saving environment, in line with a desire to minimize treatment costs, values reduced administration costs and keeping patients out of the hospital. There has been a recent increase in research into orally active antibacterial agents, such as carbapenems and cephalosporins, and non-glycopeptide natural products.