The effects of masseter muscle pain on biting performance.

The present study applied a standardized test food of known hardness to evaluate the biting performance of 20 female patients who had pain mainly in the masseter muscle during palpation. Another 20 women of a similar age group who were pain-free during examination served as controls. Electromyograms (EMG) of the masseter and sternocleidomastoid (SCM) muscles and the jaw position were recorded and measured when the subjects were biting through two types of test foods with known hardness (hard type, 20 kg hardness and extra-hard type, 60 kg hardness). Pressure-pain-threshold (PPT) values of both the patients and the normal subjects were obtained with an algometer. It was found that the PPT of the patients with pain was significantly lower and that the extra-hard food took more masseter muscle activity and more working side jaw movement in both the pain and the normal groups. During both hard and extra-hard food biting, a significantly longer duration of masseter muscle activity was found in pain patients while the total muscle activity was not significantly stronger. Strong correlation existed between SCM and masseter muscle activity during both hard and extra-hard food biting in the patient group, while such correlation was very weak in the normal group. In conclusion, painful masseter muscles required longer masseter and SCM muscle contraction time for breaking through a hard food of 20 kg and more, and co-activation of SCM and masseter muscles existed and was more evident when the food was harder or the pain was more severe.

Structural influence of hanatoxin binding on the carboxyl terminus of S3 segment in voltage-gated K(+)-channel Kv2.1.

The voltage-sensing domains of voltage-gated potassium channels Kv2.1 (drk1) contain four transmembrane segments in each subunit, termed S1 to S4. While S4 is known as the voltage sensor, the carboxyl terminus of S3 (S3C) bears a gradually broader interest concerning the site for gating modifier toxins like hanatoxin and thus the secondary structure arrangement as well as its surrounding environment. To further examine the putative three-dimensional (3-D) structure of S3C and to illustrate the residues required for hanatoxin binding (which may, in turn, show the influence on the S4 in terms of changes in channel gating), molecular simulations and dockings were performed. These were based on the solution structure of hanatoxin and the structural information from lysine-scanning results for S3C fragment. Our data suggest that several basic and acidic residues of hanatoxin are electrostatically and stereochemically mapped onto their partner residues on S3C helix, whereas some aromatic or hydrophobic residues located on the same helical fragment interact with the hydrophobic patch of the toxin upon binding. Therefore, a slight distortion of the S3C helix, in a direction toward the N-terminus of S4, may exist. Such conformational change of S3C upon toxin binding is presented as a possible explanation for the observed shift in hanatoxin binding-induced gating.

Three-dimensional modelling of the catalytic domain of Streptococcus mutans glucosyltransferase GtfB.

Glucosyltransferases (GtfB/C/D) of Streptococcus mutans, a pathogen for human dental caries, synthesize water-insoluble glucan through the hydrolysis of sucrose. Genetic and biochemical approaches have identified several active sites of these enzymes, but no three-dimensional (3D) structural evidence is yet available to elucidate the subdomain arrangement and molecular mechanism of catalysis. Based on a combined sequence and secondary structure alignment against known crystal structures of segments from closely related proteins, we propose here the 3D model of an N-terminal domain essential for the sucrose binding and splitting in GtfB. A Tim-barrel of (alpha/beta)(8) structural characteristics is revealed and the structural correlation for two peptides is described.

Voltage gating of Escherichia coli porin channels: role of the constriction loop.

In the homotrimeric OmpF porin from Escherichia coli, each channel is constricted by a loop protruding into the beta-barrel of the monomer about halfway through the membrane. The water-filled channels exist in open or closed states, depending on the transmembrane potential. For the transition between these conformations, two fundamentally different mechanisms may be envisaged: a bulk movement of the constriction loop L3 or a redistribution of charges in the channel lumen. To distinguish between these hypotheses, nine mutant proteins were constructed on the basis of the high-resolution x-ray structure of the wild-type protein. Functional changes were monitored by measuring single-channel conductance and critical voltage of channel closing. Structural alterations were determined by x-ray analysis to resolutions between 3.1 and 2.1 A. Tethering the tip of L3 to the barrel wall by a disulfide bridge (E117C/A333C), mobilizing L3 by perturbing its interaction with the barrel wall (D312N, S272A, E296L), or deleting residues at the tip of the loop (Delta116-120) did not alter appreciably the sensitivity of the channels to an external potential. A physical occlusion, due to a gross movement of L3, which would cause the channels to assume a closed conformation, can therefore be excluded.

Structural and functional characterization of OmpF porin mutants selected for larger pore size. II. Functional characterization.

The effects on the channel characteristics of four single amino acid substitutions in OmpF porin and of a deletion mutant in the constriction loop L3 have been studied. These mutations are all located in the narrow section of the channel of the protein that forms pores across the outer membrane of Escherichia coli. The single channel conductance of the deletion mutant (Delta109-114) is decreased by one third, whereas the point mutations do not exhibit significant deviations from that of the wild-type protein. The mutants exhibit drastic changes in ion selectivities. In the wild-type protein, the critical threshold potential (Vc), above which channels close reversibly, exhibits a strong pH dependence, with a titration point of approximately pH 7.7, which is abolished in all mutants studied here. Diffusion of six monosaccharides is little affected in the point mutants, while four disaccharides are taken up at highly increased rates by the deletion mutant. The functional results, presented here, are correlated to the x-ray structures of the mutants (Lou, K.-L., Saint, N., Prilipov, A., Rummel, G., Benson, S.A., Rosenbusch, J.P., and Schirmer, T. (1996) J. Biol. Chem. 271, 20669-20675). In most, but not all, cases, the structural changes explain the functional alterations observed.

Structural and functional characterization of OmpF porin mutants selected for larger pore size. I. Crystallographic analysis.

OmpF porin is a nonspecific pore protein from the outer membrane of Escherichia coli. Previously, a set of mutants was selected that allow the passage of long maltodextrins that do not translocate through the wild-type pore. Here, we describe the crystal structures of four point mutants and one deletion mutant from this set; their functional characterization is reported in the accompanying paper (Saint, N., Lou, K.-L., Widmer, C., Luckey, M., Schirmer, T., Rosenbusch, J. P. (1996) J. Biol. Chem. 271, 20676-20680). All mutations have a local effect on the structure of the pore constriction and result in a larger pore cross-section. Substitution of each of the three closely packed arginine residues at the pore constriction (Arg-42, Arg-82, and Arg-132) by shorter uncharged residues causes rearrangement of the adjacent basic residues. This demonstrates mutual stabilization of these residues in the wild-type porin. Deletion of six residues from the internal loop (Delta109-114) results in disorder of seven adjacent residues but does not alter the structure of the beta-barrel framework. Thus, the large hollow beta-barrel motif can be regarded as an autonomous structure.

Replacement of the sole histidinyl residue in OmpF porin from E. coli by threonine (H21T) does not affect channel structure and function.

The sole histidine residue in OmpF porin was replaced by threonine using site-directed mutagenesis. This exchange affected neither channel properties nor channel structure, as determined by X-ray analysis to 3.2 A. Conductance and critical voltage (Vc) were observed in the pH range 4.3-9.4, with results indistinguishable from those observed in the wild-type protein. The validity of these observations is supported by the independence of the methods used, and by the fact that mutants in residues located in the channel constriction yielded significantly different values from wild-type protein. The binding of a glycolipid molecule might be affected.

Interaction of plasminogen and fibrin in plasminogen activation.

Glu1-, Lys77-, miniplasminogens, kringle 1-3, kringle 1-5A, and kringle 1-5R were able to bind with fibrin, while microplasminogen and kringle 4 did not bind significantly. Kringle 1-5A, but not kringle 1-3, effectively inhibited the binding of Glu1-, Lys77-, and miniplasminogens with fibrin. Miniplasminogen also inhibited the binding of Glu1-plasminogen with fibrin. The binding of kringle 1-3 with fibrin was blocked by mini- or Glu1-plasminogen. It is therefore evident that there are two fibrin-binding domains in plasminogen and that the one in kringle 5 is of higher affinity than that in kringle 1-3. CNBr cleavage products of fibrinogen effectively enhanced the activation of Glu1-, Lys77-, or miniplasminogens, but not microplasminogen, by tissue-type plasminogen activator. Kringle 1-5, but not kringle 1-3, dose-dependently inhibited the enhancement by fibrinogen degradation products of Glu1-plasminogen activation by the activator. Lysine and epsilon-aminocaproic acid could inhibit the binding of plasminogens and plasminogen derivatives with fibrin and block the enhancement effect of fibrinogen degradation products on plasminogen activation. The data clearly illustrate that the binding of plasminogen with fibrin, mainly determined by kringle 5, is essential for effective activation by tissue-type plasminogen activator. However, the presence of kringle 1-4 in the plasminogen molecule is required for the full enhancing effect since the kcat/Km of miniplasminogen activation in the presence of fibrinogen degradation products was 8.2 microM-1 min-1 which is significantly less than 52.0 microM-1 min-1 of Glu1-plasminogen.