Dynamic allosteric communication pathway directing differential activation of the glucocorticoid receptor.

Allosteric communication within proteins is a hallmark of biochemical signaling, but the dynamic transmission pathways remain poorly characterized. We combined NMR spectroscopy and surface plasmon resonance to reveal these pathways and quantify their energetics in the glucocorticoid receptor, a transcriptional regulator controlling development, metabolism, and immune response. Our results delineate a dynamic communication network of residues linking the ligand-binding pocket to the activation function-2 interface, where helix 12, a switch for transcriptional activation, exhibits ligand- and coregulator-dependent dynamics coupled to graded activation. The allosteric free energy responds to variations in ligand structure: subtle changes gradually tune allostery while preserving the transmission pathway, whereas substitution of the entire pharmacophore leads to divergent allosteric control by apparently rewiring the communication network. Our results provide key insights that should aid in the design of mechanistically differentiated ligands.

Attitudes to dental care, Sweden 2003-2013, and clinical correlates of oral health-related quality of life in 2013.

OBJECTIVE: To investigate attitudes to dental care, and to assess possible associations with socio-economic and clinical variables over a period of ten years, and to investigate the association between OHRQoL assessed by oral impact on daily performance (OIDP), and socio-economic, dental care habits, smoking and oral status. MATERIALS AND METHODS: Cross-sectional studies performed in the county of Dalarna, Sweden, in 2003, 2008 and 2013. Random samples of 1,107-1,115 dentate individuals, aged 30-85 years, who answered a questionnaire and who were radiographically and clinically examined were included. RESULTS: The importance of preventive treatment, regular recalls and meeting the same caregiver as on previous visits became less important. In individuals with alveolar bone loss, meeting the same caregiver as on previous visits was important (P<.05). In individuals with manifest caries, information on treatment cost was important, while prevention became less important (P<.05). OIDP was reported by 31% of the individuals in the study, and frequent impact was reported by 10%. Individuals with manifest caries lesions, less than 20 remaining teeth, and temporomandibular disorders (TMD) reported OIDP to a significantly higher degree, compared to orally healthy individuals. CONCLUSION: Attitudes important in maintaining and improving good oral health, such as preventive care and regular recalls to dentistry, became less important during this period of 10 years. Oral impact was found to be associated with irregular dental visits and limited economy for dental care, individuals with less than 20 remaining teeth, TMD and manifest caries.

Release of calcium into the myofibrillar space in response to active shortening of striated muscle.

AIM: The study was undertaken to explore whether shortening of striated muscle during activity is associated with release of bound Ca2+ into the myofibrillar space as has previously been proposed in order to explain the depressant effect of active shortening. METHODS: The experiments were carried out on single muscle fibres isolated from the anterior tibialis muscle of Rana temporaria. The fibres were loaded with the calcium sensitive indicator Fluo-3. The fibres, stimulated to produce a partially fused isometric tetanus, were subjected to a shortening ramp or, alternatively, to a stretch ramp during activity while force, fibre length, sarcomere length and the Fluo-3 signal were recorded. RESULTS: A shortening ramp performed during a partially fused tetanus caused an increase in the myofibrillar free calcium concentration and produced, simultaneously, a decrease in active force. The isometric force recovered gradually after the shortening ramp, while the intracellular Ca2+ concentration stayed above the control level during the remainder of the stimulation period. A stretch ramp applied during a partially fused tetanus caused a considerably smaller change in the myofibrillar Ca2+ concentration. CONCLUSION: The results provide evidence that the myosin cross-bridges interact with the calcium binding sites on the thin filaments during active shortening, causing sustained release of calcium and reduced contractile strength.

Trends over 30 years in the prevalence and severity of alveolar bone loss and the influence of smoking and socio-economic factors--based on epidemiological surveys in Sweden 1983-2013.

OBJECTIVE: Epidemiological studies of the prevalence of periodontitis over an extended time using the same methodology to investigate and classify periodontitis are sparse in the literature. Smoking and socio-economic factors have been proven to increase the risk for periodontal disease. The objective of this study was to investigate 30-year time trends, using the same methodology to classify the prevalence and severity in alveolar bone loss (ABL) and to investigate the influence of tobacco and socio-economic factors. METHODS: Four cross-sectional epidemiological studies in an adult population were performed in the county of Dalarna, Sweden, in 1983, 2003, 2008 and 2013. Random samples of 787-1133 individuals aged 35-85 who answered a questionnaire about tobacco use and socio-economic factors were radiographically and clinically examined. A number of teeth, ABL and calculus visible on radiographs were registered. The severity of ABL as detected on radiographs was classified into no bone loss, moderate or severe. RESULTS: The prevalence of moderate ABL decreased from 45% in 1983 to 16% in 2008, but increased to 33% in 2013 (P < 0.05). The prevalence of severe ABL remained the same from 1983 (7%) to 2013 (6%). Calculus visible on radiographs increased from 22% in 2008 to 32% in 2013 (P < 0.05). Socio-economic factors had limited impact on the severity of ABL. CONCLUSION: Moderate ABL and calculus visible on radiographs significantly increased between 2008 and 2013. Smoking was the strongest factor associated with ABL overall.

Improved replication efficiency of echovirus 5 after transfection of colon cancer cells using an authentic 5' RNA genome end methodology.

Oncolytic virotherapy is a promising novel form of cancer treatment, but the therapeutic efficiency needs improvement. A potential strategy to enhance the therapeutic effect of oncolytic viruses is to use infectious nucleic acid as therapeutic agent to initiate an oncolytic infection, without administrating infectious viral particles. Here we demonstrate improved viral replication activation efficiency when transfecting cells with 5' end authentic in vitro transcribed enterovirus RNA as compared to genomic RNA with additional non-genomic 5' nucleotides generated by conventional cloning methods. We used echovirus 5 (E5) as an oncolytoc model virus due to its ability to replicate in and completely destroy five out of six colon cancer cell lines and kill artificial colon cancer tumors (HT29 spheroids), as shown here. An E5 infectious cDNA clone including a hammerhead ribozyme sequence was used to generate in vitro transcripts with native 5' genome ends. In HT29 cells, activation of virus replication is approximately 20-fold more efficient for virus genome transcripts with native 5' genome ends compared to E5 transcripts generated from a standard cDNA clone. This replication advantage remains when viral progeny release starts by cellular lysis 22 h post transfection. Hence, a native 5' genomic end improves infection activation efficacy of infectious nucleic acid, potentially enhancing its therapeutic effect when used for cancer treatment. The clone design with a hammerhead ribozyme is likely to be applicable to a variety of oncolytic positive sense RNA viruses for the purpose of improving the efficacy of oncolytic virotherapy.

The force-velocity relationship at negative loads (assisted shortening) studied in isolated, intact muscle fibres of the frog.

AIM: The study was undertaken to explore the force-velocity relationship under conditions where the myofilament system is subjected to an external force that serves as a negative load and assists the shortening movement. METHODS: The experiments were carried out on single muscle fibres isolated from the anterior tibialis muscle of Rana temporaria. The fibres, being operated under load-clamp control, were released to shorten during tetanic stimulation at sarcomere lengths where the fibres carried different degrees of passive tension. The shortening thus occurred while the sarcomeres were subjected to a force that may be characterized as a 'negative load', that is, a force assisting the shortening movement. RESULTS: The force-velocity relationship below zero load was found to be a smooth continuation of the force-velocity curve recorded at positive loads with the shortening velocity increasing steeply at loads below zero. A negative load amounting to merely 10% of the isometric force, thus raised the shortening velocity to a level two to three times higher than V0 , the velocity recorded at zero load. CONCLUSIONS: The results provide evidence that, even in the presence of a longitudinal compressive force, the speed of shortening of the muscle fibre is determined by the cycling rate of the interacting cross-bridges. The force-velocity relationship at negative loads may play a relevant part during fast movements of striated muscle as pointed out in the discussion.

A two-nucleotide deletion renders the mannose-binding lectin 2 (MBL2) gene nonfunctional in Danish Landrace and Duroc pigs.

The mannose-binding lectins (MBLs) are central components of innate immunity, facilitating phagocytosis and inducing the lectin activation pathway of the complement system. Previously, it has been found that certain single-nucleotide polymorphisms (SNPs) in porcine MBL1 and MBL2 (pMBL1, pMBL2) affect mRNA expression, serum concentration, and susceptibility to disease, but the combinatory effect of pMBL1 and pMBL2 genotypes needs further elucidation. In the present study, pMBL1 and pMBL2 alleles, combined pMBL haplotypes, and MBL-A concentration in serum were analyzed in purebred Landrace (N = 30) and Duroc (N = 10) pigs. Furthermore, the combined pMBL haplotypes of 89 Piètrain × (Large White × Landrace) crossbred pigs were studied, and the genotypes of 67 crossbreds challenged with Escherichia coli were compared to their individual disease records. In the purebred animals, three non-synonymous SNPs and a two-nucleotide deletion were detected in the coding sequence of pMBL2. The two-nucleotide deletion was present at a frequency of 0.88 in the Landrace pigs and 0.90 in the Duroc pigs, respectively. In the crossbreds, the T allele of the SNP G949T in pMBL1-previously shown to have profound effect on MBL-A concentration even in the heterozygote condition-was detected in 47 % of the animals. Finally, an association was found between low-producing MBL genotypes and low body weight on the day of weaning in the same animals.

Residual force enhancement after stretch in striated muscle. A consequence of increased myofilament overlap?

When skeletal muscle is stretched above optimal sarcomere length during tetanic activity there is an increase in force that stays above the isometric force level throughout the activity period. This long-lasting increase in contractile force, generally referred to as 'residual force enhancement after stretch' (FE(resid)), has been studied in great detail in various muscle preparations over more than half a century. Substantial evidence has been presented to show that non-uniform sarcomere behaviour plays a major part in the development of FE(resid). However, in a great number of recent studies the role of sarcomere non-uniformity has been challenged and alternative mechanisms have instead been proposed to explain the increase in force such as enhancement of cross-bridge function and/or strengthening of parallel elastic elements along the muscle fibres. This article presents a short review of the salient features of FE(resid) and provides evidence that non-uniform sarcomere behaviour is indeed likely to play a major role in the development of FE(resid). Electron microscopical studies of fibres rapidly fixed after active stretch demonstrate that, dispersed in the preparation, there are assymetrical length changes within the two halves of myofibrillar sarcomeres resulting in greater filament overlap in one half of the sarcomere than in the opposite sarcomere half. Sarcomere halves with increased filament overlap will consequently be in a situation where they are able to produce a greater force than that recorded in the isometric control. Weaker regions in series will be able to keep the enhanced force by recruitment of elastic elements.

Extensive polymorphism in the porcine Toll-like receptor 10 gene.

The great importance of the Toll-like receptors (TLRs) in innate immunity is well established, but one family member--TLR10--remains elusive. TLR10 is expressed in various tissues in several species, but its ligand is not known and its function is still poorly understood. The open reading frame of TLR10 was sequenced in 15 wild boars, representing three populations, and in 15 unrelated domestic pigs of Hampshire, Landrace and Large White origin. Amino acid positions corresponding to detected nonsynonymous single nucleotide polymorphisms (SNPs) were analysed in the crystal structures determined for the human TLR1-TLR2-lipopeptide complex and the human TLR10 Toll/Interleukin 1 receptor (TIR) dimer. SNP occurrence in wild boars and domestic pigs was compared, and haplotypes for the TLR10 gene and the TLR6-1-10 gene cluster were reconstructed. Despite the limited number of animals sequenced in the present study (N = 30), a larger number of SNPs were found in TLR10 than recently reported for TLR1, TLR6 and TLR2. Thirty-three SNPs were detected, of which 20 were nonsynonymous. The relative frequency of nonsynonymous (d(N) ) and synonymous (d(S) ) SNPs between wild boars and domestic pigs was higher in TLR10 than recently reported for TLR1, TLR6 and TLR2. However, the polymorphism reported in the present study seems to leave the function of the TLR10 molecule unaffected. Furthermore, no nonsynonymous SNPs were detected in the part of the gene corresponding to the hinge region of the receptor, probably reflecting rigorously acting functional constraint. The total number of SNPs and the number of nonsynonymous SNPs were significantly lower (P < 0.05) in the wild boars than in the domestic pigs, and fewer TLR10 haplotypes were present in the wild boars. The majority of the TLR6-1-10 haplotypes were specific for either wild boars or domestic pigs, probably reflecting differences in microbial environment and population history.

Contractile performance of striated muscle.

The single muscle fiber preparation provides an excellent tool for studying the mechanical behaviour of the contractile system at sarcomere level. The present article gives an overview of studies based on intact single fibers from frog and mouse skeletal muscle. The following aspects of muscle function are treated: (1) The length-tension relationship. (2) The biphasic force-velocity relationship. (3) The maximum speed of shortening, its independence of sarcomere length and degree of activation. (4) Force enhancement during stretch, its relation to sarcomere length and myofilament lattice width. (5) Residual force enhancement after stretch. (6) Force reduction after loaded shortening. (7) Deactivation by active shortening. (8) Differences in kinetic properties along individual muscle fibers.

Non-linear myofilament elasticity in frog intact muscle fibres.

The aim of the present investigation was to elucidate the elastic properties of the myofilaments during tetanic activity in striated muscle. The study was carried out on intact single muscle fibres from the anterior tibialis muscle of Rana temporaria (2.0-2.5 degrees C). The instantaneous stiffness was measured as the change in force that occurred in response to a high-frequency (2-4 kHz) length oscillation while the fibre was released to shorten against a pre-set constant load that ranged between 40 and 70% of maximum tetanic force in different experiments. Measurements of fibre stiffness were carried out, at a given load, both at 2.20 microm sarcomere length (S(2.20)), i.e. at full overlap between the thick and thin filaments, and at 2.60 microm sarcomere length (S(2.60)). The fact that the load on the fibre was constant during the stiffness measurements at the two sarcomere lengths implies that the stiffness of elastic elements, acting in series with the myofilaments, was constant at the two sarcomere lengths. The fibre stiffness was consistently lower at the extended sarcomere length, the S(2.60)/S(2.20) ratio ranging from 0.83 to 0.97 at the different loads investigated. Based on the S(2.60)/S(2.20) ratio, the compliance of the free portions of the thick and thin filaments could be calculated. The myofilament stiffness was found to increase progressively as the load was raised from 40 to 70% of maximum tetanic force. At 2.20 microm sarcomere length and at 40% of maximum load on the fibre, the calculated myofilament stiffness was approximately 2.5 times the maximum cross-bridge stiffness.

Determinants of force rise time during isometric contraction of frog muscle fibres.

Force-velocity (F-V) relationships were determined for single frog muscle fibres during the rise of tetanic contraction. F-V curves obtained using isotonic shortening early in a tetanic contraction were different from those obtained at equivalent times with isovelocity shortening, apparently because changing activation early in the contraction leads, in isovelocity experiments, to changing force and changing series elastic extension. F-V curves obtained with isotonic and with isovelocity shortening are similar if the shortening velocity in the isovelocity trials is corrected for series elastic extension. There is a progressive shift in the scaling of force-velocity curves along the force axis during the course of the tetanic rise, reflecting increasing fibre activation. The time taken for F-V curves to reach the steady-state position was quite variable, ranging from about 50 ms after the onset of contraction (1-3 degrees C) to well over 100 ms in different fibres. The muscle force at a fixed, moderately high shortening velocity relative to the force at this velocity during the tetanic plateau was taken as a measure of muscle activation. The reference velocity used was 60% of the maximum shortening velocity (V(max)) at the tetanic plateau. The estimated value of the fractional activation at 40 ms after the onset of contraction was used as a measure of the rate of activation. The rate of rise of isometric tension in different fibres was correlated with the rate of fibre activation and with V(max) during the plateau of the tetanus. Together differences in rate of activation and in V(max) accounted for 60-80% of the fibre-to-fibre variability in the rate of rise of isometric tension, depending on the measure of the force rise time used. There was not a significant correlation between the rate of fibre activation and V(max). The steady-state F-V characteristics and the rate at which these characteristics are achieved early in contraction are seemingly independent. A simulation study based on F-V properties and series compliance in frog muscle fibres indicates that if muscle activation were instantaneous, the time taken for force to rise to 50% of the plateau value would be about 60% shorter than that actually measured from living fibres. Thus about 60% of the force rise time is a consequence of the time course of activation processes and about 40% represents time taken to stretch series compliance by activated contractile material.

Contractile properties of mouse single muscle fibers, a comparison with amphibian muscle fibers.

Single fibers, 25-40 microm wide and 0.5-0.7 mm long, were isolated from the flexor digitorum brevis muscle of the mouse. Force and movement were recorded (21-27 degrees C) from the fiber as a whole and, in certain experiments, from a short marked segment that was held at constant length by feedback control. The maximum tetanic force, 368+/-57 kN/m2 (N = 10), was not significantly different from that recorded in frog muscle fibers at equal temperature. However, the rising phase of the tetanus was considerably slower in the mammalian fibers, 202+/-20 ms (N = 17) being required to reach 90% of maximum tetanic force as compared with 59+/-4 ms (N = 20) in the frog muscle fibers. Similar to the situation in frog muscle fibers, the force-velocity relation exhibited two distinct curvatures located on either side of a breakpoint near 80% of the isometric force. Maximum speed of shortening was 4.0+/-0.3 fiber lengths s(-1) (N = 6). The relationship between tetanic force and sarcomere length was studied between 1.5 and 4.0 microm sarcomere spacings, based on length-clamp recordings that were free of 'tension creep'. There was a flat maximum (plateau) of the length-tension relation between approximately 2.0 and 2.4 microm sarcomere lengths. The descending limb of the length-tension relation (linear regression) intersected the length axis (zero force) at 3.88 microm and reached maximum force at 2.40 microm sarcomere length. The slope of the descending limb is compatible with a thick filament length of 1.63 microm and an average thin filament length of 1.10 microm. These values accord well with recent electron microscope measurements of myofilament length in mammalian muscle.

Effects of intracellular acidification and varied temperature on force, stiffness, and speed of shortening in frog muscle fibers.

This study aimed to establish whether the temperature-dependent effect of acidification on maximum force observed in mammalian muscles also applies to frog muscle. Measurements of force, stiffness, and unloaded velocity of shortening in intact single muscle fibers from the anterior tibialis muscle of Rana temporaria were performed between 0 and 22 degrees C during fused tetani in H(2)CO(3)-CO(2)-buffered Ringer solution with pH adjusted to 7.0 and 6.3, respectively. The force-to-stiffness ratio increased as a rectilinear function of temperature between 0 and 20 degrees C at pH 7.0. Lowering the pH to 6.3 reduced the tetanic force by 13.5 +/- 1.2 and 11.5 +/- 1.4% at 2.8 and 20.5 degrees C, respectively, with only a minor reduction in fiber stiffness. The maximum speed of shortening was decreased by lowered pH by 12.9 +/- 1.5 and 7.8 +/- 1.1% at low and high temperature, respectively. Acidification increased the time to reach 70% of maximum force by 18.0% at approximately 2 degrees C; the same pH change performed at approximately 20 degrees C in the same fibers reduced the rise time by 24.1%. The same increase in the rate of rise of force at high temperature was also found at normal pH after the fibers were fatigued by frequent stimulation. It is concluded that, in frog muscle, the force-depressant effect of acidification does not vary significantly with temperature. By contrast, acidification affects the onset of activation in a manner that is critically dependent on temperature.

A mechanical stretch induces contractile activation in unstimulated developing rat skeletal muscle in vitro.

The effects of a stretch-release cycle (approximately 25% of the resting muscle fibre length, Lo) on both tension and [Ca2+]i in small, unstimulated, intact muscle fibre bundles isolated from adult and neonatal rats were investigated at 20 degrees C. The results show that the effects of the length change depended on the age of the rats. Thus, the length change produced three effects in the neonatal rat muscle fibre bundles, but only a single effect in the adult ones. In the neonatal fibre bundles, the length change led to an increase in resting muscle tension and to a transient increase in [Ca2+]i. The stretch-release cycle was then followed by a twitch-like tension response. In the adult fibre bundles, only the increase in resting tension was seen and both the transient increase in [Ca2+]i and the stretch-induced twitch-like tension response were absent. The amplitude of the twitch-like tension response was affected by both 2,3-butanedione monoxime and sarcomere length in the same manner as active twitch tension, suggesting that it arose from actively cycling crossbridges. It was also reversibly abolished by 25 mM K+, 1 microM tetrodotoxin and 1.5 mM lidocaine (lignocaine), and was significantly depressed (P < 0.001) by lowering [Ca2+]o. These findings suggest that a rapid stretch in neonatal rats induces a propagated impulse that leads to an increase in [Ca2+]i, and that abolishing the action potential abolishes the stretch-induced twitch-like tension response. In 5- to 7-day-old rats, the twitch-like tension response was approximately 50 % of the isometric twitch. It then decreased progressively with age and was virtually absent by the time the rats were 21 days old. Interestingly, this is the same period over which rat muscles differentiate from their neonatal to their adult types.

Exposure assessment to alpha- and beta-pinene, delta(3)-carene and wood dust in industrial production of wood pellets.

The main aim of the study was to measure the exposure to monoterpenes (alpha- and beta-pinene and Delta(3)-carene) and wood dust during industrial production of wood pellets and briquettes. Additional aims were to compare the results from wood dust sampled on a filter with real time measurements using a direct reading instrument and to identify peak exposures to dust. Twenty-four men working at six companies involved in industrial production of wood pellets and briquettes participated in the study. Monoterpenes were measured by diffusive sampling and wood dust was measured as total dust. A data logger (DataRAM) was used for continuous monitoring of dust concentration for 18 of the participants. The sampling time was approximately 8 h. The personal exposure to monoterpenes ranged from 0.64 to 28 mg/m(3) and a statistically significant (Kruskal-Wallis test, P = 0.0002) difference in levels of monoterpenes for workers at different companies was seen. In the companies the personal exposure to wood dust varied between 0.16 and 19 mg/m(3) and for 10 participants the levels exceeded the present Swedish occupational exposure limit (OEL) of 2 mg/m(3). The levels of wood dust during the morning shift were significantly (Mann-Whitney test, P = 0.04) higher compared with the afternoon shift. Continuous registration of dust concentration showed peak values for several working operations, especially cleaning of truck engines with compressed air. For 24 workers in six companies involved in industrial production of wood pellets the personal exposure to monoterpenes was low and to wood dust high compared with the present Swedish OEL and previous studies in Swedish wood industries. Since the DataRAM can identify critical working tasks with high wood dust exposure a reduction in exposure levels could probably be achieved by changes in working routines and by the use of protective equipment.

Contractile properties of isolated muscle spindles of the frog.

Force and isotonic shortening velocities were studied (0.6-4.0 degrees C) in isolated single muscle spindles from the anterior tibialis muscle of Rana temporaria using techniques that enabled measurements both from the spindle as a whole and from marked segments of the preparation. The force-velocity relationship during tetanic stimulation exhibited the same biphasic shape as previously described for extrafusal muscle fibres. However, the maximum speed of shortening of the spindle fibres was merely 0.95 +/- 0.006 lengths s(-1) (mean +/- S.E.M., n = 11), which is approximately half the value recorded in extrafusal fibres of the same muscle. The maximum tetanic force, 91 +/- 10 kN m(-2), n = 14, was likewise only approximately half that produced by extrafusal fibres. The force generated by the capsule segment was lower than that produced by the whole spindle resulting in elongation of the capsule region during a fixed-end tetanus. The intracellular calcium ion concentration reached during the plateau of the tetanus, 1.7 +/- 0.1 microM (n = 8), was substantially lower than the value attained in extrafusal fibres under equivalent conditions. In accordance, the spindle fibres did not become fully activated during supramaximal electrical stimulation as indicated by the finding that the tetanic force could be further increased by 16.6 +/- 0.04 % (n = 5) on addition of 0.5 mM caffeine. Inadequate activation may thus, to a certain extent, account for the relatively low force per cross-sectional area of the spindle fibres. The contractile properties of the intrafusal fibres should make the spindle organ suited to provide feedback control during eccentric (forced lengthening) and static (isometric) contractions and, with reduced effectiveness, during slow muscle shortening.

X-ray structure of sensory rhodopsin II at 2.1-A resolution.

Sensory rhodopsins (SRs) belong to a subfamily of heptahelical transmembrane proteins containing a retinal chromophore. These photoreceptors mediate the cascade of vision in animal eyes and phototaxis in archaebacteria and unicellular flagellated algae. Signal transduction by these photoreceptors occurs by means of transducer proteins. The two archaebacterial sensory rhodopsins SRI and SRII are coupled to the membrane-bound HtrI and HtrII transducer proteins. Activation of these proteins initiates phosphorylation cascades that modulate the flagellar motors, resulting in either attractant (SRI) or repellent (SRII) phototaxis. In addition, transducer-free SRI and SRII were shown to operate as proton pumps, analogous to bacteriorhodopsin. Here, we present the x-ray structure of SRII from Natronobacterium pharaonis (pSRII) at 2.1-A resolution, revealing a unique molecular architecture of the retinal-binding pocket. In particular, the structure of pSRII exhibits a largely unbent conformation of the retinal (as compared with bacteriorhodopsin and halorhodopsin), a hydroxyl group of Thr-204 in the vicinity of the Schiff base, and an outward orientation of the guanidinium group of Arg-72. Furthermore, the structure reveals a putative chloride ion that is coupled to the Schiff base by means of a hydrogen-bond network and a unique, positively charged surface patch for a probable interaction with HtrII. The high-resolution structure of pSRII provides a structural basis to elucidate the mechanisms of phototransduction and color tuning.

X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate.

Studies on the catalytic mechanism and inhibition of serine proteases are widely used as paradigms for teaching enzyme catalysis. Ground-breaking work on the structures of chymotrypsin and subtilisin led to the idea of a conserved catalytic triad formed by the active site Ser, His and Asp residues. An oxyanion hole, consisting of the peptide amide of the active site serine and a neighbouring glycine, was identified, and hydrogen bonding in the oxyanion hole was suggested to stabilize the two proposed tetrahedral intermediates on the catalytic pathway. Here we show electron density changes consistent with the formation of a tetrahedral intermediate during the hydrolysis of an acyl-enzyme complex formed between a natural heptapeptide and elastase. No electron density for an enzyme-product complex was observed. The structures also suggest a mechanism for the synchronization of hydrolysis and peptide release triggered by the conversion of the sp2 hybridized carbonyl carbon to an sp3 carbon in the tetrahedral intermediate. This affects the location of the peptide in the active site cleft, triggering the collapse of a hydrogen bonding network between the peptide and the beta-sheet of the active site.