Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism.

The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle.

Oscillatory activity reflects differential use of spatial reference frames by sighted and blind individuals in tactile attention.

Touch can be localized either on the skin in anatomical coordinates, or, after integration with posture, in external space. Sighted individuals are thought to encode touch in both coordinate systems concurrently, whereas congenitally blind individuals exhibit a strong bias for using anatomical coordinates. We investigated the neural correlates of this differential dominance in the use of anatomical and external reference frames by assessing oscillatory brain activity during a tactile spatial attention task. The EEG was recorded while sighted and congenitally blind adults received tactile stimulation to uncrossed and crossed hands while detecting rare tactile targets at one cued hand only. In the sighted group, oscillatory alpha-band activity (8-12Hz) in the cue-target interval was reduced contralaterally and enhanced ipsilaterally with uncrossed hands. Hand crossing attenuated the degree of posterior parietal alpha-band lateralization, indicating that attention deployment was affected by external spatial coordinates. Beamforming suggested that this posture effect originated in the posterior parietal cortex. In contrast, cue-related lateralization of central alpha-band as well as of beta-band activity (16-24Hz) were unaffected by hand crossing, suggesting that these oscillations exclusively encode anatomical coordinates. In the blind group, central alpha-band activity was lateralized, but did not change across postures. The pattern of beta-band activity was indistinguishable between groups. Because the neural mechanisms for posterior alpha-band generation seem to be linked to developmental vision, we speculate that the lack of this neural mechanism in blind individuals is related to their preferred use of anatomical over external spatial codes in sensory processing.

The effect of endodontic access preparation on the failure load resistance of a 3Y-TZP monolithic zirconia crown.

The effect of endodontic access preparation on the failure load resistance of 3Y-TZP zirconia crowns was accomplished by preparing human molars and luting monolithic zirconia crowns with a self-adhesive resin cement. Besides the intact control, teeth received endodontic access preparations and then grouped (n = 12) into a positive control (no access repair), dentin core replacement only and complete access repair groups. Specimens were axially tested until failure with results of no significant difference between the failure load of intact controls and the complete access repair group. However, the positive control and dentin replacement only groups failed at significantly lower loads. Under the conditions of this study, there was no significant failure load difference between 3Y-TZP monolithic zirconia crowns with repaired endodontic access preparations to that evidenced by an unprepared control. Although this evidence is encouraging, caution is advised and definitive recommendations cannot be made until verified by clinical studies.

Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics.

Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone's catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms.

The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation.

Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.

Alpha-band oscillations reflect external spatial coding for tactile stimuli in sighted, but not in congenitally blind humans.

We investigated the function of oscillatory alpha-band activity in the neural coding of spatial information during tactile processing. Sighted humans concurrently encode tactile location in skin-based and, after integration with posture, external spatial reference frames, whereas congenitally blind humans preferably use skin-based coding. Accordingly, lateralization of alpha-band activity in parietal regions during attentional orienting in expectance of tactile stimulation reflected external spatial coding in sighted, but skin-based coding in blind humans. Here, we asked whether alpha-band activity plays a similar role in spatial coding for tactile processing, that is, after the stimulus has been received. Sighted and congenitally blind participants were cued to attend to one hand in order to detect rare tactile deviant stimuli at this hand while ignoring tactile deviants at the other hand and tactile standard stimuli at both hands. The reference frames encoded by oscillatory activity during tactile processing were probed by adopting either an uncrossed or crossed hand posture. In sighted participants, attended relative to unattended standard stimuli suppressed the power in the alpha-band over ipsilateral centro-parietal and occipital cortex. Hand crossing attenuated this attentional modulation predominantly over ipsilateral posterior-parietal cortex. In contrast, although contralateral alpha-activity was enhanced for attended versus unattended stimuli in blind participants, no crossing effects were evident in the oscillatory activity of this group. These findings suggest that oscillatory alpha-band activity plays a pivotal role in the neural coding of external spatial information for touch.

Task demands affect spatial reference frame weighting during tactile localization in sighted and congenitally blind adults.

Task demands modulate tactile localization in sighted humans, presumably through weight adjustments in the spatial integration of anatomical, skin-based, and external, posture-based information. In contrast, previous studies have suggested that congenitally blind humans, by default, refrain from automatic spatial integration and localize touch using only skin-based information. Here, sighted and congenitally blind participants localized tactile targets on the palm or back of one hand, while ignoring simultaneous tactile distractors at congruent or incongruent locations on the other hand. We probed the interplay of anatomical and external location codes for spatial congruency effects by varying hand posture: the palms either both faced down, or one faced down and one up. In the latter posture, externally congruent target and distractor locations were anatomically incongruent and vice versa. Target locations had to be reported either anatomically ("palm" or "back" of the hand), or externally ("up" or "down" in space). Under anatomical instructions, performance was more accurate for anatomically congruent than incongruent target-distractor pairs. In contrast, under external instructions, performance was more accurate for externally congruent than incongruent pairs. These modulations were evident in sighted and blind individuals. Notably, distractor effects were overall far smaller in blind than in sighted participants, despite comparable target-distractor identification performance. Thus, the absence of developmental vision seems to be associated with an increased ability to focus tactile attention towards a non-spatially defined target. Nevertheless, that blind individuals exhibited effects of hand posture and task instructions in their congruency effects suggests that, like the sighted, they automatically integrate anatomical and external information during tactile localization. Moreover, spatial integration in tactile processing is, thus, flexibly adapted by top-down information-here, task instruction-even in the absence of developmental vision.