Global Dynamics of Yeast Hsp90 Middle and C-Terminal Dimer Studied by Advanced Sampling Simulations.

The Hsp90 protein complex is one of the most abundant molecular chaperone proteins that assists in folding of a variety of client proteins. During its functional cycle it undergoes large domain rearrangements coupled to the hydrolysis of ATP and association or dissociation of domain interfaces. In order to better understand the domain dynamics comparative Molecular Dynamics (MD) simulations of a sub-structure of Hsp90, the dimer formed by the middle (M) and C-terminal domain (C), were performed. Since this MC dimer lacks the ATP-binding N-domain it allows studying global motions decoupled from ATP binding and hydrolysis. Conventional (c)MD simulations starting from several different closed and open conformations resulted in only limited sampling of global motions. However, the application of a Hamiltonian Replica exchange (H-REMD) method based on the addition of a biasing potential extracted from a coarse-grained elastic network description of the system allowed much broader sampling of domain motions than the cMD simulations. With this multiscale approach it was possible to extract the main directions of global motions and to obtain insight into the molecular mechanism of the global structural transitions of the MC dimer.

Multidomain structure and correlated dynamics determined by self-consistent FRET networks.

We present an approach that enables us to simultaneously access structure and dynamics of a multidomain protein in solution. Dynamic domain arrangements are experimentally determined by combining self-consistent networks of distance distributions with known domain structures. Local structural dynamics are correlated with the global arrangements by analyzing networks of time-resolved single-molecule fluorescence parameters. The strength of this hybrid approach is shown by an application to the flexible multidomain protein Hsp90. The average solution structure of Hsp90's closed state resembles the known X-ray crystal structure with Angstrom precision. The open state is represented by an ensemble of conformations with interdomain fluctuations of up to 25 Å. The data reveal a state-specific suppression of the submillisecond fluctuations by dynamic protein-protein interaction. Finally, the method enables localization and functional characterization of dynamic elements and domain interfaces.

Hearing threshold estimation using concurrent measurement of distortion product otoacoustic emissions and auditory steady-state responses.

Both distortion product otoacoustic emissions (DPOAEs) and auditory steady-state responses (ASSRs) provide frequency-specific assessment of hearing. However, each method suffers from some restrictions. Hearing losses above 50 dB HL are not quantifiable using DPOAEs and their performance at frequencies below 1 kHz is limited, but their recording time is short. In contrast, ASSRs are a time-consuming method but have the ability to determine hearing thresholds in a wider range of frequencies and hearing losses. Thus, recording DPOAEs and ASSRs simultaneously at their adequate frequencies and levels could decrease the overall test time considerably. The goal of the present study was to develop a parameter-setting and test-protocol to measure DPOAEs and ASSRs binaurally and simultaneously at multiple frequencies. Ten normal-hearing and 23 hearing-impaired subjects participated in the study. The interaction of both responses when stimulated simultaneously at frequencies between 0.25 and 6 kHz was examined. Two limiting factors need to be kept. Frequency distance between ASSR carrier frequency f(c) and DPOAE primary tone f(2) needs to be at least 1.5 octaves, and DPOAEs may not be measured if the ASSR stimulus level is 70 dB SPL or above. There was a significant correlation between pure-tone and DPOAE/ASSR-thresholds in sensorineural hearing loss ears.

Binaural measurement of bone conduction click evoked otoacoustic emissions in adults and infants.

Transient evoked otoacoustic emissions (TEOAEs) are usually evoked with air conduction (AC) stimuli. Only a few reports exist about OAEs where stimuli have been delivered using bone conduction (BC) by placing a bone conductor on the forehead or the mastoid. The aims of the present study were to improve the test performance of BC-TEOAEs by using a nonlinear stimulation protocol and to find out, whether this technique can be applied in newborn hearing screening. BC-TEOAEs were measured binaurally in ten normal hearing adults and in ten infants. For measurements in infants, miniaturized probes without loudspeakers were constructed to allow a complete insertion of the probe in the infant's ear canal. It could be shown that robust and valid BC-TEOAEs can be elicited using a nonlinear stimulation protocol. Findings in adults indicated that BC-TEOAEs can be measured with properties similar to AC-TEOAEs. However, mean BC-TEOAE levels were reduced by 0.8-3.7 dB depending on frequency. In view of test time, this is compensated by performing binaural recordings. Measurements in infants indicated that the screening performance of BC-TEOAEs and AC-TEOAEs may be comparable. Further studies have to investigate, whether BC-TEOAEs are more robust than AC-TEOAEs against small conductive hearing loss.