The outer hair cell (OHC) in the mammalian ear has a unique membrane potential-dependent motility, which is considered to be important for frequency discrimination (tuning). The OHC motile mechanism is located at the cell membrane and is strongly influenced by its passive mechanical properties. To study the viscoelastic properties of OHCs, we exposed cells to a hypoosmotic solution for varying durations and then punctured them, to immediately release the osmotic stress. Using video records of the cells, we determined both the imposed strain and the strain after puncturing, when stress was reset to zero. The strain data were described by a simple rheological model consisting of two springs and a dashpot, and the fit to this model gave a time constant of 40 +/- 19 s for the relaxation (reduction) of tension during prolonged strain. For time scales much shorter or longer than this, we would expect essentially elastic behavior. This relaxation process affects the membrane tension of the cell, and because it has been shown that membrane tension has a modulatory role in the OHC's motility, this relaxation process could be part of an adaptation mechanism, with which the motility system of the OHC can adjust to changing conditions and maintain optimum membrane tension.

Animals↗

PubMed1995

Ligand binding and protein dynamics in cupredoxins.

Type 1 copper sites bind nitric oxide (NO) in a photolabile complex. We have studied the NO binding properties of the type 1 copper sites in two cupredoxins, azurin and halocyanin, by measuring the temperature dependence of the ligand binding equilibria and the kinetics of the association reaction after photodissociation over a wide range of temperature (80-280 K) and time (10(-6)-10(2) s). In both proteins, we find nonexponential kinetics below 200 K that do not depend on the NO concentration. Consequently, this process is interpreted as geminate recombination. In azurin, the rebinding can be modeled with the Arrhenius law using a single pre-exponential factor of 10(8.3) s-1 and a Gaussian distribution of enthalpy barriers centered at 22 kJ/mol with a width [full width at half-maximum (FWHM)] of 11 kJ/mol. In halocyanin, a more complex behavior is observed. About 97% of the rebinding population can also be characterized by a Gaussian distribution of enthalpy barriers at 12 kJ/mol with a width of 6.0 kJ/mol (FWHM). The pre-exponential of this population is 1.6 x 10(12) s-1 at 100 K. After the majority population has rebound, a power-law phase that can be modeled with a gamma-distribution of enthalpy barriers is observed. Between 120 and 180 K, an additional feature that can be interpreted as a relaxation of the barrier distribution toward higher barriers shows up in the kinetics. Above 200 K, a slower, exponential rebinding appears in both cupredoxins. Since the kinetics depend on the NO concentration, this process is identified as bimolecular rebinding.(ABSTRACT TRUNCATED AT 250 WORDS)

Azurin↗

PubMed1992

Conformational substates in azurin.

Azurin is a small blue copper protein in the electron transfer chain of denitrifying bacteria. It forms a photolabile complex with nitric oxide (NO) at low temperatures. We studied the temperature dependence of the ligand binding equilibrium and the kinetics of the association reaction after photodissociation over a wide range of temperature (80-280 K) and time (10(-6)-10(2) s). The nonexponential rebinding below 200 K is independent of the NO concentration and is interpreted as internal recombination. The rebinding can be modeled with the Arrhenius law by using a single preexponential factor of 6.3 x 10(8) s-1 and a Gaussian distribution of enthalpy barriers centered at 23 kJ/mol with a width of 11 kJ/mol. Above 200 K, a slower, exponential rebinding process appears. The dependence of the kinetics on the NO concentration characterizes this reaction as bimolecular rebinding. The binding kinetics of NO to azurin show impressive analogies to the binding of carbon monoxide to myoglobin. We conclude that conformational substates occur not only in heme proteins but also in proteins with different active sites and secondary structures.

Azurin↗

PubMed1991

Ligand binding to heme proteins: connection between dynamics and function.

Ligand binding to heme proteins is studied by using flash photolysis over wide ranges in time (100 ns-1 ks) and temperature (10-320 K). Below about 200 K in 75% glycerol/water solvent, ligand rebinding occurs from the heme pocket and is nonexponential in time. The kinetics is explained by a distribution, g(H), of the enthalpic barrier of height H between the pocket and the bound state. Above 170 K rebinding slows markedly. Previously we interpreted the slowing as a "matrix process" resulting from the ligand entering the protein matrix before rebinding. Experiments on band III, an inhomogeneously broadened charge-transfer band near 760 nm (approximately 13,000 cm-1) in the photolyzed state (Mb*) of (carbonmonoxy)myoglobin (MbCO), force us to reinterpret the data. Kinetic hole-burning measurements on band III in Mb* establish a relation between the position of a homogeneous component of band III and the barrier H. Since band III is red-shifted by 116 cm-1 in Mb* compared with Mb, the relation implies that the barrier in relaxed Mb is 12 kJ/mol higher than in Mb*. The slowing of the rebinding kinetics above 170 K hence is caused by the relaxation Mb*----Mb, as suggested by Agmon and Hopfield [(1983) J. Chem. Phys. 79, 2042-2053]. This conclusion is supported by a fit to the rebinding data between 160 and 290 K which indicates that the entire distribution g(H) shifts. Above about 200 K, equilibrium fluctuations among conformational substates open pathways for the ligands through the protein matrix and also narrow the rate distribution. The protein relaxations and fluctuations are nonexponential in time and non-Arrhenius in temperature, suggesting a collective nature for these protein motions. The relaxation Mb*----Mb is essentially independent of the solvent viscosity, implying that this motion involves internal parts of the protein. The protein fluctuations responsible for the opening of the pathways, however, depend strongly on the solvent viscosity, suggesting that a large part of the protein participates. While the detailed studies concern MbCO, similar data have been obtained for MbO2 and CO binding to the beta chains of human hemoglobin and hemoglobin Zürich. The results show that protein dynamics is essential for protein function and that the association coefficient for binding from the solvent at physiological temperatures in all these heme proteins is governed by the barrier at the heme.

Binding Sites↗