Protein-water interactions Water associated with proteins can be described as being one of the following three types: Free water Associated (bound) water. - презентация

1 Protein-water interactions Water associated with proteins can be described as being one of the following three types: Free water Associated (bound) water Tightly bound (structural) water The difference between association is the amount of energy it takes to remove the water. Free water removal requires little energy. Bound water removal is a difficult task. As bound water decreases a protein can move easily interact with another protein. PROTEIN HYDRATION SHELL BULK WATER exchange fast tumbling slow tumbling

2 proteinhydration water Mutual influence principle Conformation transitions in proteins result in redistribution of free/bound water ratio Observing the free/bound water state it is possible to study protein structural transitions protein hydration water

3 Dielectric permeability There are three dispersion regions for biological objects: lg f lg ' dispersion (0 to 10 Hz) - due to ion conductivity of membrane channels dispersion (10 Hz to 10 GHz) - due to relaxation of protein/membrane charges dispersion (10-20 GHz) - due to water molecule relaxation * = + i Dielectric permeability is a complex value: Debyes equation Why we measure dielectric parameters at 9.2 GHz? dielectric constant dielectric loss

4 IF: volume of dielectric (sample) << cavity resonator volume dielectric doesnt cause essential distortion of electrtic field in a cavity resonator The theory of small disturbances can be applied to calculate * f/f 0 where f - shift of resonance frequency of a cavity resonator upon sample entry = tg where tg =A/Q 0 (Q 0 /Q E -1), Q 0 and Q E - storage factors of resonator without and with sample. So to determine * one needs to: measure the resonance frequency and storage factors for cavity resonator without and with sample, calculate and tg. Q=f P / F, where f P - resonance frequency, F - width of resonance curve There are two ways to determine tg : take a resonance curve with each sample at each temperature OR measure of energy loss upon sample entry. In this case Q 0 /Q E =10 n/20, n - attenuation

5 Objects of investigation: albumin globular protein - albumin fibrinogen fibrillar protein - fibrinogen Experimental conditions: 0.15 M NaCl, pH , temperature C

6 Block diagram of UHF-dielectrometer UHF-power supply attenuation meter attenuation meter reference cavity resonator measuring cavity resonator detector automatic frequency control indicato r wattmeter © O.Nikolov, S.Gatash

7 Cylindrical cavity resonator with axial sample location capillary (diameter 2 mm) thermostabilisation shell cavity resonator

8 Dependencies of and on temperature for deionized water and 0.15 M NaCl. Dependencies of f d and S on temperature for deionized water and 0.15 M NaCl.

9 Dependencies of and on temperature for fibrinogen in 0.15 M NaCl

10 Dependencies of on temperature for albumin in 0.15 M NaCl

11 (t 0 ) and (t 0 ) dependencies are reversible

12 Dependencies of and on temperature for native and denatured fibrinogen in 0.15 M NaCl

13 Calculation of protein hydration from UHF-dielectrometry data In cm-wavelength diapason protein molecules and molecules of bound water have less mobility than free water molecules and values for solutions are less than and values for free water S = S water - S solution Protein hydration Dependencies of statical dielectric permeability decrement ( S ) on temperature for albumin in 0.15 M NaCl where S - statical dielectric permeability

14 Conclusions: The temperature dependencies of the dielectric parameters of fibrinogen and albumin solutions have a number of peculiarities at 8-10°C, 18-22°C and 35°C. At these temperatures redistribution of free and bound water in protein-water system occurs due to structural changes in protein molecules. The mechanism of proteins thermal stabilisation in physiological temperatures interval has similar character for fibrillar and globular proteins. The stabilisation of H-bonds network occurs in these temperature regions. Protein conformation changes observed can be responsible for high thermostability of proteins in physiological interval of temperatures.