Protein structural change at the cytoplasmic surface as the cause of cooperativity in the bacteriorhodopsin photocycle

The effects of excitation light intensity on the kinetics of the bacteriorhodopsin photocycle were investigated. The earlier reported intensity-dependent changes at 410 and 570 nm are explained by parallel increases in two of the rate constants, for proton transfers to D96 from the Schiff base and from the cytoplasmic surface, without changes in the others, as the photoexcited fraction is increased. Thus, it appears that the pKa of D96 is raised by a cooperative effect within the purple membrane. This interpretation of the wild-type kinetics was confirmed by results with several mutant proteins, where the rates are well separated in time and a model-dependent analysis is unnecessary. Based on earlier results that demonstrated a structural change of the protein after deprotonation of the Schiff base that increases the area of the cytoplasmic surface, and the effects of high hydrostatic pressure and lowered water activity on the photocycle steps in question, we suggest that the pKa of D96 is raised by a lateral pressure that develops when other bacteriorhodopsin molecules are photoexcited within the two-dimensional lattice of the purple membrane. Expulsion of no more than a few water molecules bound near D96 by this pressure would account for the calculated increase of 0.6 units in the pKa

Picosecond and Nanosecond Components in Bacteriorhodopsin Light-Induced Electric Response Signal

Numerous investigations on the primary events of the bacteriorhodopsin photocycle indicate that the first steps of the energy transformation process take place in the 500 fs-5 ps region. These processes are known to be followed by others in the μs and ms regions. Recent observations indicate also the existence of nanosecond intermediate(s). Here we are reporting on direct measurements of the light-induced electric response signal of purple membrane carried out in the ps and ns regions. The laser flash-induced electric response of dried oriented purple membrane samples were detected by an ultrafast sampling oscilloscope. The measured kinetic curves were analyzed by exponential fitting and by a simulation-optimization method taking into account the time characteristics of the measuring setup. This analysis revealed a two phase real charge separation process. The first phase (tau = 21±2 ps) coincides well with the overall bR-[unk] K transition. The second phase (tau = 6±0.5 ns) can be correlated with the nanosecond optical transitions reported by several workers, or may be an optically silent charge movement inside the protein moiety or on the surface of the membrane

Characterization of the Proton-Transporting Photocycle of Pharaonis Halorhodopsin

AbstractThe photocycle of pharaonis halorhodopsin was investigated in the presence of 100mM NaN3 and 1M Na2SO4. Recent observations established that the replacement of the chloride ion with azide transforms the photocycle from a chloride-transporting one into a proton-transporting one. Kinetic analysis proves that the photocycle is very similar to that of bacteriorhodopsin. After K and L, intermediate M appears, which is missing from the chloride-transporting photocycle. In this intermediate the retinal Schiff base deprotonates. The rise of M in halorhodopsin is in the microsecond range, but occurs later than in bacteriorhodopsin, and its decay is more accentuated multiphasic. Intermediate N cannot be detected, but a large amount of O accumulates. The multiphasic character of the last step of the photocycle could be explained by the existence of a HR′ state, as in the chloride photocycle. Upon replacement of chloride ion with azide, the fast electric signal changes its sign from positive to negative, and becomes similar to that detected in bacteriorhodopsin. The photocycle is enthalpy-driven, as is the chloride photocycle of halorhodopsin. These observations suggest that, while the basic charge translocation steps become identical to those in bacteriorhodopsin, the storage and utilization of energy during the photocycle remains unchanged by exchanging chloride with azide

Biológiai membránok elektromos térszerkezetének vizsgálata = Investigation of the charge structure of biological membranes

A pályázat célja a biológiai membránok fizikai tulajdonságainak vizsgálata volt, különös tekintettel a töltéseloszlás és - ezzel összefüggésben - az elektromos térszerkezet funkcionális szerepére. Modellobjektumunk a protonpumpáló bakteriorodopszin (bR) fehérjét kristályos rendben tartalmazó bíbormembrán. Kutatásainkat és a velük kapcsolatos módszertani fejlesztőmunkát három téma köré csoportosítottuk: 1. A molekulán belüli elektromos töltéseloszlás funkcionális leírása 2. A határfelületi vízréteg szerepe a fehérjék szerkezetében és működésében 3. A bR nemlineáris optikai tulajdonságainak jellemzése Eredmények: 1.Megmutattuk, hogy az általunk kifejlesztett módszer a molekula működését kísérő szerkezetváltozásokat érzékenyen jellemzi, és lehetőséget teremt az elektromos töltésátrendeződések funkcionális értelmezésére. 2.Rámutattunk, hogy a semleges sók anionjainak tulajdonított Hofmeister-effektus a fehérje-víz határfelületi réteg szerkezetváltozásaival magyarázható. Elméletünk elsőként ad teljeskörű kvalitatív leírást a 120 éves problémakörre. 3.Integrált optikai módszerekkel az eddigieknél érzékenyebben detektáltuk a bR molekulában - a lokális elektromos tér változásai következtében ? fellépő abszorpció- és törésmutató-változásokat. Megmutattuk, hogy a molekula gerjesztését követő spektrális változások alkalmasak fénykapcsolásra és -modulálásra. Az eredmények gyakorlati alkalmazása az optoelektronikában és a bioszenzorikában várható. | In the framework of the project, we investigated some physical properties of biological membranes, with special respect to the physiological role of the electric charge distribution (hence the electric field structure). Our model object was purple membrane, containing the paradigmatic proton pumping molecule bacteriorhodopsin (bR) in a quasi crystalline structure. Our research work and the related methodological developments were centered round three topics: 1.Functional description of the intra-molecular charge distribution 2.The role of interfacial water in the structure and function of proteins 3.Characterization of the nonlinear optical properties of bR Results: 1.We showed that our method represents a sensitive test for the characterization of structural changes associated to molecular function, and allows interpretation of charge rearrangements. 2.We pointed out that the Hofmeister effect, attributed to the anions of neutral salts, can be interpreted according to structural changes in the protein-water interface. Our theory gives a full-spectrum qualitative description, solving a 120-years old puzzle. 3.Using integrated optical (IO) techniques, we could sensitively characterize the nonlinear optical properties of bR films, and showed that IO light switching and modulation can be performed based on the light-induced spectral changes of bR. Practical utilization of the results is expected in optoelectronics and sensorics