Hydroxylamine as a thermal destabiliser of bacteriorhodopsin.
The light-catalysed reaction of hydroxylamine (HA) with retinal is one of the basic features of bacteriorhodopsin (BR). Surprisingly, according to recent results, neither the photocycle and proton pumping of BR, nor the trans-cis isomerisation of retinal is prerequisite for photobleaching of BR in the presence of HA. How, then, is the accessibility of retinal to HA enhanced on illumination? We studied whether local thermal denaturation of BR, proposed recently, could provide an explanation for HA-promoted bleaching. According to our results, HA does not alter the absorption spectrum and the photocycle kinetics of BR substantially at room temperature, even at molar concentrations, but grossly affects the temperature of thermal denaturation. At pH 7, the presence of 0.5 M: HA reduces the denaturation temperature from 100°C to as low as 72°C. The decrease is proportional to the logarithm of the HA concentration over more than three orders of magnitude, and even 0.5 mM: HA has a significant effect. In addition, photobleaching becomes considerably faster with increasing temperature in the presence of HA, it takes a few seconds at 50-60°C. Our results suggest that photobleaching of BR in the presence of HA can be explained by overall destabilisation of the structure of the protein and local thermal denaturation that has already accounted for the photobleaching of the HA-free BR at elevated temperatures. These results further support the importance of thermooptic effects in protein photoreactions and identify HA as a thermal destabiliser of BR.
Tokaji
Zsolt
Z
Institute of Biophysics, Biological Research Centre, P.O.B. 521, Szeged, 6701, Hungary. tokaji@brc.hu
Fodor
Elfrieda
E
Szabó-Nagy
Andrea
A
Páli
Tibor
T
D001436:Bacteriorhodopsins / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects; D004305:Dose-Response Relationship, Drug; D006217:Halobacterium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine / Q000494:pharmacology*; D007536:Isomerism; D007700:Kinetics; D038761:Photobleaching / Q000187:drug effects*; D011489:Protein Denaturation / Q000187:drug effects* / Q000528:radiation effects; D012172:Retinaldehyde / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D019811:Hydroxylamine; D001436:Bacteriorhodopsins; D012172:Retinaldehyde