Stray light correction in the optical spectroscopy of crystals.

It has long been known in spectroscopy that light not passing through a sample, but reaching the detector (i.e., stray light), results in a distortion of the spectrum known as absorption flattening. In spectroscopy with crystals, one must either include such stray light or take steps to exclude it. In the former case, the derived spectra are not accurate. In the latter case, a significant amount of the crystal must be masked off and excluded. In this paper, we describe a method that allows use of the entire crystal by correcting the distorted spectrum.

Further studies with isolated absolute infrared spectra of bacteriorhodopsin photocycle intermediates: conformational changes and possible role of a new proton-binding center.

We recently published procedures describing the isolation of absolute infrared spectra for the intermediates of the bacteriorhodopsin (BR) photocycle and from these, obtaining transitional difference spectra between consecutive intermediates. In that work, we concentrated mainly on proton-binding centers and the route of proton transport across the membrane. In the current study, we used isolated spectra for the amide I, amide II, and amide III envelopes to obtain quantitative information on the extent of conformational change accompanying each transition in the photocycle. Our main finding was that most of the conformational changes occur in the conversion of the M(F) intermediate to N. In our earlier publication, a new proton acceptor, absorbing at 1650 cm(-1) was identified, which appeared to accept a proton from Asp96COOH during the transformation of BR† to L. Below, we present evidence that supports this interpretation and propose a possible role for this new component.

Infrared and visible absolute and difference spectra of bacteriorhodopsin photocycle intermediates.

We have used new kinetic fitting procedures to obtain infrared (IR) absolute spectra for intermediates of the main bacteriorhodopsin (bR) photocycle(s). The linear-algebra-based procedures of Hendler et al. (J. Phys. Chem. B, 105, 3319-3228 (2001)) for obtaining clean absolute visible spectra of bR photocycle intermediates were adapted for use with IR data. This led to isolation, for the first time, of corresponding clean absolute IR spectra, including the separation of the M intermediate into its M(F) and M(S) components from parallel photocycles. This in turn permitted the computation of clean IR difference spectra between pairs of successive intermediates, allowing for the most rigorous analysis to date of changes occurring at each step of the photocycle. The statistical accuracy of the spectral calculation methods allows us to identify, with great confidence, new spectral features. One of these is a very strong differential IR band at 1650 cm(-1) for the L intermediate at room temperature that is not present in analogous L spectra measured at cryogenic temperatures. This band, in one of the noisiest spectral regions, has not been identified in any previous time-resolved IR papers, although retrospectively it is apparent as one of the strongest L absorbance changes in their raw data, considered collectively. Additionally, our results are most consistent with Arg82 as the primary proton-release group (PRG), rather than a protonated water cluster or H-bonded grouping of carboxylic residues. Notably, the Arg82 deprotonation occurs exclusively in the M(F) pathway of the parallel cycles model of the photocycle.

The ability of actinic light to modify the bacteriorhodopsin photocycle revisited: heterogeneity vs photocooperativity.

In 1995, evidence both for photocooperativity and for heterogeneity as possible explanations for the ability of actinic light to modify the kinetics and pathways of the bacteriorhodopsin (BR) photocycle was reviewed ( Shrager, R. I., Hendler, R. W., and Bose, S. (1995) Eur. J. Biochem. 229, 589-595 ). Because both concepts could be successfully modeled to experimental data and there was suggestive published evidence for both, it was concluded that both photocooperativity and heterogeneity may be involved in the adaptation of the BR photocycle to different levels of actinic light. Since that time, more information has become available and it seemed appropriate to revisit the original question. In addition to the traditional models based on all intermediates in strict linear sequences, we have considered both homogeneous and heterogeneous models with branches. It is concluded that an explanation based on heterogeneity is more likely to be the true basis for the variation of the properties of the photocycle caused by changes in actinic light intensity. On the basis of new information presented here, it seems that a heterogeneous branched model is more likely than one with separate linear sequences.

Electrogenic proton-pumping capabilities of the m-fast and m-slow photocycles of bacteriorhodopsin.

The parallel model for the bacteriorhodopsin (BR) photocycle at neutral pH and a temperature near 20 degrees C contains an M-fast cycle with steps BR-->K-->L-->Mf-->N-->O-->BR and an M-slow cycle which contains steps BR-->K-->L-->Ms-->BR. With increasing actinic laser strength, the M-fast cycle at first rises faster than the M-slow cycle, but reaches saturation sooner and at a lower level than the M-slow cycle. The O-intermediate shows the same saturation behavior as Mf. In this paper, we show that the peak current of proton flux and the apparent voltages developed by this flux show the same saturation behavior as Ms, which is very different from that of both M f and O. It is further shown that most of the proton-charge displacement is connected with the step Ms-->BR. The optical and electrical data in these studies were collected simultaneously by a newly designed and built spectrometer which is described separately.

Simultaneous measurements of fast optical and proton current kinetics in the bacteriorhodopsin photocycle using an enhanced spectrophotometer.

A one-of-a-kind high speed optical multichannel spectrometer was designed and built at NIH and described in this journal in 1997 [J.W. Cole, R.W. Hendler, P.D. Smith, H.A. Fredrickson, T.J. Pohida, W.S. Friauf. A high speed optical multichannel analyzer. J Biochem Biophys Methods 1997;35:16-174.]. The most unique aspect of this instrument was the ability to follow an entire time course from a single activation using a single sample. The instrument has been used to study rapid kinetic processes in the photon-driven bacteriorhodopsin photocycle and electron transport from cytochrome c to cytochrome aa3 and from cytochrome aa3 to oxygen. The present paper describes a second generation instrument with a number of important enhancements which significantly improve its capabilities for multichannel kinetic studies. An example application is presented in which the kinetics of photon-induced proton flow across the biological membrane is measured simultaneously with the individual steps of the photocycle determined optically. Matching the time constants for the two processes indicates which molecular transformations are associated with major proton movements.

An apparent general solution for the kinetic models of the bacteriorhodopsin photocycles.

For the past decade, the field of Bacteriorhodopsin (BR) research has been influenced by a kinetic view of the photocycle as a reversible, homogeneous, model (RHM) with a linear sequence of intermediates. More recently, we proposed a much different model which consists of essentially unidirectional, parallel (i.e., heterogeneous) cycles (UPM) (Hendler, R. W.; Shrager, R. I.; Bose, S. J. Phys. Chem. B 2001, 105, 3319-3328). It is important to try to resolve which of the two models is more likely to be correct, because models influence and provide a basis for further experimentation. Therefore, in this communication, we reexamine the basis for the RHM with a focus on the most recent and complete description of this model (van Stokkum, I., H., M.; Lozier, R. J. Phys. Chem. B 2002, 106, 3477-3485) vis a vis the UPM in an in-depth study. We show that (i) the tested RHM does not really work for the data of van Stokkum and Lozier nor ours; (ii) no previously published RHM model has been shown to work for data under any conditions; (iii) there are many published observations that are difficult if not impossible to explain by RHM, but are readily explained by parallel cycles. It is also shown that either a UPM or a parallel cycle model with limited reversibility correctly describes photocycle data collected at pH 5, 7, and 9 and at 10, 20, and 30 degrees and is consistent with all known experimental observations.

Interconversions among four M-intermediates in the bacteriorhodopsin photocycle.

Halobacterium salinarum displays four distinct kinetic forms of M-intermediate in its bacteriorhodopsin photocycle. In wild-type, there are mainly two species with time constants near 2 and 5 ms. Under various kinds of stress, two other species arise with time constants near 10 and 70 ms. We show that these four species are interconvertible. Increases in membrane hydrophobicity convert the slower to faster forms. Perturbations caused by Triton X-100 or mutations convert faster to slower forms. The fastest form requires a hydrophobic membrane environment near a ring of four charged aspartate residues in the trimer, namely Asp36, Asp38, Asp102, and Asp104 in the cytoplasmic loop regions. Interconversions of the 2-ms and 5-ms species of the wild-type are accomplished by pH-changes. The potential significance of these findings is discussed.

Purple membrane lipid control of bacteriorhodopsin conformational flexibility and photocycle activity.

Specific lipids of the purple membrane of Halobacteria are required for normal bacteriorhodopsin structure, function, and photocycle kinetics [Hendler, R.W. & Dracheva, S. (2001) Biochemistry (Moscow)66, 1623-1627]. The decay of the M-fast intermediate through a path including the O intermediate requires the presence of a hydrophobic environment near four charged aspartic acid residues within the cytoplasmic loop region of the protein (R. W. Hendler & S. Bose, unpublished results). On the basis of the unique ability of squalene, the most hydrophobic purple membrane lipid, to induce recovery of M-fast activity in Triton-treated purple membrane, we proposed that this uncharged lipid modulates an electrostatic repulsion between the membrane surface of the inner trimer space and the nearby charged aspartic acids of the cytoplasmic loop region to promote transmembrane alpha-helical mobility with a concomitant increase in the speed of the photocycle. We examined Triton-treated purple membranes in various stages of reconstitution with native lipid suspensions using infrared spectroscopic techniques. We demonstrate a correlation between the vibrational half-width parameter of the protein alpha-helical amide I mode at 1660 cm-1, reflecting the motional characteristics of the transmembrane helices, and the lipid-induced recovery of native bacteriorhodopsin properties in terms of the visible absorbance maxima of ground state bacteriorhodopsin and the mean decay times of the photocycle M-state intermediates.