Isotopically labeled tyrosines have been selectively incorporated into bacteriorhodopsin (bR). A comparison of the low-temperature bR570 to K Fourier transform infrared-difference spectra of these samples and normal bR provides information about the role of tyrosine in the primary phototransition. Several tyrosine contributions to the difference spectrum are found. These results and comparison with the spectra of model compounds suggest that a tyrosinate group protonates during the bR570 to K transition. This conclusion is strongly supported by the results of UV difference spectroscopy.Elucidation of the mechanism by which bacteriorhodopsin (bR), a light-driven proton pump in the purple membrane of Halobacterium halobium functions remains an important problem in biology (1). Two molecular events have been implicated in the bR primary phototransition. (i) An all-trans to 13-cis isomerization of the retinal chromophore has been deduced from resonance Raman measurements (2). (ii) Movement of a proton has been surmised from picosecond visible absorption data (3). It is not known, however, which bR groups are involved in proton transfer or how this event is coupled to retinal chromophore isomerization.Recently, our group and others (4-12) have begun to study the molecular alterations occurring during the bR photocycle with Fourier transform infrared (FTIR) spectroscopy. It has been demonstrated by this work that FTIR is sufficiently sensitive to detect changes occurring in single chemical groups in both the bR chromophore and the protein component. It is important for further progress that contributions to the FTIR-difference spectrum from specific amino acid residues be identified. This can be accomplished by selectively incorporating in bR isotopically labeled amino acids such as [E-15N]lysine (13). Such an approach was recently used by Englehard et al., who were able to identify the 1760 cm-1 carboxyl vibration (4, 7) as due to an aspartic acid residue (11).We report here the results of FTIR measurements on bR samples containing L-ring-deuterated tyrosine and L-[ring-4-13C]tyrosine (carbon label nearest hydroxyl group). We compare these difference spectra with FTIR measurements on model tyrosine compounds at high and low pH and p2H. Our results indicate that one or more tyrosines change during the bR570 to K phototransition and suggest that a tyrosinate group in bR570 becomes protonated by K. These conclusions are strongly supported by UV difference measurements, which also suggest the possible involvement of tryptophan at this stage of the photocycle. (14) and was purified by recrystallization. All isotope substitutions were verified by NMR spectroscopy. MATERIALS AND METHODSHalobacterium halobium R1 was grown in a synthetic medium like that of Gochnauer and Kushner (15) Purple membrane was isolated by the method of Oesterhelt and Stoeckenius (16). Specific activity measurements indicated that 50-80% of the tyrosine residues were labeled in various preparations. Amino acid analysis showed that <10% of t...

We have obtained room-temperature transient infrared difference spectra of the M412 photoproduct of bacteriorhodopsin (bR) by using a "rapid-sweep" Fouriertransform infrared (FT-IR) technique that permits the collection of an entire spectrum (extending from 1000 to 2000 cm-' with 8-cm'1 resolution) in 5 ms. These spectra exhibit <10-4 absorbance unit of noise, even utilizing wet samples containing -40 pmol of bR in the measuring beam. The bR --M transient difference spectrum is similar to FT-IR difference spectra previously obtained under conditions where M decay was blocked (low temperature or low humidity). In particular, the transient spectrum exhibits a set of vibrational difference bands that were previously attributed to protonation changes of several tyrosine residues on the basis of isotopic derivative spectra of M at low temperature. Our rapid-sweep FT-IR spectra demonstrate that these tyrosine/tyrosinate bands are also present under more physiological conditions. Despite the overall similarity to the low-temperature and low-humidity spectra, the room-temperature bR --M transient difference spectrum shows significant additional features in the amide I and amide II regions. These features' presence suggests that a small alteration of the protein backbone accompanies M formation under physiological conditions and that this conformational change is inhibited in the absence of liquid water.Infrared difference spectroscopy is a useful technique for measuring protein structural changes. Every residue has infrared-active group vibrations that are potentially sensitive to changes in covalent bonding (e.g., conformation, protonation state) and in noncovalent interactions with the surrounding environment (e.g., hydrogen bonding, steric hindrance). Although the presence of many IR-active groups in a large protein leads to a very complex IR spectrum, careful null measurements make it possible to observe only the small subset of vibrations that change during a biochemical transformation.The photoreactive proteins bacteriorhodopsin (bR) and rhodopsin are ideally suited for observing such difference spectra. By photolyzing these proteins inside a spectrometer, it has been possible to make very precise measurements of the resulting IR absorbance changes (1-12). These IR difference spectra have provided a wealth of information. For example, it has been shown that during the photoreaction of bR (Xmax = 568 nm) to M (Xmax = 412 nm), an aspartate residue becomes protonated (1, 7); additional protonation changes of carboxylic acid residues occur at other steps in the bR photocycle (7, 9, 10). More recently, IR difference spectra (along with UV difference spectra) have detected changes in protonation of several tyrosines in the photointermediates between bR and M (8-10). Such spectra provide important experimental tests of proposed proton-translocation mechanisms for bR.Early IR difference spectra of bR and rhodopsin photoproducts were obtained by using flash photolysis techniques and single-wavelength transient m...

The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR570----M412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labelling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M412. One group undergoes a tyrosinate----tyrosine conversion during the BR570----K630 transition. A second tyrosine group deprotonates between L550 and M412. Low-temperature UV difference spectra in the 220--350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbation(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR570 and M412, as indicated by infrared absorption changes in the 1770--1720-cm-1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups.

We present results on using cooperative interactions to shield liposomes by incorporating multiple hydrophobic anchoring sites on polyethylene glycol (PEG) polymers. The hydrophobically-modified PEGs (HMPEGs) are comb-graft polymers with strictly alternating monodisperse PEG blocks (M(w)=6, 12, or 35 kDa) bonded to C18 stearylamide hydrophobes. Cooperativity is varied by changing the degree of oligomerization at a constant ratio of PEG to stearylamide. Fusogenic liposomes prepared from N-C12-DOPE:DOPC 7:3 (mol:mol) were equilibrated with HMPEGs. Affinity for polymer association to liposomes increases with the degree of oligomerization; equilibrium constants (given as surface coverage per equilibrium concentration of free polymer) for 6 kDa PEG increased from 6.1+/-0.8 (mg/m(2))/(mg/ml) for 2.5 loops to 78.1+/-12.2 (mg/m(2))/(mg/ml) for 13 loops. In contrast, the equilibrium constant for distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG5k) was 0.4+/-0.1 (mg/m(2))/(mg/ml). The multi-loop HMPEGs demonstrate higher levels of protection from complement binding than DSPE-PEG5k. Greater protection does not correlate with binding strength alone. The best shielding was by HMPEG6k-DP3 (with three 6 kDa PEG loops), suggesting that PEG chains with adequate surface mobility provide optimal protection from complement opsonization. Complement binding at 30 min and 12 h demonstrates that protection by multi-looped PEGs is constant whereas DSPE-PEG5k initially protects but presumably partitions off of the surface at longer times.

Aluminum adjuvants are commonly used in prophylactic vaccines to enhance antigen immunogenicity through induction of high-titer antibody responses. Three major forms of aluminum adjuvants with substantially different physical and chemical properties have been described: aluminum phosphate (AlPO(4)), aluminum hydroxide (AlOH) and amorphous aluminum hydroxyphosphate sulfate (AAHS). Here we describe the effect of these different aluminum adjuvants on the formulation and subsequent immunogenicity in mice of virus-like particles (VLPs) consisting of the L1 protein of Human Papillomavirus (HPV) Type 16. Electron microscopy demonstrated that the physical appearance of the phosphate-containing aluminum adjuvants was markedly different from that of aluminum hydroxide. All three aluminum adjuvants were found to display unique surface charge profiles over a range of pH, while AAHS demonstrated the greatest inherent capacity for adsorption of L1 VLPs. These differences were associated with differences in immunogenicity: anti-HPV L1 VLP responses from mice immunized with AAHS-formulated HPV16 vaccine were substantially greater than those produced by mice immunized with the same antigen formulated with aluminum hydroxide. In addition, HPV L1 VLPs formulated on AAHS also induced a substantial interferon-gamma secreting T cell response to L1 peptides indicating the potential for an enhanced memory response to this antigen. These results indicate that the chemical composition of aluminum adjuvants can have a profound influence on the magnitude and quality of the immune response to HPV VLP vaccines.