Exploring tryptophan dynamics in acid-induced molten globule state of bovine α-lactalbumin: a wavelength-selective fluorescence approach

The relevance of partially ordered states of proteins (such as the molten globule state) in cellular processes is beginning to be understood. Bovine α-lactalbumin (BLA) assumes the molten globule state at acidic pH. We monitored the organization and dynamics of the functionally important tryptophan residues of BLA in native and molten globule states utilizing the wavelength-selective fluorescence approach and fluorescence quenching. Quenching of BLA tryptophan fluorescence using quenchers of varying polarity (acrylamide and trichloroethanol) reveals varying degrees of accessibility of tryptophan residues, characteristic of native and molten globule states. We observed red edge excitation shift (REES) of 6 nm for the tryptophans in native BLA. Interestingly, we show here that BLA tryptophans exhibit REES (3 nm) in the molten globule state. These results constitute one of the early reports of REES in the molten globule state of proteins. Taken together, our results indicate that tryptophan residues in BLA in native as well as molten globule states experience motionally restricted environment and that the regions surrounding at least some of the BLA tryptophans offer considerable restriction to the reorientational motion of the water dipoles around the excited-state tryptophans. These results are supported by wavelength-dependent changes in fluorescence anisotropy and lifetime for BLA tryptophans. These results could provide vital insight into the role of tryptophans in the function of BLA in its molten globule state in particular, and other partially ordered proteins in general.     

Organization and dynamics of tryptophans in the molten globule state of bovine α-lactalbumin utilizing wavelength-selective fluorescence approach: Comparisons with native and denatured states

Bovine α-lactalbumin (BLA) is known to be present in molten globule form in its apo-state (i.e., Ca²⁺ depleted state). We explored the organization and dynamics of the functionally important tryptophan residues of BLA in native, molten globule and denatured states utilizing the wavelength-selective fluorescence approach. We observed red edge excitation shift (REES) of 7 nm for the tryptophans in native BLA. Interestingly, we show here that BLA tryptophans exhibit considerable REES (8 nm) in its molten globule state. Taken together, these results indicate that tryptophan residues in BLA in native as well as molten globule states experience motionally restricted environment. We further show that even the denatured form of BLA exhibits a modest REES of 3 nm, indicating that the tryptophans are shielded from bulk solvent, even when denatured, due to the presence of residual structure around tryptophan(s). This is further supported by wavelength-dependent changes in fluorescence anisotropy and lifetime for BLA tryptophans. These novel results constitute one of the first reports of REES in the molten globule state of proteins, and could provide vital insight into the role of tryptophans in the function of BLA in its molten globule state in particular, and other partially ordered proteins in general.     

Multisite fluorescence in proteins with multiple tryptophan residues. Apomyoglobin natural variants and site-directed mutants

Time-resolved fluorescence experiments were carried out on a variety of apomyoglobins with one or two tryptophan (Trp) residues located at invariant positions 7 and 14 in the primary sequence. In all cases, the Trp fluorescence kinetics were resolved adequately into two discrete lifetime domains, and decay-associated spectra (DAS) were obtained for each decay component. The DAS resolved for unfolded proteins were indistinguishable by position of the emission maxima and the spectral shapes. The folded proteins revealed noticeable differences in the DAS, which relate to the diverse local environments around the Trp residues in the individual proteins. Furthermore, the DAS of wild-type protein possessing two Trp residues were simulated well by that of one Trp mutants either in the native, molten globule, or unfolded states. Overall, employing Trp fluorescence and site-directed mutagenesis allowed us to highlight the conformational changes induced by the single amino acid replacement and generate novel structural information on equilibrium folding intermediates. Specifically, it was found that conformational fluctuations in the local cluster around the evolutionarily conserved Trp(14) are very similar in the native and molten globule states of apomyoglobins. This result indicates that residues in the E and B helices contributing to this cluster are most likely involved in the stabilization of the overall architecture of the structured molten globule intermediate.     

Structure and dynamics of the alpha-lactalbumin molten globule: fluorescence studies using proteins containing a single tryptophan residue

The fluorescence properties of three variants of alpha-lactalbumin (alpha-LA) containing a single tryptophan residue were investigated under native, molten globule, and unfolded conditions. These proteins have levels of secondary structure and stability similar to those of the wild type. The fluorescence signal in the native state is dominated by that of W104, with the signal of W60 and W118 significantly quenched by the disulfide bonds in their vicinity. In the molten globule state, the magnitude of the fluorescence signal of W60 and W118 increases, due to the loss of rigid, specific side chain packing. In contrast, the magnitude of the signal of W104 decreases in the molten globule state, perhaps due to the protonation of H107 or quenching by D102 or K108. The solvent accessibilities of individual tryptophan residues were investigated by their fluorescence emission maximum and by acrylamide quenching studies. In the native state, the order of solvent accessibility is as follows: W118 > W60 > W104. This order changes to W60 > W104 > W118 in the molten globule state. Remarkably, the solvent accessibility of W118 in the alpha-LA molten globule is lower than that in the native state. The dynamic properties of the three tryptophan residues were examined by time-resolved fluorescence anisotropy decay studies. The overall rotation of the molecule can be observed in both the native and molten globule states. In the molten globule state, there is an increase in the extent of local backbone fluctuations with respect to the native state. However, the fluctuation is not sufficient to result in complete motional averaging. The three tryptophan residues in the native and molten globule states have different degrees of motional freedom, reflecting the folding pattern and dynamic heterogeneity of these states. Taken together, these studies provide new insight into the structure and dynamics of the alpha-LA molten globule, which serves as a prototype for partially folded proteins.     

Molten globule versus variety of intermediates: influence of anions on pH-denatured apomyoglobin.

The molten globule state was shown to be the third thermodynamic state of protein molecules in addition to their native and unfolded states. On the other hand, it was reported that optical and hydrodynamic properties of pH-denatured apomyoglobin depend on the nature of anions added to the protein solution. This observation was used to conclude that there are many 'partly folded' intermediates between the native and unfolded states rather than one distinct molten globule state. However, little is known on the structures of pH-denatured apomyoglobin in the presence of different anions. Two tyrosine residues in horse apomyoglobin have been successively modified by the reaction with tetranitromethane. This approach was employed to measure the distances between tryptophans and modified tyrosines in different states of apomyoglobin by the method of direct energy transfer. Experimental data show that the distance between the middle of the A-helix and the beginning of the G-helix and/or the end of the H-helix in 'anion-induced' states are very close to those in the native holo- and apomyoglobins. This suggests that the AGH helical complex, being the most structured part of apomyoglobin in the molten globule state, exists also in pH-denatured apomyoglobin in the presence of different anions. Consequently, all non-native forms of apomyoglobin studied so far share the common important feature of its native structure.     

Dynamics of the core tryptophan during the formation of a productive molten globule intermediate of barstar

The evolution of the nanosecond dynamics of the core tryptophan, Trp53, of barstar has been monitored during the induction of collapse and structure formation in the denatured D form at pH 12, by addition of increasing concentrations of the stabilizing salt Na(2)SO(4). Time-resolved fluorescence methods have been used to monitor the dynamics of Trp53 in the intermediates that are populated during the salt-induced transition of the D form to the molten globule B form. The D form approximates a random coil and displays two rotational correlation times. A long rotational correlation time of 2.54 ns originates from segmental mobility, and a short correlation time of 0.26 ns originates from independent motion of the tryptophan side chain. Upon addition of approximately 0.1 M Na(2)SO(4), the long rotational correlation time increases to approximately 6.4 ns, as the chain collapses and the segmental motions merge to reflect the global tumbling motion of a pre-molten globule P form. The P form exists as an expanded form with approximately 30% greater volume than the native (N) state. The persistence of an approximately 50% contribution to anisotropy decay by the short rotational correlation time suggests that the core of the P form is highly molten and permits free rotation of the Trp side chain. With increasing salt concentrations, tight core packing is achieved before secondary and tertiary structure formation is complete, an observation which agrees well with earlier kinetic folding studies. Thus, the equilibrium model developed here for describing the formation of structure during folding faithfully captures snapshots of transient kinetic intermediates observed on the folding pathway of barstar. A comparison of the refolding kinetics at pH 7, when refolding is initiated from the D, P, and B forms, suggests that formation of a collapsed state with a rigid core and approximately 30% secondary and tertiary structure, which presumably defines a coarse native-like topology, constitutes the intrinsic barrier in the folding of barstar.     

Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies.

Steady-state and lifetime-resolved fluorescence anisotropy measurements of protein fluorescence were used to investigate the depolarizing motions of tryptophan residues in proteins. Lifetime resolution was achieved by oxygen quenching. The proteins investigated were carbonic anhydrase, carboxypeptidase A, alpha-chymotrypsin, trypsin, pepsin, and bovine and human serum albumin. When corrected for overall protein rotation, the steady state anisotropies indicate that, on the average, the tryptophan residues in these proteins rotate 29 degrees +/- 6 degrees during the unquenched excited state lifetimes of these proteins, which range from 1.7 to 6.1 ns. The lifetime-resolved anisotropies reveal correlation times for these displacements ranging from 1 to 12 ns. On the average these correlation times are tenfold shorter than that expected for overall protein rotation. We conclude that the tryptophan residues in these proteins display remarkable freedom of motion within the protein matrix, which implies that these matrices are highly flexible on the nanosecond time scale.     

Fluorescence determination of tryptophan side-chain accessibility and dynamics in triple-helical collagen-like peptides

We report tryptophan fluorescence measurements of emission intensity, iodide quenching, and anisotropy that describe the environment and dynamics at X and Y sites in stable collagen-like peptides of sequence (Gly-X-Y)(n). About 90% of tryptophans at both sites have similar solvent exposed fluorescence properties and a lifetime of 8.5-9 ns. Analysis of anisotropy decays using an associative model indicates that these long lifetime populations undergo rapid depolarizing motion with a 0.5 ns correlation time; however, the extent of fast motion at the Y site is considerably less than the essentially unrestricted motion at the X site. About 10% of tryptophans at both sites have a shorter ( approximately 3 ns) lifetime indicating proximity to a protein quenching group; these minor populations are immobile on the peptide surface, depolarizing only by overall trimer rotation. Iodide quenching indicates that tryptophans at the X site are more accessible to solvent. Side chains at X sites are more solvent accessible and considerably more mobile than residues at Y sites and can more readily fluctuate among alternate intermolecular interactions in collagen fibrils. This fluorescence analysis of collagen-like peptides lays a foundation for studies on the structure, dynamics, and function of collagen and of triple-helical junctions in gelatin gels.     

Fluorescence lifetime and spectral study of the acid expansion of bovine serum albumin

The fluorescence lifetimes of the tryptophan residues of bovine serum albumin were measured in the native and acid-expanded conformation. A three-exponential process is required to fit the fluorescence decay data. The results are interpreted empirically in terms of two emitting species. The emission at longer wavelength (360 nm) has slower rates of decay than that at shorter wavelength (325 nm). For both emitting species the average lifetime decreases when the N-F transition occurs and shortens further when the protein expands. Rotational correlation times, derived from the decay of the fluorescence anisotropy of the tryptophan residues, suggest that longer emission wavelengths are associated with somewhat shorter correlation times. There is no certain indication of any independent motion of the tryptophans in any conformation, although some very fast process, perhaps Raman scattering, appears to occur. On acid expansion the long correlation times decrease to around 10 ns in the fully expanded form. Static quenching experiments using I- or acrylamide suggest a greater average exposure of the tryptophans when the protein is most greatly expanded. This is despite the fact that the fluorescence emission maximum shifts to shorter wavelength under these conditions. Also, there is no difference in accessibility to quenching between the longer and shorter wavelength emissions.     

Two steps in the transition between the native and acid states of bovine α-lactalbumin detected by circular polarization of luminescence: Evidence for a premolten globule state?

A few studies indirectly support the existence of an intermediate in the transition of Ca(2+)-saturated bovine alpha-lactalbumin (alpha-LA) from the native (N) to the acidic (A) state, known as the molten globule state. However, direct experimental evidence for the appearance of this intermediate has not been obtained. The signal of circular polarization of luminescence (CPL) is sensitive to fine conformational transitions because of its susceptibility to changes in the environmental asymmetry of fluorescent chromophores in their excited electronic states. In the present study, CPL measurements were applied using the intrinsic tryptophan fluorescence of alpha-LA as well as the fluorescence of 8-anilino-1-naphthalenesulfonic acid (ANS) bound to alpha-LA. CPL of tryptophan and ANS was measured in the pH range of 2.5-6 in order to find direct experimental evidence for the proposed intermediate. CPL (characterized by the emission anisotropy factor, g(em)) depends on the asymmetry of the protein molecular structure in the environment of the tryptophan and the ANS chromophores in the excited electronic state. The pH dependence of both the gab, absorption anisotropy factor determined by CD, and the ANS steady state fluorescence, showed a single transition at pH 3-3.7 as already reported elsewhere. This transition was interpreted as being a result of a change of the alpha-LA tertiary structure, which resulted in a loss of asymmetry of the environment of both the tryptophan residues and the ANS hydrophobic binding sites. The pH dependence of the tryptophan and ANS g(em) showed an additional conformational transition at pH 4-5, which coincided with the pKa of Ca2+ dissociation (pKa 5), as predicted by Permyakov et al. (1981, Biochem Biophys Res Commun 100:191-197). The titration curve showed that there is a pH range between 3.7 and 4.1 in which alpha-LA exists in an intermediate state between the N- and A-state. We suggest that the intermediate is the premolten globule state characterized by a reduced Ca2+ binding to the alpha-LA, native-like tertiary structure, and reduced asymmetric fluctuation of the tertiary structure on the nanosecond time scale. This intermediate resembles the "critical activated state" theoretically deduced by Kuwajima et al. (1989, J Mol Biol 206:547-561). The present study demonstrates the power of CPL measurements for the investigation of folding/unfolding transitions in proteins.     

Direct energy transfer to study the 3D structure of non-native proteins: AGH complex in molten globule state of apomyoglobin.

The direct energy transfer technique was modified and applied to probe the relative localization of apomyoglobin A-, G- and H-helixes, which are partly protected from deuterium exchange in the equilibrium molten globule state and in the molten globule-like kinetic intermediate. The non-radiative transfer of tryptophan electronic energy to 3-nitrotyrosine was studied in different conformational states of apomyoglobin (native, molten globule, unfolded) and interpreted in terms of average distances between groups of the protein chain. The experimental data show that the distance between the middle of A-helix and the N-terminus of G-helix as well as the distance between the middle of the A-helix and the C-terminus of the H-helix in the molten globule state are close to those in the native state. This is a strong argument in favor of similarity of the overall architecture of the molten globule and native states.     

Increased exposure of hydrophobic surface in molten globule state of alpha-lactalbumin. Fluorescence and hydrophobic photolabeling studies.

The involvement of molten globule state as a distinct intermediate in the denaturation process in proteins is well documented. However, the structural characterization of such an intermediate is far from complete. We have, using fluorescence and fluorescence quenching, studied the molten globule state of bovine alpha-lactalbumin. Unlike the native state, where all the 4 tryptophans are buried in the protein, 2 tryptophans are exposed in the molten globule state. Using the hydrophobic photoactivable reagent [3H]diazofluorene, we observe an increased hydrophobic exposure in the molten globule state. These structural characteristics conform to the current views on the molten globule state, i.e. it has similar secondary structure but a poorly defined tertiary structure. Our fluorescence studies indicate the involvement of a premolten globule state in the native to molten globule state transition. This premolten globule state exists at pH 5.0 and has a very compact structure involving increased hydrophobic interactions in the protein interior. These results are also supported by circular dichroism studies.     

Defining the core structure of the alpha-lactalbumin molten globule state.

Molten globules are partially folded states of proteins which are generally believed to mimic structures formed during the folding process. In order to determine the minimal requirements for the formation of a molten globule state, we have prepared a set of peptide models of the molten globule state of human alpha-lactalbumin (alphaLA). A peptide consisting of residues 1-38 crosslinked, via the native 28-111 disulfide bond, to a peptide corresponding to residues 95-120 forms a partially folded state at pH 2.8 which has all of the characteristics of the molten globule state of alphaLA as judged by near and far UV CD, fluorescence, ANS binding and urea denaturation experiments. The structure of the peptide construct is the same at pH 7.0. Deletion of residues 95-100 from the construct has little effect. Thus, less than half the sequence is required to form a molten globule. Further truncation corresponding to the selective deletion of the A (residues 1-19) or D (residues 101-110) helices or the C-terminal 310 helix (residues 112-120) leads to a significant loss of structure. The loss of structure which results from the deletion of any of these three regions is much greater than that which would be expected based upon the non-cooperative loss of local helical structure. Deletion of residues corresponding to the region of the D helix or C-terminal 310 helix region results in a peptide construct which is largely unfolded and contains no more helical structure than is expected from the sum of the helicity of the two reduced peptides. These experiments have defined the minimum core structure of the alphaLA molten globule state.     

Identification and thermodynamic characterization of molten globule states of periplasmic binding proteins

Molten globule-like intermediates have been shown to occur during protein folding and are thought to be involved in protein translocation and membrane insertion. However, the determinants of molten globule stability and the extent of specific packing in molten globules is currently unclear. Using far- and near-UV CD and intrinsic and ANS fluorescence, we show that four periplasmic binding proteins (LBP, LIVBP, MBP, and RBP) form molten globules at acidic pH values ranging from 3.0 to 3.4. Only two of these (LBP and LIVBP) have similar sequences, but all four proteins adopt similar three-dimensional structures. We found that each of the four molten globules binds to its corresponding ligand without conversion to the native state. Ligand binding affinity measured by isothermal titration calorimetry for the molten globule state of LIVBP was found to be comparable to that of the corresponding native state, whereas for LBP, MBP, and RBP, the molten globules bound ligand with approximately 5-30-fold lower affinity than the corresponding native states. All four molten globule states exhibited cooperative thermal unfolding assayed by DSC. Estimated values of DeltaCp of unfolding show that these molten globule states contain 28-67% of buried surface area relative to the native states. The data suggest that molten globules of these periplasmic binding proteins retain a considerable degree of long range order. The ability of these sequentially unrelated proteins to form highly ordered molten globules may be related to their large size as well as an intrinsic property of periplasmic binding protein folds.     

Application of a reference convolution method to tryptophan fluorescence in proteins. A refined description of rotational dynamics

A reference method for the deconvolution of polarized fluorescence decay data is described. Fluorescence lifetime determinations for p-terphenyl, p-bis[2-(5-phenyloxazolyl)]benzene and N-acetyltryptophanamide (AcTrpNH2) show that with this method more reliable fits of the decays can be made than with the scatterer method, which is most frequently used. Analysis of the AcTrpNH2 decay with p-terphenyl as the reference compound yields an excellent fit with lifetimes of 2.985 ns for AcTrpNH2 and 1.099 ns for p-terphenyl (20 degrees C), whereas the AcTrpNH2 decay cannot be satisfactorily fitted when the scatterer method is used. The frequency of the detected photons is varied to determine the conditions where pulse pile-up starts to affect the measured decays. At detection frequencies of 5 kHz and 15 kHz, which corresponds to 1.7% and 5% respectively of the rate of the excitation photons no effects are found. Decays measured at 30 kHz (10%) are distorted, indicating that pile-up effects play a role at this frequency. The fluorescence and fluorescence anisotropy decays of the tryptophan residues in the proteins human serum albumin, horse liver alcohol dehydrogenase and lysozyme have been reanalysed with the reference method. The single tryptophan residue of the albumin is shown to be characterized by a triple-exponential fluorescence decay. The anisotropy decay of albumin was found to be mono-exponential with a rotational correlation time of 26 ns (20 degrees C). The alcohol dehydrogenase has two different tryptophan residues to which single lifetimes are assigned. It is found that the rotational correlation time for the dehydrogenase changes with excitation wavelength (33 ns for lambda ex = 295 nm and 36 ns for lambda ex = 300 nm at 20 degrees C), indicating a nonspherical protein molecule. Lysozyme has six tryptophan residues, which give rise to a triple-exponential fluorescence decay. A single-exponential decay with a rotational correlation time of 3.8 ns is found for the anisotropy. This correlation time is significantly shorter than that arising from the overall rotation and probably originates from intramolecular, segmental motion.     

The molten globule state of a chimera of human alpha-lactalbumin and equine lysozyme.

The molten globule state of equine lysozyme is more stable than that of alpha-lactalbumin and is stabilized by non-specific hydrophobic interactions and native-like hydrophobic interactions. We constructed a chimeric protein which is produced by replacing the flexible loop (residues 105-110) in human alpha-lactalbumin with the helix D (residues 109-114) in equine lysozyme to investigate the possible role of the helix D for the high stability and native-like packing interaction in the molten globule state of equine lysozyme. The stability of the molten globule state formed by the chimeric protein to guanidine hydrochloride-induced unfolding is the same as that of equine lysozyme and is substantially greater than that of human alpha-lactalbumin, although only six residues come from equine lysozyme. Our results also suggest that the non-native interaction in the molten globule state of alpha-lactalbumin changes to the native-like packing interaction due to helix substitution. The solvent-accessibility of the Trp residues in the molten globule state of the chimeric protein is similar to that in the molten globule state of equine lysozyme in which packing interaction around the Trp residues in the native state is partially preserved. Therefore, the helix D in equine lysozyme is one of the contributing factors to the high stability and native-like packing interaction in the molten globule state of equine lysozyme. Our results indicate that the native-like packing interaction can stabilize the rudimentary intermediate which is stabilized by the non-specific hydrophobic interactions.     

Detection and characterization of an ovine placental lactogen stable intermediate in the urea-induced unfolding process.

The urea-induced equilibrium unfolding of ovine placental lactogen, purified from ovine placenta, was followed by size-exclusion chromatography, far-UV CD, and intrinsic tryptophan fluorescence. The data obtained by each of these methods showed a poor fit to a two-state model involving only a native and an unfolded form. A satisfactory fit required, instead, a model that involved a stable, partially folded form in addition to the native and unfolded ones. The results obtained from the best-fitting theoretical curves for the three-state model indicated that this intermediate state, which is the predominant species in solution at 3.6 M of urea activity, is compact, largely alpha-helical, and changes considerably the native-like tertiary packing around its tryptophan residues. These findings suggest that this stable intermediate exhibits properties similar to those that characterize the molten globule state.     

Lipid binding specificity of bovine α-lactalbumin: A multidimensional approach

Many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. Bovine α-lactalbumin (BLA) represents an excellent prototype for monitoring membrane interaction due to its conformational plasticity. In this work, we comprehensively monitored the interaction of apo-BLA with zwitterionic and negatively charged membranes utilizing a variety of approaches. We show that BLA preferentially binds to negatively charged membranes at acidic pH with higher binding affinity. This is supported by spectral changes observed with a potential-sensitive membrane probe and fluorescence anisotropy measurements of a hydrophobic probe. Our results show that BLA exhibits a molten globule conformation when bound to negatively charged membranes. We further show, using the parallax approach, that BLA penetrates the interior of negatively charged membranes, and tryptophan residues are localized at the membrane interface. Red edge excitation shift (REES) measurements reveal that the immediate environment of tryptophans in membrane-bound BLA is restricted, and the restriction is dependent on membrane lipid composition. We envision that understanding the mechanism of BLA–membrane interaction would help in bioengineering of α-lactalbumin, and to address the mechanism of tumoricidal and antimicrobial activities of BLA–oleic acid complex.     

Calorimetric determination of the energetics of the molten globule intermediate in protein folding: apo-alpha-lactalbumin

High-sensitivity differential scanning calorimetry has been used to characterize the energetics of the molten globule state of apo-alpha-lactalbumin. This characterization has been possible by performing temperature scans at different guanidine hydrochloride (GuHCl) concentrations in order to experimentally define the temperature-GuHCl stability surface of the protein. Multidimensional analysis of the heat capacity surface has allowed simultaneous resolution of the energetics of the unfolded and molten globule states. These experiments indicate that the intrinsic enthalpy difference (i.e., excluding additional contributions such as those arising from differential GuHCl binding) between the unfolded and native states is 31.8 kcal/mol at 25 degrees C whereas that of the molten globule and native states is only 7.7 kcal/mol. At the same temperature, the entropy changes are 99.2 and 23.7 cal/K.mol and the heat capacity changes are 1821 and 326 cal/K.mol, respectively. Analysis of the thermodynamic data indicates that in passing from the native to the molten globule state only approximately 19% of the hydrogen bonds are broken. In addition, the magnitude of delta Cp for the molten globule suggests that water does not largely penetrate into the interior of the molten globule, implying that significant hydrophobic interactions are still present in this state. These parameters provide precise energetic constraints to the allowed structural conformations of the molten globule.     

Rhodopsin and its thermal intermediates: Fast structural fluctuations in their protein component

The tryptophan residues of bovine rhodopsin have been employed as intrinsic fluorescent probes for investigating the dynamics of rhodopsin and of the thermal intermediates of its bleaching at low temperature. A fast depolarization of the fluorescence of the tryptophans on the nanosecond time scale has been observed for all cases. The fluorescence spectra for the various intermediates shift progressively to the red as a function of time following excitation with nanosecond light pulses. It is inferred that rhodopsin possesses flexibility in the vicinity of its tryptophans that permits rapid structural fluctuations to take place.