Co-rebinding in myoglobin as seen by time-resolved X-ray absorption spectroscopy

The dynamics of selected conformational coordinates, key roles in the understanding of the CO-rebinding process, are investigated in horse heart carbonmonoxy myoglobin (MbCO) through time-resolved X-ray absorption spectroscopy. We present here the results obtained at 90 K in the second time scale. The approach of the CO molecule towards the Fe atom in the active site pocket is speculated to act as a natural precursor to the Fe displacement with the consequent undoming of the protein porphyrin plane. The arrangement of the Fe-C-O bonding angle geometry follows and the final MbCO active site configuration is completely reached within 1 min.     

Detection of a pH-dependent conformational change in azurin by time-resolved phosphorescence.

Azurin, a blue copper protein from the bacterial species Pseudomonas aeruginosa, contains a single tryptophan residue. Previous fluorescence measurements indicate that this residue is highly constrained and unusually inaccessible to water. In the apoprotein this residue also possesses a long-lived room-temperature phosphorescence (RTP), the nonexponential decay of which can be resolved into two major components associated with lifetimes of 417 and 592 ms, which likely originate from at least two conformations of the protein. The relative weights of these two decay components change with pH in good correlation with a change in protonation of His-35, which has been studied in Cu(II) azurin. Interestingly, the structural changes characterized in earlier work have little effect on the fluorescence decay and appear to occur away from the tryptophan residue. However, in the present work, the two RTP lifetimes suggest conformations with different structural rigidities in the vicinity of the tryptophan residue. The active conformation that predominates below a pH of 5.6 has the shorter lifetime and is less rigid. Phosphorescence decays of several metal derivatives of azurin were also measured and revealed strong similarities to that of apoazurin, indicating that the structural constraints upon the metal-binding site are imposed predominately by the protein.     

Picosecond structural dynamics of myoglobin following photodissociation of carbon monoxide as revealed by ultraviolet time-resolved resonance Raman spectroscopy

Picosecond protein dynamics of myoglobin in response to structural changes in heme upon CO dissociation were observed in a site-specific fashion for the first time using time-resolved UV resonance Raman spectroscopy. Transient UV resonance Raman spectra showed several phases of intensity changes in both tryptophan and tyrosine Raman bands. Five picoseconds after dissociation, the W18, W16, and W3 bands of tryptophan residues and the Y8a band of tyrosine residues decreased in intensity, followed by recovery of the Y8a band intensity in hundreds of picoseconds and recovery of the tryptophan bands in nanoseconds. These spectral changes suggest that the change in heme structure impulsively drives concerted movement of the EF helical section and that rearrangements toward a deoxy structure occur in the heme vicinity and in the A helix within a time frame of sub-nanoseconds to nanoseconds.     

Characterization of transient protein folding intermediates during myoglobin reconstitution by time-resolved electrospray mass spectrometry with on-line isotopic pulse labeling

A novel technique for studying protein folding kinetics is presented. It is based on a continuous-flow setup that is coupled to an electrospray (ESI) mass spectrometer and allows initiation of a folding reaction, followed by isotopic pulse labeling. The protein is electrosprayed "quasi-instantaneously" after exposure to the deuterated solvent. This approach yields structural information from the ESI charge state distribution and from the H/D exchange levels of individual protein states, while at the same time noncovalent interactions can be monitored. This technique is used to study the reconstitution of holomyoglobin (hMb) from unfolded apomyoglobin (aMb) and free heme. MS/MS is used to establish that a short-lived folding intermediate with two heme groups attached represents a protein-bound heme dimer. This state appears to have a compactness close to that of native hMb; however, isotopic labeling indicates a significantly perturbed structure. Another intermediate is bound to a single heme group and shows a charge state distribution similar to that of unfolded aMb. Exchange levels exhibited by this state are lower than for unfolded aMb, indicating that fewer hydrogens are exposed to the solvent and/or that more of them are involved in hydrogen bonding. Native hMb leads to the formation of low charge state ions (hMb(9+), hMb(8+)) and shows low exchange levels. However, early during reconstitution, a slightly unfolded form of the heme-protein complex contributes to the observed hMb(9+) ions. A peak width analysis reveals that the structural heterogeneity of some of the observed protein species decreases as reconstitution proceeds.     

Protein conformational relaxation and ligand migration in myoglobin: a nanosecond to millisecond molecular movie from time-resolved Laue X-ray diffraction.

A time-resolved Laue X-ray diffraction technique has been used to explore protein relaxation and ligand migration at room temperature following photolysis of a single crystal of carbon monoxymyoglobin. The CO ligand is photodissociated by a 7.5 ns laser pulse, and the subsequent structural changes are probed by 150 ps or 1 micros X-ray pulses at 14 laser/X-ray delay times, ranging from 1 ns to 1.9 ms. Very fast heme and protein relaxation involving the E and F helices is evident from the data at a 1 ns time delay. The photodissociated CO molecules are detected at two locations: at a distal pocket docking site and at the Xe 1 binding site in the proximal pocket. The population by CO of the primary, distal site peaks at a 1 ns time delay and decays to half the peak value in 70 ns. The secondary, proximal docking site reaches its highest occupancy of 20% at approximately 100 ns and has a half-life of approximately 10 micros. At approximately 100 ns, all CO molecules are accounted for within the protein: in one of these two docking sites or bound to the heme. Thereafter, the CO molecules migrate to the solvent from which they rebind to deoxymyoglobin in a bimolecular process with a second-order rate coefficient of 4.5 x 10(5) M(-1) s(-1). Our results also demonstrate that structural changes as small as 0.2 A and populations of CO docking sites of 10% can be detected by time-resolved X-ray diffraction.     

Probing local structural fluctuations in myoglobin by size-dependent thiol-disulfide exchange

All proteins undergo local structural fluctuations (LSFs) or breathing motions. These motions are likely to be important for function but are poorly understood. LSFs were initially defined by amide hydrogen exchange (HX) experiments as opening events, which expose a small number of backbone amides to ¹H/²H exchange, but whose exchange rates are independent of denaturant concentration. Here, we use size-dependent thiol-disulfide exchange (SX) to characterize LSFs in single cysteine-containing variants of myoglobin (Mb). SX complements HX by providing information on motions that disrupt side chain packing interactions. Most importantly, probe reagents of different sizes and chemical properties can be used to characterize the size of structural opening events and the properties of the open state. We use thiosulfonate reagents (126–274 Da) to survey access to Cys residues, which are buried at specific helical packing interfaces in Mb. In each case, the free energy of opening increases linearly with the radius of gyration of the probe reagent. The slope and the intercept are interpreted to yield information on the size of the opening events that expose the buried thiol groups. The slope parameter varies by over 10-fold among Cys positions tested, suggesting that the sizes of breathing motions vary substantially throughout the protein. Our results provide insight to the longstanding question: how rigid or flexible are proteins in their native states?     

The primary event in vision investigated by time-resolved fluorescence spectroscopy.

The picosecond fluorescence kinetics and quantum yield from bovine rhodopsin were measured in the 5-40 degrees K range. The fluorescence rise and decay times are faster than our resolution of 15 ps (full width at half maximum) over this entire temperature range. The size of the observed emission was also temperature independent, and we find that the upper limit of rhodopsin's fluorescence quantum yield to be phi f approximately equal to 10(-5). Replacing all of rhodopsin's exchangeable protons with deuterons by suspending rhodopsin in D2O had no effect on either the kinetics of the emission or the value of the quantum yield. Our data provide strong confirmation of the idea that the first step in the visual process is an excited-state cis-to-trans isomerization about the C11-C12 double bond of retinal.     

Multiple conformational states in myoglobin revealed by frequency domain fluorometry

The tryptophanyl fluorescence decays of two myoglobins, i.e., sperm whale and tuna myoglobin, have been examined in the frequency domain with an apparatus which utilizes the harmonic content of a mode-locked laser. Data analysis was performed in terms of continuous distribution of lifetime having a Lorentzian shape. Data relative to sperm whale myoglobin, which possesses two tryptophanyl residues, i.e., Trp-A-5 and -A-12, provided a broad lifetime distribution including decay rates from a few picoseconds to about 10 ns. By contrast, the tryptophanyl lifetime distribution of tuna myoglobin, which contains only Trp-A-12, showed two well-separated and narrow Lorentzian components having centers at about 50 ps and 3.37 ns, respectively. In both cases, the chi 2 obtained from distribution analysis was lower than that provided by a fit using the sum of exponential components. The long-lived components present in the fluorescence decay of the two myoglobins do not correspond to any of those observed for the apoproteins at neutral pH. The tryptophanyl lifetime distribution of sperm whale apomyoglobin consists of two separated Lorentzian components centered at 2.25 and 5.4 ns, whereas that of tuna apomyoglobin consists of a single Lorentzian component, whose center is at 2.19 ns. Acidification of apomyoglobin to pH 3.5 produced a shift of the distribution centers toward longer lifetimes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic analysis of efficient heme rotation in myoglobin by NMR spectroscopy.

Sperm whale myoglobin was reconstituted with 1,4,5,8-tetramethylhemin. The hyperfine-shifted proton NMR signals from the prosthetic group exhibit remarkable pattern changes around 15 degrees C, while the globin resonances are normal to obey the Curie law. The NMR anomaly specifically observed for the heme signals suggests a slow to rapid rotational transition of the hemin about the iron-histidine bond. The temperature-dependent pattern changes were quantitatively analyzed by a dynamic NMR method. Two sets of analyses with the heme-methyl and pyrrole-proton lines consistently afforded delta H not equal to = 16.3 kcal/mol, delta S not equal to = 14.0 e.u., delta G not equal to = 12.1 kcal/mol at 298 K, and a frequency of 90 degrees heme rotation 5600 s-1 at 20 degrees C. The relatively large activation entropy suggests that structural rearrangements at the direct heme vicinity are involved and that efficient heme rotation is accomplished by a number of fluctuative local heme-globin contacts within a conserved crevice structure.     

Monitoring the conformational changes of photoactivated rhodopsin from microseconds to seconds by transient fluorescence spectroscopy

The transient changes of the tryptophan fluorescence of bovine rhodopsin in ROS membranes were followed in time from 1 micros to 10 s after flash excitation of the photoreceptor. Up to about 100 micros the fluorescence did not change, suggesting that the tryptophan lifetimes in rhodopsin and the M(I) intermediate are similar. The fluorescence then decreases on the millisecond time scale with kinetics that match the rise of the M(II) state as measured on the same sample by the transient absorption increase at 360 nm. Both the sign and kinetics of the fluorescence change strongly suggest that it is due to an increase in energy transfer to the retinylidene chromophore caused by the increased spectral overlap in M(II). Calculation of the Forster radius of each tryptophan from the high-resolution crystal structure suggests that W265 and W126 are already completely quenched in the dark, whereas W161, W175, and W35 are located at distances from the retinal chromophore that are comparable to their Forster radii. The fluorescence from these residues is thus sensitive to an increase in energy transfer in M(II). Similar results were obtained at other temperatures and with monomeric rhodopsin in dodecyl maltoside micelles. A large light-induced transient fluorescence increase was observed with ROS membranes that were selectively labeled with Alexa594 at cysteine 316 in helix 8. Using transient absorption spectroscopy the kinetics of this structural change at the cytoplasmic surface was compared to the formation of the signaling state M(II) (360 nm) and to the kinetics of proton uptake as measured with the pH indicator dye bromocresol purple (605 nm). The fluorescence kinetics lags behind the deprotonation of the Schiff base. The proton uptake is even further delayed. These observations show that in ROS membranes (at pH 6) the sequence of events is Schiff base deprotonation, structural change, and proton uptake. From the temperature dependence of the kinetics we conclude that the Schiff base deprotonation and the transient fluorescence have comparable activation energies, whereas that of proton uptake is much smaller.     

Breathing mode of conformational fluctuations in globular proteins.

Globular proteins in the native state are assumed to behave as continuous elastic spheres in the low frequency breathing motions. Reasonable values of Young's modulus E = 10(11) dyne/cm2 and the radius of the sphere ro = 20 A, yield a wave number of 26 cm-1 for the fundamental vibration of the sphere. The peak at around 30 cm-1 in the laser Raman spectra of native alpha-chymotrypsin and pepsin observed by Brown et al. might be assigned to the breathing motion which the native proteins undergo as continuous elastic bodies.     

Solvent effects on myoglobin conformational substates as studied by electron paramagnetic resonance.

Electronic paramagnetic resonance spectra of frozen horse myoglobin solutions at two different pH values and with different added organic solvents are analyzed by computer simulation in terms of Gaussian distributions of some ferric ion crystal field parameters. The mean values and the corresponding variances of these distributions, thought as arising from a distribution of the protein conformational substates, are found to be affected by both the pH and the addition of organic solvents. The significant narrowing of the conformational substate distribution, induced by large addition of glycerol, is discussed.     

Picosecond dynamics of bacteriorhodopsin, probed by time-resolved infrared spectroscopy.

The photoinduced reaction cycle of bacteriorhodopsin (BR) has been studied by means of a recently developed picosecond infrared spectroscopic method at ambient temperature. BR - K difference spectra between 1560 and 1700 cm-1 have been recorded at delay times from 100 ps to 14 ns. The spectrum remains unchanged during this period. The negative difference OD band at 1660 cm-1 indicates the peptide backbone responds within 50 ps. A survey in the region of carboxylic side chain absorption around 1740 cm-1 reveals that perturbations of those groups, present in low-temperature FTIR spectra, are not observable within 10 ns, suggesting a slow conformational change.     

Immunological measurements of conformational motility in regions of the myoglobin molecule.

The conformational motilities of three regions of the sperm whale myoglobin molecule and of an isolated peptide of myoglobin have been examined by measuring the equilibrium constant for the native equilibrium nonnative transition. The immunological approach of Furie et al. (Furie, B., Schechter, A.N., Sachs D., and Anfinsen, C.B. (1975), J. Mol. Biol.92, 497-506) was used with convenient modifications. Antibodies specific to the nonnative conformations were used in assaying for competition between the radioactively labeled peptide and native myoglobin. Labeling was by 125I iodination of the peptide or its 3-(4-hydroxyphenyl)propionyl derivative, and separation of the immune complex from the free peptide was either by ammonium sulfate precipitation or by centrifugation of the antibodies immobilized on Agarose beads. For the antigenic regions of the sequence (1-55), the measured conformational equilibrium constant was 840 +/- 200 at 22 degrees C; the value for the C-terminal region (132-153) was 280 +/- 120 at 25 degrees C, while that for the region (66-76) adjacent to the heme group was greater than 2.5 x 10(6). Measurements on the isolated peptide (132-153) indicated that 1% of the molecules adopt native-type folding in aqueous solution at 36 degrees C.     

Absorption spectra indicate conformational alteration of myoglobin adsorbed on polydimethylsiloxane.

To assess the effects of adsorption on protein structure, ultraviolet optical absorption spectra of myoglobin (Mb) bound to polydimethylsiloxane (PDMS) were measured. A flow cell, which enabled adsorption under controlled hydrodynamic conditions, was used in conjunction with a conventional spectrophotometer to obtain the spectra. Adsorption to PDMS reduced significantly the absorbance in the Soret region of the Mb spectrum, whereas the spectrum in the region near 280 nm was essentially unaffected. This result showed that disruption of the native structure of Mb occurs following interaction with PDMS. Furthermore, the change in the absorption spectrum may indicate loss of heme from the heme pocket of the adsorbed protein. Mb structure was altered from its solution configuration within fifteen min of contact with the surface. Exchange of adsorbed Mb with Mb in solution had little or no effect on the absorption spectrum of the surface-confined protein, indicating that exchange occurs only between conformationally altered species or between native species.     

Real-time monitoring of yeast cell division by dielectric spectroscopy.

To assay cell cycle progression in synchronized culture of yeast we have applied dielectric spectroscopy to its real-time monitoring. The dielectric monitoring is based on the electromagnetic induction method, regarded as a nonelectrode method, which has resolved the problems encountered in measurements with metal electrodes, namely electrode polarization and bubble formation on electrodes. In the synchronized culture with temperature-sensitive cell division cycle mutants, the permittivity of the culture broth showed cyclic changes at frequencies below 300 kHz. The increase and decrease in the cyclic changes of the relative permittivity correspond to the increase in cell length and bud size and to the septum formation between mother and daughter cells, respectively.     

Observation of small conformational changes in the sperm-whale myoglobin by the far-infrared spectra

Small conformational changes in a molecule of sperm-whale myoglobin in its native solid state for different pH values at room temperature as well as during heat denaturation in alkali medium at different stages of unfolding of the globule were observed by using far-infrared spectroscopy in the region from 30 to 600 cm^?1. The changes appeared in the absorption bands near 420 and 470 cm^?1 ascribed to the side-chain vibrations of helical segments of the myoglobin molecule. For the first time the high structural sensitivity of the far-infrared region of the skeletal vibrations has been confirmed experimentally and the applicability of this technique to globular proteins demonstrated.     

Real-time observation of fluid flows in tissue during stress relaxation using Raman spectroscopy

This paper outlines a technique to measure fluid levels in articular cartilage tissue during an unconfined stress relaxation test. A time series of Raman spectrum were recorded during relaxation and the changes in the specific Raman spectral bands assigned to water and protein were monitored to determine the fluid content of the tissue. After 1000 s unconfined compression the fluid content of the tissue is reduced by an average of 3.9% ± 1.7%. The reduction in fluid content during compression varies between samples but does not significantly increase with increasing strain. Further development of this technique will allow mapping of fluid distribution and flows during dynamic testing making it a powerful tool to understand the role of interstitial fluid in the functional performance of cartilage.     

Chromophore structure in lumirhodopsin and metarhodopsin I by time-resolved resonance Raman microchip spectroscopy.

Time-resolved resonance Raman microchip flow experiments have been performed on the lumirhodopsin (Lumi) and metarhodopsin I (Meta I) photointermediates of rhodopsin at room temperature to elucidate the structure of the chromophore in each species as well as changes in protein-chromophore interactions. Transient Raman spectra of Lumi and Meta I with delay times of 16 micros and 1 ms, respectively, are obtained by using a microprobe system to focus displaced pump and probe laser beams in a microfabricated flow channel and to detect the scattering. The fingerprint modes of both species are very similar and characteristic of an all-trans chromophore. Lumi exhibits a relatively normal hydrogen-out-of-plane (HOOP) doublet at 951/959 cm(-1), while Meta I has a single HOOP band at 957 cm(-1). These results suggest that the transitions from bathorhodopsin to Lumi and Meta I involve a relaxation of the chromophore to a more planar all-trans conformation and the elimination of the structural perturbation that uncouples the 11H and 12H wags in bathorhodopsin. Surprisingly, the protonated Schiff base C=N stretching mode in Lumi (1638 cm(-1)) is unusually low compared to those in rhodopsin and bathorhodopsin, and the C=ND stretching mode shifts down by only 7 cm(-1) in D2O buffer. This indicates that the Schiff base hydrogen bonding is dramatically weakened in the bathorhodopsin to Lumi transition. However, the C=N stretching mode in Meta I is found at 1654 cm(-1) and exhibits a normal deuteration-induced downshift of 24 cm(-1), identical to that of the all-trans protonated Schiff base. The structural relaxation of the chromophore-protein complex in the bathorhodopsin to Lumi transition thus appears to drive the Schiff base group out of its hydrogen-bonded environment near Glu113, and the hydrogen bonding recovers to a normal solvated PSB value but presumably a different hydrogen bond acceptor with the formation of Meta I.