A dynamical investigation of acrylodan-labeled mutant phosphate binding protein

The static and dynamical behavior of a fluorescently labeled mutant of the Escherichia coli periplasmic phosphate binding protein (PBP) was investigated through steady-state and time-resolved fluorescence spectroscopy. As a means of developing a biorecognition element for inorganic phosphate (P(i)), alanine-197 of PBP was replaced with a cysteine. This site was then labeled with an environmentally sensitive fluorophore. The fluorescence emission of the mutant PBP labeled with acrylodan (MPBP-AC) proved to be sensitive to micromolar concentrations of P(i), as indicated by a 50% increase in the steady-state emission intensity. Steady-state results indicated that the labeling protocol was specific for cys-197 only and did not label the wild-type PBP; thus, a site-selective labeling protocol was developed. Time-resolved measurements were used to determine the influence of the dynamics of MPBP-AC on the process of signal transduction. Time-resolved anisotropy measurements revealed that rotational dynamics were best described by a model with two independent motions: the global motion of the protein and the local motion of the acrylodan probe. The rates of both global and local rotational reorientation of MPBP-AC were faster when the protein was P(i)-bound rather than P(i)-free. This was a result of structural changes involving or surrounding both the P(i)-binding site (global changes) and the residues in near proximity to the fluorescent reporter group (local changes). Recovery of the semiangle (theta) indicated that local structural changes in MPBP-AC took place when P(i) was bound to the protein. Acrylodan gained mobility when MPBP-AC bound P(i), as indicated by the fact that theta increased by approximately 5 degrees. In addition, dynamic quenching measurements confirmed that structural changes occurred locally near the cys-197. Acrylodan became more accessible to iodide when MPBP-AC bound P(i), as demonstrated by the 35% increase in the value of the bimolecular quenching constant.     

Accessibility of the fluorescent reporter group in native, silica-adsorbed, and covalently attached acrylodan-labeled serum albumins

Fluorescence quenching techniques are used to investigate the accessibility of a model biorecognition element-reporter group system when in buffer, surface-adsorbed, and covalently attached to a silica surface. The site-selective fluorescent reporter group, 6-acryloyl(dimethylamino)naphthalene (acrylodan, Ac), is attached covalently (at cysteine-34) to bovine and human serum albumin (BSA and HSA, respectively) and serves as a surrogate recognition element-reporter group system. Molecular oxygen is used to quench the Ac fluorescence and the accessibility, in the form of bimolecular rate constants (kq), in each model system is quantified. Although one might expect these systems to exhibit similar behavior, differences in quenching characteristics are observed, such as wavelength dependency of the Stern-Volmer quenching constant (KSV) for the native proteins in buffer. BSA-Ac exhibits wavelength dependent KSV values as well as a blue-shifted emission spectrum on O2 addition. Physisorption of BSA-Ac onto a fused-silica optical fiber lowers the accessibility of Ac to O2, whereas covalent attachment of BSA-Ac to APTES/glutaraldehyde-modified silica enhances the accessibility of the Ac reporter group to O2.     

On the mobility of riboflavin 5'-phosphate in Megasphaera elsdenii flavodoxin as studied by 13C-nuclear-magnetic-resonance relaxation

The mobility of the isoalloxazine ring of the prosthetic group of Megasphaera elsdenii flavodoxin was investigated by a 13C relaxation study of the non-protonated ring atoms 2, 4, 4a and 10a. In this study a selectively enriched (greater than 90% 13C) prosthetic group was bound to the apoprotein. T1 and T2 values were determined at two magnetic field strengths, i.e. 8.46 T (90.5 MHz) and 5.88 T (62.8 MHz). Values of nuclear Overhauser effects (NOE) were determined at 5.88 T. It is shown that both the dipole-dipole interaction and the chemical shift anisotropy are important relaxation sources for all the carbon atoms investigated. The results are in agreement with a spectral density function of the isoalloxazine ring in which only the overall reorientational motion of the protein is accounted for. From this it is concluded that the isoalloxazine ring is tightly associated with the apoprotein. The protein-bound isoalloxazine ring does not exhibit large fluctuations on the nanosecond time scale, although small amplitude fluctuations cannot be excluded. This information was obtained by a combination of field-dependent T1 and NOE measurements. T2 values are in agreement with these results. On the basis of the dipolar part of the overall T1 values, the distance between the carbon investigated and the nearest proton was calculated and found to be in fair agreement with the crystallographic results of the related flavodoxin from Clostridium MP. In addition, it is shown that, based on the chemical shift anisotropy as a relaxation source, information on the internal mobility is difficult to obtain. The main reason for this is the low precision in the determination of the chemical shift anisotropy tensor.     

Functional differences between keratins of stratified and simple epithelia

We present a simple method for extracting interference effects between chemical shift anisotropy (CSA) and dipolar coupling from spin relaxation measurements in macromolecules, and we apply this method to extracting cross-correlation rates involving interference of amide 15N CSA and 15N-1H dipolar coupling and interference of carbonyl 13C' CSA and 15N-13C' dipolar coupling, in a small protein. A theoretical basis for the interpretation of these rates is presented. While it proves difficult to quantitatively separate the structural and dynamic contributions to these cross-correlation rates in the presence of anisotropic overall tumbling and a nonaxially symmetric chemical shift tensor, some useful qualitative correlations of data with protein structure can be seen when simplifying assumptions are made.     

Characterization of an inwardly rectifying potassium channel in the rabbit superior lacrimal gland

We have investigated the underlying assumptions in estimating cross-correlation rates between chemical shift anisotropy (CSA) and dipolar coupling mechanisms in a scalar-coupled two-spin IS system, from laboratory frame relaxation experiments. It has ben shown that for an arbitrary relaxation delay, the difference in relaxation rates of the individual components of an in-phase (or antiphase) doublet is not related to the CSA-dipolar coupling cross-correlation rate in a simple way. This is especially true in the case where the difference in the decay rates of the in-phase and antiphase terms of the density matrix becomes comparable to the magnitude of the scalar coupling between the two spins. Improved means of extracting cross-correlation rates in these cases are presented.     

The main-chain dynamics of the dynamin pleckstrin homology (PH) domain in solution: analysis of 15N relaxation with monomer/dimer equilibration

The backbone dynamics of the pleckstrin homology (PH) domain from dynamin were studied by 15N NMR relaxation (R1 and R2) and steady state heteronuclear 15N [1H] nuclear Overhauser effect measurements at 500 and 600 MHz, at protein concentrations of 1.7 mM and 300 microM, and by molecular dynamics (MD) simulations. The analysis was performed using the model-free approach. The method was extended in order to account for observed partial (equilibrium) dimerization of the protein at NMR concentrations. A model is developed that takes into account both rapid monomer-dimer exchange and anisotropy of the over-all rotation of the dimer. The data show complex dynamics of the dynamin PH domain. Internal motions in elements of the secondary structure are restricted, as inferred from the high value of the order parameter (S2 approximately 0.9) and from the local correlation time < 100 ps. Of the four extended loop regions that are disordered in the NMR-derived solution structure of the protein, loops beta 1/beta 2 and beta 5/beta 6 are involved in a large-amplitude (S2 down to 0.2 to 0.3) subnanosecond to nanosecond time-scale motion. Reorientation of the loops beta 3/beta 4 and beta 6/beta 7, in contrast, is restricted, characterized by the values of order parameter S2 approximately 0.9 more typical of the protein core. These loops, however, are involved in much slower processes of motion resulting in a conformational exchange on a microsecond to submillisecond time scale. The motions of the terminal regions (residues 1 to 10, 122 to 125) are practically unrestricted (S2 down to 0.05, characteristic times in nanosecond time scale), suggesting that these parts of the sequence do not participate in the protein fold. The analysis shows a larger sensitivity of the 15N relaxation data to protein microdynamic parameters (S2, tau loc) when protein molecular mass (tau c) increases. The use of negative values of the steady state 15N[1H] NOEs as an indicator of the residues not belonging to the folded structure is suggested. The amplitudes of local motion observed in the MD simulation are in a good-agreement with the NMR data for the amide NH groups located in the protein core.     

A diazo-2 study of relaxation mechanisms in frog and barnacle muscle fibres: effects of pH, MgADP, and inorganic phosphate

The aim of this study was to compare the effects of increased concentrations of MgADP, inorganic phosphate (Pi) and H+ ([MgADP], [Pi] and [H+], respectively) on the rate of relaxation in two different muscle types: skinned muscle fibres from the frog Rana temporaria and myofibrillar bundles from the giant Pacific acorn barnacle Balanus nubilus. Relaxation transients are produced by the photolysis of diazo-2 and are well fitted with a double exponential curve, giving two rate constants: k1 [5.6+/-0.1 s-1 for barnacle, n=30; 26.3+/-0.7 s-1 for frog, n=14 (mean+/-SEM)] and k2 [0.6+/-0.1 s-1 in barnacle, n=30; 10.4+/-1.0 s-1 in frog, n=14 (mean+/-SEM)], at 10 degrees C. Decreasing the pH by 0.5 pH units did not significantly affect k1 for barnacle relaxation [5.6+/-0.1 s-1 (mean+/-SEM), n=15] compared to the decrease in k1 of 40% seen in frog. Use of the Ca2+-sensitive fluorescent label acrylodan on barnacle wild-type troponin C demonstrated that decreasing the pH from 7.0 to 6.6 only alters the pCa50 value by 0.23 in the cuvette, while stopped-flow experiments with acrylodan revealed no significant change in koff from the labelled protein [322+/-32 s-1 at pH 7.0 and 381+/-24 s-1 (mean+/-SEM) at pH 6.6]. Increasing [MgADP] by 20 microM (50 microM added ADP) from control values of 50 microM in frog decreased k1 to 12.3+/-0.4 s-1 (mean+/-SEM, n=8), and at 400 microM MgADP, k1=9.6+/-0.1 s-1 (mean+/-SEM, n=12). In barnacle, 500 microM MgADP had a much smaller effect on k1 (4.0+/-0. 9 s-1, mean+/-SEM, n=8). Increasing the free [Pi] from the contaminant level of 0.36 mM to 1.9 mM slowed k1 by approximately 15% in barnacle [4.8+/-0.8 s-1, mean+/-SEM, n=7], compared to a approximately 30% reduction seen in frog. We conclude that the differences between barnacle and frog seen here are most probably due to different isomers of the contractile proteins, and that events underlying the crossbridge cycle are the same or similar. We interpret our results according to a model of crossbridge transitions during relaxation.     

Characterizing semilocal motions in proteins by NMR relaxation studies

The understanding of protein function is incomplete without the study of protein dynamics. NMR spectroscopy is valuable for probing nanosecond and picosecond dynamics via relaxation studies. The use of 15N relaxation to study backbone dynamics has become virtually standard. Here, we propose to measure the relaxation of additional nuclei on each peptide plane allowing for the observation of anisotropic local motions. This allows the nature of local motions to be characterized in proteins. As an example, semilocal rotational motion was detected for part of a helix of the protein Escherichia coli flavodoxin.     

Glyoxalase I (GLO I, EC 4.4.1.5) polymorphism in hemolysates and bloodstains

Oxidized and reduced forms of high-potential iron-sulfur protein (HiPIP) from the purple non-sulfur photosynthetic bacterium Rhodoferax fermentans have been characterized using 1H-NMR spectroscopy. Pairwise and sequence-specific assignments of hyperfine-shifted 1H-NMR signals to protons of cysteine residues bound to the [4Fe-4S]3+/2+ cluster have been performed using one-dimensional NOE and exchange spectroscopy experiments. 1H-NMR hyperfine shifts and relaxation rates of cluster-bound Cys beta-CH2 protons indicate that in the [4Fe-4S]3+ cluster one iron ion can be formally described as Fe(III), while electron density corresponding to one electron is unevenly delocalized onto the remaining three iron ions. This delocalization is effected by means of two different electronic distributions interconverting rapidly on the NMR time scale. The mechanism of paramagnetic proton relaxation, studied by analyzing longitudinal relaxation rates of Cys beta-CH2 protons in HiPIPs from six different sources as a function of the Fe-S-C beta-C alpha dihedral angle, indicate that the major contribution is due to a dipolar metal-centered mechanism, with a non-negligible contribution from a ligand-centered dipolar mechanism which involves the 3p orbital of the Cys sulfur atom. A semi-quantitative tool for extracting structural information from relaxation time measurements is proposed.     

A "parallel plate" electrostatic model for bimolecular rate constants applied to electron transfer proteins

A "parallel plate" model describing the electrostatic potential energy of protein-protein interactions is presented that provides an analytical representation of the effect of ionic strength on a biomolecular rate constant. The model takes into account the asymmetric distribution of charge on the surface of the protein and localized charges at the site of electron transfer that are modeled as elements of a parallel plate condenser. Both monopolar and dipolar interactions are included. Examples of simple (monophasic) and complex (biphasic) ionic strength dependencies obtained from experiments with several electron transfer protein systems are presented, all of which can be accommodated by the model. The simple cases do not require the use of both monopolar and dipolar terms (i.e., they can be fit well by either alone). The biphasic dependencies can be fit only by using dipolar and monopolar terms of opposite sign, which is physically unreasonable for the molecules considered. Alternatively, the high ionic strength portion of the complex dependencies can be fit using either the monopolar term alone or the complete equation; this assumes a model in which such behavior is a consequence of electron transfer mechanisms involving changes in orientation or site of reaction as the ionic strength is varied. Based on these analyses, we conclude that the principal applications of the model presented here are to provide information about the structural properties of intermediate electron transfer complexes and to quantify comparisons between related proteins or site-specific mutants. We also conclude that the relative contributions of monopolar and dipolar effects to protein electron transfer kinetics cannot be evaluated from experimental data by present approximations.     

Solution structure and dynamics of linked cell attachment modules of mouse fibronectin containing the RGD and synergy regions: comparison with the human fibronectin crystal structure

We report the three-dimensional solution structure of the mouse fibronectin cell attachment domain consisting of the linked ninth and tenth type III modules, mFnFn3(9,10). Because the tenth module contains the RGD cell attachment sequence while the ninth contains the synergy region, mFnFn3(9,10) has the cell attachment activity of intact fibronectin. Essentially complete signal assignments and approximately 1800 distance and angle restraints were derived from multidimensional heteronuclear NMR spectra. These restraints were used with a hybrid distance geometry/simulated annealing protocol to generate an ensemble of 20 NMR structures having no distance or angle violations greater than 0.3 A or 3 degrees. Although the beta-sheet core domains of the individual modules are well-ordered structures, having backbone atom rmsd values from the mean structure of 0.51(+/-0.12) and 0.40(+/-0.07) A, respectively, the rmsd of the core atom coordinates increases to 3.63(+/-1.41) A when the core domains of both modules are used to align the coordinates. The latter result is a consequence of the fact that the relative orientation of the two modules is not highly constrained by the NMR restraints. Hence, while structures of the beta-sheet core domains of the NMR structures are very similar to the core domains of the crystal structure of hFnFn3(9,10), the ensemble of NMR structures suggests that the two modules form a less extended and more flexible structure than the fully extended rod-like crystal structure. The radius of gyration, Rg, of mFnFn3(9,10) derived from small-angle neutron scattering measurements, 20.5(+/-0.5) A, agrees with the average Rg calculated for the NMR structures, 20.4 A, and is ca 1 A less than the value of Rg calculated for the X-ray structure. The values of the rotational anisotropy, D ||/D perpendicular, derived from an analysis of 15N relaxation data, range from 1.7 to 2.1, and are significantly less than the anisotropy of 2.67 predicted by hydrodynamic modeling of the crystal coordinates. In contrast, hydrodynamic modeling of the NMR coordinates yields anisotropies in the range of 1.9 to 2.7 (average 2.4(+/-0.2)), with NMR structures bent by more than 20 degrees relative the crystal structure having calculated anisotropies in best agreement with experiment. In addition, the relaxation parameters indicate that several loops in mFnFn3(9,10), including the RGD loop, are flexible on the nanosecond to picosecond time-scale. Taken together, our results suggest that, in solution, the limited set of interactions between the mFnFn3(9,10) modules position the RGD and synergy regions to interact specifically with cell surface integrins, and at the same time permit sufficient flexibility that allows mFnFn3(9,10) to adjust for some variation in integrin structure or environment.     

A rapid method for dry weight determination of proteins on a micro scale with an electrobalance

A method to determine the dry weight (0.25-2 mg) of aqueous protein solutions within 1 h, using an electrobalance, is described. The drying of 50-200 mul solution pipetted onto a glass fiber disc is carried out in vacuo at 70 degrees C until the recorded dry weight becomes constant (within 25-40 min). It has been shown that the dried residue can subsequently be used for other purposes, such as quantitative amino acid analyses. The method is also suitable for the determination of moisture content in lyophilized protein samples.     

Determination of peptide phi angles in solids by relayed anisotropy correlation NMR

A solid state NMR method is presented for determination of a backbone dihedral angle phi in peptides, being based on the previously reported method, relayed anisotropy correlation (RACO) NMR [Y. Ishii et al., Chem. Phys. Lett. 256 (1996) 133]. In the present method, the 15N-1H and the 13C-1H dipolar tensors in the 1H-15N-13C-1H system are two-dimensionally (2D) correlated via polarization transfer from 15N to 13C under magic angle spinning (MAS). This method was applied to N-acetyl[1,2-13C,15N]D,L-valine, and the H-C-N-H dihedral angle was determined to be 154.0 +/- 1.4 degrees or 206.0 +/- 1.4 degrees, the former agreeing with the X-ray value of 154 +/- 5 degrees.     

Principles of psychoneuroendocrinology

We present the results of studies of an aqueous sample of a highly [15N,2H] enriched protein, the SH3 domain from Fyn. Measurements of 1H relaxation and interactions between H2O solvent and exchangeable protons are given, as well as a method for increasing the effective longitudinal relaxation of solvent exchangeable proton resonances. The long-range isotope shifts are measured, for 1H and 15N, which arise due to perdeuteration. Simulations, which employed a 7 or 8 spin relaxation matrix analysis, were compared to the experimental data from a time series of 2D NOESY datasets for some resonances. The agreement between experiment and simulation suggest that, with this 1H dilute sample, relatively long mixing times (up to 1.2 s) can be used to detect specific dipolar interactions between amide protons up to about 7A apart. A set of 155 inter-amide NOEs and 7 side chain NOEs were thus identified in a series of 3D HSQC-NOESY-HSQC experiments. These data, alone and in combination with previously collected restraints, were used to calculate sets of structures using X-PLOR. These results are compared to the available X-ray and NMR structures of the Fyn SH3 domain.     

Selective steady-state and time-resolved fluorescence spectroscopy of an HLA-A2-peptide complex

The human class I major histocompatibility complex (MHC) encoded molecule HLA-A2 loaded with the high-affinity peptide GILGRVFTL (p790), was studied by means of steady-state and picosecond fluorescence intensity and fluorescence anisotropy methods. The large number of tryptophan residues (W) (10 W/heavy chain, 2 W/beta 2m) as well as their fluorescence sensitivity to the microenvironment, determine the emission of the studied complex. The HLA-A2/peptide complex exhibits a considerable static inhomogeneous broadening (IB) of the W electronic spectra, which results in a dependence of the steady-state fluorescence spectrum on the excitation wavelength. The high concentration of W's chromophores and the spectral IB cause a directed non-radiative migration of electronic excitation energy by Foerster's mechanism from 'blue' W residues to 'red' ones. This phenomenon manifests itself in a nanosecond fluorescence spectral shift and an accelerated fluorescence depolarization at the red edge of the emission spectrum. Selective excitation at the red edge of the W absorption band (310 nm) provided a space selective reduction in the number of excited chromophores and enabled resolution of the emission of the 'red' subset of the protein's tryptophans. This avoided the non-radiative homo-energy transfer and enabled to study the fluorescence anisotropy decay kinetics of these residues without a distortion by the energy transfer (ET) process. Under these experimental conditions the fluorescence anisotropy decays practically from the limiting anisotropy value (0.3) for W in a bi-exponential process. The longer decay constant has a value larger than that expected for a global rotation of the HLA-A2/peptide complex suggesting that the protein molecules exist in an oligomeric form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oligomeric state of human erythrocyte band 3 measured by fluorescence resonance energy homotransfer

The oligomeric state of the erythrocyte anion exchange protein, band 3, has been assayed by resonance energy homotransfer. Homotransfer between oligomeric subunits, labeled with eosin-5-maleimide at Lys430 in the transmembrane domain, has been demonstrated by steady-state and time-resolved fluorescence spectroscopy, and is readily observed by its depolarization of the eosin fluorescence. Polarized fluorescence measurements of HPLC-purified band 3 oligomers indicate that eosin homotransfer increases progressively with increasing species size. This shows that homotransfer also occurs between labeled band 3 dimers as well as within the dimers, making fluorescence anisotropy measurements sensitive to band 3 self-association. Treatment of ghost membranes with either Zn2+ or melittin, agents that cluster band 3, significantly decreases the anisotropy as a result of the increased homotransfer within the band 3 clusters. By comparison with the anisotropy of species of known oligomeric state, the anisotropy of erythrocyte ghost membranes at 37 degrees C is consistent with dimeric and/or tetrameric band 3, and does not require postulation of a fraction of large clusters. Proteolytic removal of the cytoplasmic domain of band 3, which significantly increases the rotational mobility of the transmembrane domain, does not affect its oligomeric state, as reported by eosin homotransfer. These results support a model in which interaction with the membrane skeleton restricts the mobility of band 3 without significantly altering its self-association state.     

Effect of variability of plasma interferences on the accuracy of drug immunoassays

Three bond proton-proton vicinal coupling constants are of potential value for analyzing sugar conformations in DNA. However, self-cancellation in antiphase cross peaks and modulation of peak splittings by transverse cross relaxation can alter the apparent coupling constants such that they do not accurately reflect the sugar conformations. Transverse cross relaxation is most effective between strongly coupled geminal proton pairs. Here we report the use of stereospecific deuteration at the H2" position in the A5 and A6 residues in the 12 base pair DNA sequence [d(CGCGAATTCGCG)2] as a means of investigating the effect of transverse cross relaxation on P.E.COSY type cross peaks. Deuteration of the H2" proton is expected to reduce the transverse cross relaxation rate by the square of ratio of the proton to deuteron gyromagnetic ratios, i.e., by a factor of 42. Additionally, a striking eight- to ninefold increase in the signal intensity was observed for cross peaks involving the remaining H2' proton resulting from diminished dipolar relaxation. Further improvements in signal-to-noise ratio were realized by collecting P.E.COSY spectra in strips, using an experiment referred to as stripe-COSY, employing selective excitation pulses which reduced the number of required t1 increments by a factor of four. A final improvement was achieved by employing selective time-shared homonuclear decoupling during the acquisition period, in an experiment referred to as superstripe-COSY, to collapse splittings due to passive couplings. Collectively, these approaches provide P.E. COSY-type spectra with two to three orders of magnitude increased sensitivity per unit time and that are relatively free from artifacts.     

Main chain and side chain dynamics of peptides in liquid solution from 13C NMR: melittin as a model peptide

Peptide backbone and lysine and tryptophan side chain mobilities in the synthetic, 26-residue peptide melittin (MLT) enriched with 13C were investigated in liquid solution by 13C T1 and steady state nuclear Overhauser effect measurements at two magnetic fields and by Trp fluorescence anisotropy measurements and were analyzed using the Lipari and Szabo model-free approach. The overall rotational correlation times at 20 degrees C were 1.28, 1.4, 2.8, and 4.2 ns for monomeric random coil MLT, for monomeric helical MLT (in CD3OD), for tetrameric MLT in neat D2O, and for the tetramer in 50 mM phosphate buffer, respectively. Motion of the backbone in the interior of the sequence was most restricted in the monomeric helix and least restricted in the tetramer. In the monomeric disordered peptide, relatively less restricted backbone motion extending from the N terminus to the fourth residue was observed. Such "end effects" continued only to the third residue in the monomeric helix and were observed just in the amino terminus glycine in the tetramer. The three Lys side chains showed the least restricted motion in the monomers and a differential restriction in the tetramers consistent with the tetramer structure. The motion of the Trp side chain was more restricted than that of Lys side chains and generally as restricted as that of the interior backbone atoms. The effective correlation times for the local motion of the backbone atoms were in the motional narrowing limit and showed distinct patterns. Agreement between NMR relaxation and Trp fluorescence anisotropy data was good for the monomer but not for the tetramer. Implications of these results for peptide dynamics in general are examined.     

Kinetic hole burning, hole filling, and conformational relaxation in heme proteins: direct evidence for the functional significance of a hierarchy of dynamical processes

Band III is a disorder and conformation-sensitive near-infrared (approximately 760 nm) charge transfer absorption band characteristic of equilibrium and nonequilibrium five coordinate ferrous high-spin hemes. The time evolution of this absorption band subsequent to photodissociation of six coordinate ferrous hemoglobin or myoglobin can provide detailed information regarding conformational relaxation, including the thermally driven fluctuations that result in the transition from inhomogeneous to homogeneous ligand rebinding kinetic. Such time-resolved measurements over a range of temperatures are difficult due to long sample recovery times at cryogenic temperatures. A new restoring technique that allows for the rapid movement of a large optically accessible cryostat is used in combination with nanosecond time-resolved near-infrared absorption spectroscopy to generate band III as a function of time for the photoproducts of the carbon monoxide derivative of adult human hemoglobin (COHbA) and, to a more limited extent, horse myoglobin (COMb). The measurements are made over a wide range of temperatures extending from well below the solvent (75% glycerol:water) glass transition at approximately 180 K to ambient temperatures. Three temperature- and/or viscosity-dependent phenomena are observed. At the highest temperatures, only conformational relaxation is observed for the 75% glycerol sample. At very high viscosity (> or = 400 cp), conformational relaxation slows dramatically, and both kinetic hole burning followed by the filling in of the "hole" (dynamic hole filling) are observed. As the temperature is lowered, conformational relaxation slows and finally ceases. Kinetic hole burning and dynamic hole filling as well as additional broadening of band III are observed down to 140 K. The observation of kinetic hole burning (KHB) is indicative of the sample being inhomogeneous on the time scale of the ligand rebinding giving rise to KHB. The onset of hole filling is a direct manifestation of the thermal homogenization of the initial inhomogeneous distribution of conformational substates responsible for KHB. The observed dynamics are used to explain the inverse temperature effect associated with the non-Arrhenius slow down of geminate rebinding above approximately 180 K. The inverse temperature effect appears to arise not only from the onset of conformational relaxation but also from the increase in the rate on thermal averaging of the initial inhomogeneous distribution of conformational substates.