Temperature-induced Conformational Changes Research Articles

Characterization of Mutations in the GTP-binding Domain of IF2 Resulting in Cold-sensitive Growth of Escherichia coli

The infB gene encodes translation initiation factor IF2. We have determined the entire sequence of infB from two cold-sensitive Escherichia coli strains IQ489 and IQ490. These two strains have been isolated as suppressor strains for the temperature-sensitive secretion mutation secY24. The mutations causing the suppression phenotype are located within infB. The only variations from the wild-type (wt) infB found in the two mutant strains are a replacement of Asp409 with Glu in strain IQ489 and an insertion of Gly between Ala421 and Gly422 in strain IQ490. Both positions are located in the GTP-binding G-domain of IF2. A model of the G-domain of E. coli IF2 is presented in Figure 1. Physiological quantities of the recombinant mutant proteins were expressed in vivo in E. coli strains from which the chromosomal infB gene has been inactivated. At 42 °C, the mutants sustained normal cell growth, whereas a significant decrease in growth rate was found at 25 °C for both mutants as compared to wt IF2 expressed in the control strain. Circular dichroism spectra were recorded of the wt and the two mutant proteins to investigate the structural properties of the proteins. The spectra are characteristic of α-helix dominated structure, and reveal a significant different behavior between the wt and mutant IF2s with respect to temperature-induced conformational changes. The temperature-induced conformational change of the wt IF2 is a two-state process. In a ribosome-dependent GTPase assay in vitro the two mutants showed practically no activity at temperatures below 10 °C and a reduced activity at all temperatures up to 45 °C, as compared to wt IF2. The results indicate that the amino acid residues, Asp409 and Gly422, are located in important regions of the IF2 G-domain and demonstrate the importance of GTP hydrolysis in translation initiation for optimal cell growth.

Increased tolerance to thermal inactivation of oxygen evolution in spinach Photosystem II membranes by substitution of the extrinsic 33-kDa protein by its homologue from a thermophilic cyanobacterium

Photosynthetic oxygen evolution is an extremely heat-sensitive process and incubation of spinach Photosystem II (PSII) membranes at 40 °C for only several minutes leads to its complete inactivation. Substitution experiments of the spinach 33-kDa manganese stabilizing protein by a homologue protein, isolated either from the thermophilic cyanobacterium Phormidium laminosum, or from Escherichia coli as a recombinant thermophilic cyanobacterial protein, showed a significant increase in tolerance to heat inactivation of the oxygen-evolving activity. The results allow us to suggest that thermal inactivation of oxygen evolution in higher plant PSII membranes is due to dissociation of the 33-kDa protein as a consequence of temperature-induced conformational changes, and stabilization can be provided by substitution by a thermostable homologue whose secondary structure and binding to PSII remain unaltered at moderately high temperatures.

Polarization IR spectra of hydrogen bonded pyrazole crystals: self-organization effects in proton and deuteron mixture systems. Long-range H/D isotopic effects

This paper deals with experimental studies of the polarization IR spectra of solid-state pyrazole H1345, as well as of its H1D345, D1H345 and D1345 deuterium derivatives. Spectra were measured for the ν NH and ν ND band frequency ranges at temperatures of 298 and 77 K. The spectra were found to strongly change their intensity distribution and their polarization properties with the decrease of temperature. These effects were ascribed to some temperature-induced conformational changes in the hydrogen bond lattices. The studies reported allowed the finding of new kind of isotopic effects H/D in the open-chain hydrogen bond systems, i.e. the self-organization effects. It was found that the spectrally active aggregates of hydrogen bonds remain unchanged despite the growing isotope H/D exchange rate. This statement was supported by analysis of the residual polarized ν NH and ν ND band properties, measured for the isotopically diluted crystalline samples. Analysis of the band shapes of the four hydrogen isotope derivative crystals proved the existence of another kind of H/D isotopic effect, i.e. the long-range isotopic effect. It depends on an influence of the pyrazole ring hydrogen atoms onto the ν NH and ν ND band widths and onto the band fine structures.

Self-Assembly of Rod−Coil Diblock Oligomers Based on α-Helical Peptides

A detailed study of the supramolecular organization of two series of peptide-based diblock oligomers is reported. The diblock oligomers are comprised of an α-helical oligopeptide rod, which is either based on γ-benzyl-l-glutamate or ε-benzyloxycarbonyl-l-lysine, and a short oligo(styrene) coil. The unique feature of these rod−coil molecules is that the conformation of the α-helical peptide rod is sensitive to temperature. As a result, the supramolecular organization of these rod−coil oligomers can be manipulated not only via changes in the block length ratio or chemical composition but also via temperature-induced conformational changes in the rod segments. This makes these molecules attractive building blocks for the development of stimuli-sensitive self-assembled materials.

Temperature-induced conformational changes in amyloid β(1-40) peptide investigated by simultaneous FT-IR microspectroscopy with thermal system

Temperature-dependent secondary structures of the amyloid β(1-40) peptide in the solid state were studied by simultaneous Fourier transform infrared/differential scanning calorimetry (FT-IR/DSC) microspectroscopic system with the heating-cooling-reheating cycle. The result indicates that a thermal transition temperature at 45°C was easily obtained from the three-dimensional plot of the transmission FT-IR spectra as a function of temperature. Furthermore, the thermal-dependent conformational transformations, due to denaturation and aggregation, of solid amyloid β(1-40) were mainly evidenced by reducing the compositions from 37 to 20–24% for α-helical and random coil structures but increasing the components from 27 to 45% for intermolecular β-sheet structures. Thermal-irreversible behavior and a poor thermal stability of solid amyloid β(1-40) were also observed from the poor restoration of the secondary conformational changes in the heated sample.

Thermal stability studies of a globular protein in aqueous poly(ethylene glycol) by 1H NMR

The reversible folding destabilization of hen lysozyme has been confirmed by a melting temperature (T(m)) decrease in aqueous poly(ethylene glycol) (PEG). The percent denatured, extracted from the histidine 15 C2H (H15 C2H) native and denatured peak areas from 500-MHz one-dimensional proton nuclear magnetic resonance (1D (1)H NMR) spectra in D(2)O, was analyzed through denaturation temperatures at 0% and 20% (w/w) PEG 1000. The lysozyme (3.5 mM) T(m) decreased by 4.2 degrees C and 7.1 degrees C in 20% (w/w) PEG 1000 at pH 3.8 and 3.0, respectively. The T(m) decreased with increasing lysozyme concentration. Additionally, the temperature-induced resonance migrations of 17 protons from 8 residues indicate that the native lysozyme structure undergoes temperature-induced conformational changes. The changes were essentially identical in both 0% and 20% (w/w) PEG 1000 at both pH 3.0 and 3.8. This small, local restructuring of the hydrophobic box region may be a manifestation of temperature-dependent solution hydrophobicity, whereas active-site cleft fluctuations may be due to the inherent active-site flexibility. The lysozyme structure in PEG at 35 degrees C was determined to be essentially native from the (1)H nuclear Overhauser effect spectroscopy (NOESY) fingerprint regions. Additionally, lysozyme chemical shifts, from 1D spectra, in PEG 200, 300, and 1000 at 35 degrees C and various concentrations were essentially identical, further confirming that the conformation remains native in various PEG solutions. (c) 1996 John Wiley & Sons, Inc.

Recombinant factor VIII SQ — the influence of formulation parameters on structure and surface adsorption

The main aim of this paper was to investigate the influence of temperature, pH and ionic interactions on the structural stability and surface adsorption of a recombinant factor VIII product, r-VIII SQ. The interaction of r-VIII SQ with glass and air interfaces, and possible means of increasing the stability of the formulation, were also investigated. The stability of r-VIII SQ was followed by measuring the biological activity (VIII:C), by circular dichroism (CD) studies and by the measurement of surface tension using the pendant drop method. The results show that the surface tension decreased exponentially with time; this decrease was more pronounced above 20°C, indicating increased conformational flexibility of the protein with increased temperatures. Far UV CD spectra were not influenced in the range 5–55°C and near UV CD measurements did not indicate structural changes below 45°C. During agitation at 25°C, VIII:C was lost rapidly in formulations without a macromolecular additive. Nonionic surfactants such as polysorbate 80 and polysorbate 20 protected VIII:C to an equally high degree against surface adsorption. Albumin was less effective, but it is possible that this is because it is a protein itself and may have been affected by the agitation. The addition of 300 mg/ml of sucrose improved the long term stability of VIII:C, a finding most likely explained by the theory of preferential hydration. Near UV CD spectra at acidic or basic pH mainly indicated changes around 242 nm, especially at low ionic strength. Addition of 10 mM EDTA at pH 7 resulted in similar changes. This effect was completely reversed by the addition of an excess of Ca 2+, Sr 2+ or Mg 2+ ions. In conclusion, CD spectra and surface tension measurements of r-VIII SQ did not reveal any temperature-induced conformational changes in the temperature range 5–20°C; changes were first noted at elevated temperatures. Surface adsorption of r-VIII SQ during agitation was prevented by the addition of a nonionic surfactant. Preferential hydration improved the storage stability of the protein but did not directly prevent its surface adsorption. The structural integrity of the molecule was preserved at pH 7, at an increased ionic strength and in the presence of some divalent metal ions (Ca 2+, Sr 2+ or Mg 2+).

Temperature-controlled interaction of thermosensitive polymer-modified cationic liposomes with negatively charged phospholipid membranes

To obtain cationic liposomes of which affinity to negatively charged membranes can be controlled by temperature, cationic liposomes consisting of 3β-[ N-( N′, N′-dimethylaminoethane)carbamoyl]cholesterol and dioleoylphosphatidylethanolamine were modified with poly( N-acryloylpyrrolidine), which is a thermosensitive polymer exhibiting a lower critical solution temperature (LCST) at ca. 52°C. The unmodified cationic liposomes did not change its zeta potential between 20–60°C. The polymer-modified cationic liposomes revealed much lower zeta potential values below the LCST of the polymer than the unmodified cationic liposomes. However, their zeta potential increased significantly above this temperature. The unmodified cationic liposomes formed aggregates and fused intensively with anionic liposomes consisting of egg yolk phosphatidylcholine and phosphatidic acid in the region of 20–60°C, due to the electrostatic interaction. In contrast, aggregation and fusion of the polymer-modified cationic liposomes with the anionic liposomes were strongly suppressed below the LCST. However, these interactions were enhanced remarkably above the LCST. In addition, the polymer-modified cationic liposomes did not cause leakage of calcein from the anionic liposomes below the LCST, but promoted the leakage above this temperature as the unmodified cationic liposomes did. Temperature-induced conformational change of the polymer chains from a hydrated coil to a dehydrated globule might affect the affinity of the polymer-modified cationic liposomes to the anionic liposomes.

Differential effect of pressure and temperature on the catalytic behaviour of wild-type human butyrylcholinesterase and its D70G mutant.

The combined action of temperature (10-35 degrees C) and pressure (0. 001-2 kbar) on the catalytic activity of wild-type human butyrylcholinesterase (BuChE) and its D70G mutant was investigated at pH 7.0 using butyrylthiocholine as the substrate. The residue D70, located at the mouth of the active site gorge, is an essential component of the peripheral substrate binding site of BuChE. Results showed a break in Arrhenius plots of wild-type BuChE (at Tt approximately 22 degrees C) whatever the pressure (dTt/dP = 1.6 +/- 1.5 degrees C.kbar-1), whereas no break was observed in Arrhenius plots of the D70G mutant. These results suggested a temperature-induced conformational change of the wild-type BuChE which did not occur for the D70G mutant. For the wild-type BuChE, at around a pressure of 1 kbar, an intermediate state, whose affinity for substrate was increased, appeared. This intermediate state was not seen for the mutant enzyme. The wild-type BuChE remained active up to a pressure of 2 kbar whatever the temperature, whereas the D70G mutant was found to be more sensitive to pressure inactivation (at pressures higher than 1.5 kbar the mutant enzyme lost its activity at temperatures lower than 25 degrees C). The results indicate that the residue D70 controls the conformational plasticity of the active site gorge of BuChE, and is involved in regulation of the catalytic activity as a function of temperature.

Temperature controlled syn-anti conformational switching in zinc containing porphyrin dimers via ligand assistance

Unique temperature dependent syn-anti conformational changes in bis- and monozinc ethane-bridged porphyrin dimers take place in alcohol containing solvents; these dimers adopt a syn conformation at room temperature, while a decrease in temperature results in the gradual shift of the conformational equilibrium towards the ant/conformer. © 1999 Elsevier Science Ltd. All rights reserved. Environmentally induced conformational changes of natural enzymatic and photosynthetic systems that contain porphyrinoid prosthetic groups or chromophores play a crucial role in natural processes. In addition, these conformational variations offer an attractive, simple mechanism for modulating a wide range of physical and chemical properties of porphyrin derivatives, both in vitro and in vivo. 1 Furthermore, porphyrin or porphyrin-driven conformational switching can also serve as a possible basis for various molecular electronic devices. 2 Such eonformational effects can be induced by synthetic manipulations and/or by axial ligation, hydrogen binding, and through the application of external physico-chemical factors such as temperature, pH, concentration, etc. Herein, we report the first observation of temperature-induced and ligand assisted syn-anti conformational changes for his-zinc and zinc-free base ethane-bridged porphyrin dimers (1 and 2). 3a,h Recently it was shown that dimers 1 and 2 adopt a syn conformation at room temperature in a variety of solvents, 3 and this conformation does not break apart, even upon heating up to 110 °C.3d The spectral characteristics of the syn isomer are typical of the porphyrin's face-to-face orientation. 3c,4 The presence of zinc porphyrin is obviously an important factor in holding porphyrins together, since other ethane-bridged porphyrin dimers that do not contain zinc ions are found only in the anti form in various solvents. Also the aggregation behavior of monomeric 5 and doubly strapped dimeric 6 zinc porphyrins via x-x interaction mechanism has been reported. Variable temperature (VT) UV-vis and VT IH NMR experiments revealed dramatic changes in the initial spectral patterns of dimers 1 and 2 in alcohol containing solutions as the temperature decreased (see Figures 1 and 2), while no such changes were observed in alcohol free solutions (Figure 1, Inset, solid line). Thus, the Soret band at 397 nm which is assigned to the corresponding syn conformer 3 and observed for dimer 1 in a mixture of n-hexane:EtOH (24:1) at room temperature gradually decreases. Instead, a new, bathochromically shifted, split Sorer band appeared at 420 nm, while the positions of the Qx0o and QXOl bands remained

Temperature control of biotin binding and release with A streptavidin-poly(N-isopropylacrylamide) site-specific conjugate.

The many laboratory and diagnostic applications utilizing streptavidin as a molecular adaptor rely on its high affinity and essentially irreversible interaction with biotin. However, there are many situations where recovery of the biotinylated molecules is desirable. We have previously shown that poly(N-isopropylacrylamide) (PNIPAAm), a temperature-sensitive polymer, can reversibly block biotin association as the polymer's conformation changes at its lower critical solution temperature (LCST). Here, we have constructed a streptavidin-PNIPAAm conjugate which is able to bind biotin at room temperature or lower and release bound biotin at 37 degrees C. The conjugate can repeatedly bind and release biotin as temperature is cycled through the LCST. A genetically engineered streptavidin mutant, E116C, which has only one cysteine residue, was conjugated site specifically via the sulfhydryl groups with a PNIPAAm that has pendent sulfhydryl-reactive vinyl sulfone groups. The conjugation site is near the tryptophan 120 residue, which forms a van der Waals contact with biotin that is important in generating the large binding free energy. The temperature-induced conformational change of the polymer at position 116 may lead to structural changes in the region of tryptophan 120 that are responsible for the reversible binding between biotin and the conjugated streptavidin.

Temperature-induced conformational changes of native lysozyme in aqueous solution studied by dielectric spectroscopy

We report dielectric measurements at radiofrequencies on lysozyme in aqueous solution at two values of pH (3.5 and 6) as a function of temperature in the interval 5–55°C. From the analysis of the dielectric relaxation of the protein solution, the effective hydrodynamic radius r and the electric dipole moment μ of the protein were calculated. The results show that temperature causes continuous gradual changes of r and μ with a maximum at 25–30°C where the Gibbs free energy for native lysozyme shows an analogous trend. We suggest that the gradual variations of r and μ are the manifestation of a redistribution of microscopic state populations of the protein within the same macroscopic native state.

Temperature-induced conformational changes in prosomatostatin-II: implications for processing.

Somatostatin (SRIF) is a 14-residue peptide hormone synthesized in the hypothalamus and pancreatic islets. SRIF-14 and an N-terminally extended form, SRIF-28, are generated by the proteolytic processing of an approx. 102-residue precursor prosomatostatin (proSRIF) at a single set of paired basic residues (Arg-Lys) and at a monobasic (Arg) site respectively. Previous work in our laboratory demonstrated that the propeptide of SRIF mediates intracellular sorting; we suggested that this information resides in the prohormone structure. To identify putative sorting domains we have investigated structural features of recombinant anglerfish proSRIF-II purified from Escherichia coli. Two species of proSRIF-II were obtained: a monomeric form and a disulphide-linked dimer. CD analyses revealed that monomeric proSRIF-II lacks appreciable periodic secondary structure; however, on slow heating (2 degrees C/min) and cooling, it assumed a predominantly alpha-helical conformation. When subjected to a second heating-and-cooling cycle, the alpha-helical conformation was maintained. In contrast, the dimeric form of proSRIF-II was predominantly alpha-helical and its helicity did not increase in response to heating and recooling. Our results suggest that proSRIF-II might exist in several different folding intermediate states.

The preparation and physico-chemical properties of poly( N-ethylacrylamide) microgels

The preparation and physico-chemical characterisation of a novel thermosensitive microgel dispersion of anionic poly( N-ethylacrylamide), poly(NEAM), is reported. Dispersions were prepared by the single-step emulsion polymerisation of N-ethylacrylamide in water, in the presence of N,N′-methylenebisacrylamide as a cross-linking agent and ammonium persulfate as the initiator. Transmission electron micrographs of the microgel dispersions showed that the reaction time greatly influenced the physical characteristics of the resultant microgel particles, with monodisperse spheres, having a mean diameter of 414 ± 21 nm, being obtained following a 24-h preparation. Using high sensitivity differential scanning calorimetry (HSDSC) a kinetically limited phase transition was observed for a 2.6% (w/w) dispersion, having an excess specific heat capacity maximum at 78.2°C (at a scan rate of 60 K h −1). The thermodynamic parameters and turbidimetric changes (as a function of temperature, pH and electrolyte concentration) associated with this transition are reported. In the presence of 1 mol dm −3 sodium chloride, the temperature at which this conformational transition occurs decreases with a corresponding increase in the calorimetric enthalpy of the transition. A reversible aggregation of the microgel particles was also observed on heating a 0.7% (w/w) dispersion to 80°C in the presence of 3 mol dm −3 sodium chloride. Experimental measurements of the reduced viscosity, η red, of a 3% (w/w) dispersion, as a function of temperature and electrolyte concentration, provided further evidence of a temperature-induced conformational change with respect to the poly(NEAM) chains, resulting in a more compact conformation.

2D FT-NIR and FT-IR correlation analysis of temperature-induced changes of nylon 12

FT-NIR and FT-IR spectra of nylon 12 were recorded from 25 to 200°C. Two-dimensional (2D) correlation spectroscopy was applied to analyse the experimental data. A heterospectral dynamical correlation between NIR and IR regions was performed. The correlation is found to be useful for making band assignments in the NIR region. It is shown that the temperature-induced conformational changes in the hydrocarbon chain proceed faster than the dissociation of hydrogen-bonded amide groups. Owing to the spectral resolution enhancement in the 2D correlation spectra, splitting of the symmetric stretching mode of the methylene unit was found. It suggests that, in the temperature range studied, nylon 12 exist both in crystalline and amorphous forms.

Colloidal copolymer microgels of N-isopropylacrylamide and acrylic acid: pH, ionic strength and temperature effects

Aqueous colloidal microgels have been prepared, based on poly(N-isopropylacrylamide)[poly(NIPAM)], cross-linked with bisacrylamide, containing 5% w/w acrylic acid (AAc) as a comonomer. Transmission electron micrographs of the microgels show that the copolymer microgels are monodisperse spheres. The size of the microgel particles containing 5% AAc has been studied, using dynamic light scattering, as a function of pH (3–10), ionic strength (10–4–10–1M NaCl) and temperature (20–75 °C). The hydrodynamic diameter of the copolymer microgels decrease both with increasing ionic strength (at pH 6 and 25 °C) and reversibly with increasing temperature at pH values of 2.6, 3.4 and 6.5. However, under isothermal conditions in 10–3M NaCl at 25 °C, the hydrodynamic diameter increases in going from pH 3.3–9.4. In addition, the temperature-induced conformational changes in the polymer chains have been followed using high-sensitivity differential scanning calorimetry (HSDSC). A comparison is made with the behaviour of poly(NIPAM) microgel particles, not containing AAc. Explanations are offered to account for the pronounced difference in the physico-chemical properties observed.

The Effect of Temperature on the Structure of Vinblastine-induced Polymers of Purified Tubulin: Detection of a Reversible Conformational Change

Addition of the antimitotic drug vinblastine to solutions of purified tubulin induces the formation of helical polymers whose structure and type of aggregation is determined by the concentration of magnesium. While paracrystalline arrangements of single coils are observed at low concentrations of the ion, for concentrations higher than 6 mM free double-coiled spirals are obtained, which are indistinguishable from those obtained in the presence of microtubule-associated proteins (MAPs). This result is consistent with a similar effect of magnesium and MAPs in neutralizing negative charges on the tubulin molecule and so allowing for lateral contacts between protofilaments. The effects that temperature has on the structure of both types of polymers, free spirals or paracrystals, have been monitored using time-resolved X-ray solution scattering. This study shows that a temperature increase: (1) affects the length and lateral aggregation of the spirals in the paracrystalline sample; (2) induces a reversible increase of the helical pitch in both types of polymers that closely follows the temperature change; (3) produces an irreversible aggregation of some of the protein in both types of polymers; and (4) can induce a reversible transformation from one type of structure to the other when the concentration of Mg 2+is in the boundary between the two ranges. We suggest that the changes in pitch are due to a temperature-induced conformational change of the tubulin molecule. This effect may be related to the structural modifications that result in the temperature-induced assembly of microtubules in vitrounder normal conditions of assembly.

Cold-induced conformational changes of ribonuclease A as investigated by subzero transverse temperature gradient gel electrophoresis

Subzero temperature gradient gel electrophoresis is a new approach which allows to measure the transition temperature of low temperature-induced subtle conformational changes of proteins and to detect the different conformational states, including unfolded states. Using this technique under destabilizing conditions, i.e., in the presence of 4 M urea, bovine pancreas ribonuclease A exhibited two transitions: (i) a continuous transition with a midpoint temperature of −14°C corresponding to a rapid equilibrium between the initial enzyme state and a conformational state more compact than the initial one; (ii) a discontinuous transition at −22.5°C from intermediate to a non migrating species. Under reducing conditions this second transition was shifted toward high temperatures (−18.5°C). We attempted to detect these two transitions by differential scanning calorimetry, UV spectrophotometry and circular dichroism measurements. These transitions have been ascribed to subtle cold-induced conformational changes.

Assembly of Atlantic Cod ( Gadus morhua) Brain Microtubules at Different Temperatures: Dependency of Microtubule-Associated Proteins Is Relative to Temperature

Isolated cod ( Gadus morhua) brain microtubules were found to have a broad temperature interval for assembly. In contrast to mammalian microtubules they assembled even at as low temperatures as 14°C. Evidence was found that temperature alters the dependency of microtubule-associated proteins (MAPs) for assembly. The assembly was MAPs-dependent at low, but not at higher temperatures. Assembly at +18°C was inhibited by both NaCl and estramustine phosphate. These compounds are well known to inhibit the binding of MAPs to tubulin. At higher temperatures there was no MAPs dependency for assembly, despite that MAPs bound to the microtubules. Cow MAPs had the same effect as cod MAPs, suggesting that despite differences in MAP composition, the effect is not caused by the unusual composition of cod MAPs. The results therefore suggest that these differences in MAPs dependency are due to intrinsic properties of cod tubulin or iubulin-to-tubulin interactions. Small temperature-induced conformational changes of tubulin and a slight enrichment of acetylated and detyrosinated tubulin in microtubules assembled at +30°C as compared to +15°C, were observed. The ability to alter the assembly stimulating effect of MAPs may be important for the cell to regulate microtubule dynamics and stability. In addition, changes in tubulin conformation and composition of tubulin isoforms may reflect adaptations for microtubule assembly at low temperatures.

Oxygen diffusion-concentration product in rhodopsin as observed by a pulse ESR spin labeling method

Permeation of molecular oxygen in rhodopsin, an integral membrane protein, has been investigated by monitoring the bimolecular collision rate between molecular oxygen and the nitroxide spin label using a pulse electron spin resonance (ESR) T1 method. Rhodopsin was labeled by regeneration with the spin-labeled 9-cis retinal analogue in which the beta-ionone ring of retinal is replaced by the nitroxide tetramethyl-oxypyrrolidine ring. The bimolecular collision rate was evaluated in terms of an experimental parameter W(x), defined as T1(-1)(air,x)--T1(-1)(N2,x) where T1's are the spin-lattice relaxation times of the nitroxide in samples equilibrated with atmospheric air and nitrogen respectively, which is proportional to the product of local oxygen concentration and local diffusion coefficient (transport). W-values at the beta-ionone binding site in spin-labeled rhodopsin are in the range of 0.02-0.13 microseconds-1, which are 10-60 times smaller than W's in water and 1.1-20 times smaller than in model membranes in the gel phase, indicating that membrane proteins create significant permeation resistance to transport of molecular oxygen inside and across the membrane. W(thereby the oxygen diffusion-concentration product) is larger in the meta II-enriched sample than in rhodopsin, indicating light-induced conformational changes of opsin around the beta-ionone binding site. W decreases with increase of temperature for both rhodopsin and meta II-enriched samples, suggesting that temperature-induced conformational changes take place in both samples. These changes were not observable using conventional ESR spectroscopy. It is concluded that W is a sensitive monitor of conformational changes of proteins.