Brain lateralization probed by water diffusion at the atomic to micrometric scale.

Combined neutron scattering and diffusion nuclear magnetic resonance experiments have been used to reveal significant interregional asymmetries (lateralization) in bovine brain hemispheres in terms of myelin arrangement and water dynamics at micron to atomic scales. Thicker myelin sheaths were found in the left hemisphere using neutron diffraction. 4.7 T dMRI and quasi-elastic neutron experiments highlighted significant differences in the properties of water dynamics in the two hemispheres. The results were interpreted in terms of hemisphere-dependent cellular composition (number of neurons, cell distribution, etc.) as well as specificity of neurological functions (such as preferential networking).

Anomalous water dynamics in brain: a combined diffusion magnetic resonance imaging and neutron scattering investigation.

Water diffusion is an optimal tool for investigating the architecture of brain tissue on which modern medical diagnostic imaging techniques rely. However, intrinsic tissue heterogeneity causes systematic deviations from pure free-water diffusion behaviour. To date, numerous theoretical and empirical approaches have been proposed to explain the non-Gaussian profile of this process. The aim of this work is to shed light on the physics piloting water diffusion in brain tissue at the micrometre-to-atomic scale. Combined diffusion magnetic resonance imaging and first pioneering neutron scattering experiments on bovine brain tissue have been performed in order to probe diffusion distances up to macromolecular separation. The coexistence of free-like and confined water populations in brain tissue extracted from a bovine right hemisphere has been revealed at the micrometre and atomic scale. The results are relevant for improving the modelling of the physics driving intra- and extracellular water diffusion in brain, with evident benefit for the diffusion magnetic resonance imaging technique, nowadays widely used to diagnose, at the micrometre scale, brain diseases such as ischemia and tumours.

Observing heme doming in myoglobin with femtosecond X-ray absorption spectroscopy.

We report time-resolved X-ray absorption measurements after photolysis of carbonmonoxy myoglobin performed at the LCLS X-ray free electron laser with nearly 100 fs (FWHM) time resolution. Data at the Fe K-edge reveal that the photoinduced structural changes at the heme occur in two steps, with a faster (∼70 fs) relaxation preceding a slower (∼400 fs) one. We tentatively attribute the first relaxation to a structural rearrangement induced by photolysis involving essentially only the heme chromophore and the second relaxation to a residual Fe motion out of the heme plane that is coupled to the displacement of myoglobin F-helix.

The boson peak of amyloid fibrils: probing the softness of protein aggregates by inelastic neutron scattering.

Proteins and polypeptides are characterized by low-frequency vibrations in the terahertz regime responsible for the so-called "boson peak". The shape and position of this peak are related to the mechanical properties of peptide chains. Amyloid fibrils are ordered macromolecular assemblies, spontaneously formed in nature, characterized by unique biological and nanomechanical properties. In this work, we investigate the effects of the amyloid state and its polymorphism on the boson peak. We used inelastic neutron scattering to probe low-frequency vibrations of the glucagon polypeptide in the native state and in two different amyloid morphologies in both dry and hydrated sample states. The data show that amyloid fibril formation and hydration state affect the softness of the polypeptide not only by changing the distribution of vibrational modes but also, and most significantly, the dissipative mechanisms of collective low-frequency vibrations provided by water-protein and protein-protein interactions. We show how the morphology of the fibril is able to tune these effects. Atomic fluctuations were also measured by elastic neutron scattering. The data confirm that any effect of protein aggregation on fluctuation amplitudes is essentially due to changes in surface exposure to hydration water. The results demonstrate the importance of protein-protein and protein-water interactions in the dynamics and mechanics of amyloid fibrils.

Unveiling the timescale of the R-T transition in human hemoglobin.

Time-resolved wide-angle X-ray scattering, a recently developed technique allowing to probe global structural changes of proteins in solution, was used to investigate the kinetics of R-T quaternary transition in human hemoglobin and to systematically compare it to that obtained with time-resolved optical spectroscopy under nearly identical experimental conditions. Our data reveal that the main structural rearrangement associated with the R-T transition takes place approximately 2 mus after the photolysis of hemoglobin at room temperature and neutral pH. This finding suggests that the 20-mus step observed with time-resolved optical spectroscopy corresponds to a small and localized structural change.

Interfacial water structure controls protein conformation.

A phenomenological theory of salt-induced Hofmeister phenomena is presented, based on a relation between protein solubility in salt solutions and protein-water interfacial tension. As a generalization of previous treatments, it implies that both kosmotropic salting out and chaotropic salting in are manifested via salt-induced changes of the hydrophobic/hydrophilic properties of protein-water interfaces. The theory is applied to describe the salt-dependent free energy profiles of proteins as a function of their water-exposed surface area. On this basis, three classes of protein conformations have been distinguished, and their existence experimentally demonstrated using the examples of bacteriorhodopsin and myoglobin. The experimental results support the ability of the new formalism to account for the diverse manifestations of salt effects on protein conformation, dynamics, and stability, and to resolve the puzzle of chaotropes stabilizing certain proteins (and other anomalies). It is also shown that the relation between interfacial tension and protein structural stability is straightforwardly linked to protein conformational fluctuations, providing a keystone for the microscopic interpretation of Hofmeister effects. Implications of the results concerning the use of Hofmeister effects in the experimental study of protein function are discussed.

Impulsive solvent heating probed by picosecond x-ray diffraction.

The time-resolved diffraction signal from a laser-excited solution has three principal components: the solute-only term, the solute-solvent cross term, and the solvent-only term. The last term is very sensitive to the thermodynamic state of the bulk solvent, which may change during a chemical reaction due to energy transfer from light-absorbing solute molecules to the surrounding solvent molecules and the following relaxation to equilibrium with the environment around the scattering volume. The volume expansion coefficient alpha for a liquid is typically approximately 1 x 10(-3) K(-1), which is about 1000 times greater than for a solid. Hence solvent scattering is a very sensitive on-line thermometer. The decomposition of the scattered x-ray signal has so far been aided by molecular dynamics (MD) simulations, a method capable of simulating the solvent response as well as the solute term and solute/solvent cross terms for the data analysis. Here we present an experimental procedure, applicable to most hydrogen containing solvents, that directly measures the solvent response to a transient temperature rise. The overtone modes of OH stretching and CH3 asymmetric stretching in liquid methanol were excited by near-infrared femtosecond laser pulses at 1.5 and 1.7 microm and the ensuing hydrodynamics, induced by the transfer of heat from a subset of excited CH3OH* to the bulk and the subsequent thermal expansion, were probed by 100 ps x-ray pulses from a synchrotron. The time-resolved data allowed us to extract two key differentials: the change in the solvent diffraction from a temperature change at constant density, seen at a very short time delay approximately 100 ps, and a term from a change in density at constant temperature. The latter term becomes relevant at later times approximately 1 mus when the bulk of liquid expands to accommodate its new temperature at ambient pressure. These two terms are the principal building blocks in the hydrodynamic equation of state, and they are needed in a self-consistent reconstruction of the solvent response during a chemical reaction. We compare the experimental solvent terms with those from MD simulations. The use of experimentally determined solvent differentials greatly improved the quality of global fits when applied to the time-resolved data for C2H4I2 dissolved in methanol.

Structure and dynamics of water confined in silica hydrogels: X-ray scattering and dielectric spectroscopy studies.

We have used a sol-gel technique to obtain optically transparent hydrogels in which water is confined within a 3D silica matrix. In this work we report X-ray scattering and dielectric spectroscopy measurements on samples having different aging times and compare them with previously obtained results with near-infrared (NIR) absorption spectroscopy. X-ray scattering at room temperature enables to characterize the structure and size of the matrix pores and the non-uniform distribution of water inside the hydrogel. Broad band dielectric spectroscopy in the temperature range 130-280 K enables to study water dynamics. In aged hydrogels two relaxations are clearly evident and show characteristic temperature dependence. The faster relaxation has an Arrhenius behavior in the whole temperature range investigated with an activation enthalpy of approximately 50 kJ/mol; it is attributed to water molecules strongly interacting with the silica matrix. The slower relaxation has a markedly non-Arrhenius behavior which can be fitted with a Vogel-Fulcher-Tamman (VFT) relation with critical temperature of approximately 100 K and activation enthalpies of 35 and 95 kJ/mol at 300 and 170 K respectively; it is attributed to water molecules within the pores that do not interact strongly with the matrix and behave collectively. The VFT temperature dependence of the dielectric relaxation time suggests that this water does not crystallize, in agreement with previous results from NIR spectroscopy.

Local dynamic properties of the heme pocket in native and solvent-induced molten-globule-like states of cytochrome c.

We report the Soret absorption band, down to cryogenic temperature, of native and molten-globule-like state of horse heart cytochrome c. The band profile is analyzed in terms of vibronic coupling of the heme normal modes to the electronic transition in the framework of the Franck-Condon approximation. From the temperature dependence of the Gaussian broadening and of the peak position, we obtain information on the 'bath' of low frequency harmonic motions of the heme group within the heme pocket. The reported data indicate that, compared to the native state, the less rigid tertiary structure of the molten globule is reflected in a higher flexibility of the heme pocket and in greater conformational disorder, allowing the transduction of large-amplitude motion of the protein to the dynamics of the heme pocket.

Heme pocket disorder in myoglobin: reversal by acid-induced soft refolding.

The protein folding process of heme proteins entails generation of not only a correct global polypeptide structure, but also a correct, functionally competent heme environment. We employed a variety of spectroscopic approaches to probe the structure and dynamics of the heme pocket of a recombinant sperm whale myoglobin. The conformational characteristics were examined by circular dichroism, time-resolved fluorescence spectroscopy, FTIR spectroscopy, and optical absorption spectroscopy in the temperature range 300-20 K. Each of these spectroscopic probes detected modifications confined exclusively to the heme pocket of the expressed myoglobin relative to the native protein. The functional properties were examined by measuring the kinetics of CO binding after flash-photolysis. The kinetics of the expressed myoglobin were more heterogeneous than those of the native protein. Mild acid exposure of the ferric derivative of the recombinant protein resulted in a protein with "nativelike" spectroscopic properties and homogeneous CO binding kinetics. The heme pocket modifications observed in this recombinant myoglobin do not derive from inverted heme. In contrast, when native apomyoglobin is reconstituted with the heme in vitro, the heme pocket disorder could be attributed exclusively to 180 degrees rotation of the bound heme [La Mar, G. N., Toi, H., and Krishnamoorthi, R. (1984) J. Am. Chem. Soc. 106, 6395-6401; Light, W. R., Rohlfs, R. J., Palmer, G., and Olson, J. S. (1987) J. Biol. Chem. 262, 46-52]. We conclude that exposure to low pH decreases the affinity of globin for the heme and allows an extended conformational sampling or "soft refolding" to a nativelike conformation.

Heme symmetry, vibronic structure, and dynamics in heme proteins: ferrous nicotinate horse myoglobin and soybean leghemoglobin.

We report the visible and Soret absorption bands, down to cryogenic temperatures, of the ferrous nicotinate adducts of native and deuteroheme reconstituted horse heart myoglobin in comparison with soybean leghemoglobin-a. The band profile in the visible region is analyzed in terms of vibronic coupling of the heme normal modes to the electronic transition in the framework of the Herzberg-Teller approximation. This theoretical approach makes use of the crude Born-Oppenheimer states and therefore neglects the mixing between electronic and vibrational coordinates; however, it takes into account the vibronic nature of the visible absorption bands and allows an estimate of the vibronic side bands for both Condon and non-Condon vibrational modes. In this framework, an x-y splitting of the Q transition for native and deuteroheme reconstituted horse myoglobin is clearly assessed and attributed to electronic perturbations that, in turn, are caused by a reduction of the typical D(4h) symmetry of the system due to heme distortions of B(1g)-type symmetry and/or to an x-y asymmetric position of the nicotinate ring; in deuteroheme reconstituted horse myoglobin the asymmetric heme peripheral substituents add to the above effect(s). On the contrary, in leghemoglobin-a no spectral splitting upon nicotinate binding is observed, pointing to a planar heme configuration in which only distortions of A(1g)-type symmetry are effective and to which the nicotinate ring is bound in an x - y symmetric position. The local dynamic properties of the heme pocket of the three proteins are investigated through the temperature dependence of spectral line broadening. Leghemoglobin-a behaves as a softer matrix with respect to horse myoglobin, thus validating the hypothesis of a looser heme pocket conformation in the former protein, which allows a nondistorted heme configuration and a symmetric binding of the bulky nicotinate ligand.

Structural and dynamic properties of the homodimeric hemoglobin from Scapharca inaequivalvis Thr-72-->Ile mutant: molecular dynamics simulation, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy studies.

Molecular dynamics simulations, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy have been performed on a mutant of the Scapharca inaequivalvis homodimeric hemoglobin, where residue threonine 72, at the subunit interface, has been substituted by isoleucine. Molecular dynamics simulation indicates that in the Thr-72-->Ile mutant several residues that have been shown to play a role in ligand binding fluctuate around orientations and distances similar to those observed in the x-ray structure of the CO derivative of the native hemoglobin, although the overall structure remains in the T state. Visible absorption spectroscopy data indicate that in the deoxy form the Soret band is less asymmetric in the mutant than in the native protein, suggesting a more planar heme structure; moreover, these data suggest a similar heme-solvent interaction in both the liganded and unliganded states of the mutant protein, at variance with that observed in the native protein. The "conformation sensitive" band III of the deoxy mutant protein is shifted to lower energy by >100 cm-1 with respect to the native one, about one-half of that observed in the low temperature photoproducts of both proteins, indicating a less polar or more hydrophobic heme environment. Resonance Raman spectroscopy data show a slight shift of the iron-proximal histidine stretching mode of the deoxy mutant toward lower frequency with respect to the native protein, which can be interpreted in terms of either a change in packing of the phenyl ring of Phe-97, as also observed from the simulation, or a loss of water in the heme pocket. In line with this latter interpretation, the number of water molecules that dynamically enters the intersubunit interface, as calculated by the molecular dynamics simulation, is lower in the mutant than in the native protein. The 10-ns photoproduct for the carbonmonoxy mutant derivative has a higher iron-proximal histidine stretching frequency than does the native protein. This suggests a subnanosecond relaxation that is slowed in the mutant, consistent with a stabilization of the R structure. Taken together, the molecular dynamics and the spectroscopic data indicate that the higher oxygen affinity displayed by the Thr-72-->Ile mutant is mainly due to a local perturbation in the dimer interface that propagates to the heme region, perturbing the polarity of the heme environment and propionate interactions. These changes are consistent with a destabilization of the T state and a stabilization of the R state in the mutant relative to the native protein.

Properties of human hemoglobins with increased polarity in the alpha- or beta-heme pocket. Carbonmonoxy derivatives.

The spectroscopic, conformational, and functional properties of mutant carbonmonoxy hemoglobins in which either the beta-globin Val67(E11) or the alpha-globin Val62(E11) is replaced by threonine have been investigated. The thermal evolution of the Soret absorption band and the stretching frequency of the bound CO were used to probe the stereodynamic properties of the heme pocket. The functional properties were investigated by kinetic measurements. The spectroscopic and functional data were related to the conformational properties through molecular analysis. The effects of this nonpolar-to-polar isosteric mutation are: (i) increase of heme pocket anharmonic motions, (ii) stabilization of the A0 conformer in the IR spectrum, (iii) increased CO dissociation rates. The spectroscopic data indicate that for the carbonmonoxy derivatives, the Val --> Thr mutation has a larger conformational effect on the beta-subunits than on the alpha-subunits. This is at variance with the deoxy derivatives where the conformational modification was larger in the heme pocket of the alpha-subunit (Cupane, A., Leone, M., Militello, V., Friedman, R. K., Koley, A. P., Vasquez, G. P., Brinigar, W. S., Karavitis, M., and Fronticelli, C. (1997) J. Biol. Chem. 272, 26271-26278). These effects are attributed to a different electrostatic interaction between Ogamma of Thr(E11) and the bound CO molecule. Molecular analysis indicates a more favorable interaction of the bound CO with Thr Ogamma in the beta-subunit heme pocket.

Structural factors controlling ligand binding to myoglobin: a kinetic hole-burning study.

Using temperature-derivative spectroscopy in the temperature range below 100 K, we have studied the dependence of the Soret band on the recombination barrier in sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation at 12 K. The spectra were separated into contributions from the photodissociated species, Mb*CO, and CO-bound myoglobin. The line shapes of the Soret bands of both photolyzed and liganded myoglobin were analyzed with a model that takes into account the homogeneous bandwidth, coupling of the electronic transition to vibrational modes, and static conformational heterogeneity. The analysis yields correlations between the activation enthalpy for rebinding and the model parameters that characterize the homogeneous subensembles within the conformationally heterogeneous ensemble. Such couplings between spectral and functional parameters arise when they both originate from a common structural coordinate. This effect is frequently denoted as "kinetic hole burning." The study of these correlations gives direct insights into the structure-function relationship in proteins. On the basis of earlier work that assigned spectral parameters to geometric properties of the heme, the connections with the heme geometry are discussed. We show that two separate structural coordinates influence the Soret line shape, but only one of the two is coupled to the enthalpy barrier for rebinding. We give evidence that this coordinate, contrary to widespread belief, is not the iron displacement from the mean heme plane.

Fourier transform infrared analysis of the interaction of azide with the active site of oxidized and reduced bovine Cu,Zn superoxide dismutase.

Binding of azide to the native and arginine-modified bovine Cu,Zn superoxide dismutase in the oxidized and reduced form and to the copper-free derivative has been investigated by Fourier transform infrared spectroscopy. The antisymmetric stretching band of the azide is shifted to higher energy upon coordination to the copper atom of the oxidized form of the native enzyme. Similar spectral changes occur upon interaction of the anion with the Cu-diethylenetriamine model compound. On the other hand, interaction of azide with the native reduced form of the enzyme results in a band shift toward lower energy with respect to the free anion band. The same shift is observed after reaction of the azide with free lysine or arginine but not when it is reacted with other amino acid residues. The antisymmetric band of the azide is not perturbed by addition of the reduced arginine-modified enzyme; it is likely shifted toward higher energy upon addition of oxidized arginine-modified enzyme while it is again shifted toward lower energy in the presence of the copper-free derivative of the unmodified enzyme. It is concluded that azide does not directly coordinate to the copper in the reduced form of Cu,Zn superoxide dismutase but it remains in the active-site pocket in electrostatic interaction with the guanidinium group of Arg141, which is an invariant residue in this class of enzymes.

Modification of alpha-chain or beta-chain heme pocket polarity by Val(E11) --> thr substitution has different effects on the steric, dynamic, and functional properties of human recombinant hemoglobin. Deoxy derivatives.

The dynamic and functional properties of mutant deoxyhemoglobins in which either the beta-globin Val67(E11) or the alpha-globin Val62(E11) is replaced by threonine have been investigated through the thermal evolution of the Soret absorption band in the temperature range 300 to 20 K and through the kinetics of CO rebinding after flash photolysis at room temperature. The conformational properties of the modified alpha chain and beta chain distal heme pockets were also studied through x-ray crystallography and molecular modeling. The data obtained with the various techniques consistently indicate that the polar isosteric mutation in the distal side of the alpha chain heme pocket has a larger effect on the investigated properties than the analogous mutation on the beta chain. We attribute the observed differences to the presence of a water molecule in the distal heme pocket of the modified alpha chains, interacting with the hydroxyl of the threonine side chain. This is indicated by molecular modeling which showed that the water molecule present in the alpha chain distal heme pocket can bridge by H bonding between Thr62(E11) and His58(E7) without introducing any unfavorable steric interactions. Consistent with the dynamic and functional data, the presence of a water molecule in the distal heme pocket of the modified beta chains is not observed by x-ray crystallography.

Local dynamics of DNA probed with optical absorption spectroscopy of bound ethidium bromide.

We have studied the local dynamics of calf thymus double-helical DNA by means of an "optical labeling" technique. The study has been performed by measuring the visible absorption band of the cationic dye ethidium bromide, both free in solution and bound to DNA, in the temperature interval 360-30 K and in two different solvent conditions. The temperature dependence of the absorption line shape has been analyzed within the framework of the vibronic coupling theory, to extract information on the dynamic properties of the system; comparison of the thermal behavior of the absorption band of free and DNA-bound ethidium bromide gave information on the local dynamics of the double helix in the proximity of the chromophore. For the dye free in solution, large spectral heterogeneity and coupling to a "bath" of low-frequency (soft) modes is observed; moreover, anharmonic motions become evident at suitably high temperatures. The average frequency of the soft modes and the amplitude of anharmonic motions depend upon solvent composition. For the DNA-bound dye, at low temperatures, heterogeneity is decreased, the average frequency of the soft modes is increased, and anharmonic motions are hindered. However, a new dynamic regime characterized by a large increase in anharmonic motions is observed at temperatures higher than approximately 280 K. The DNA double helix therefore appears to provide, at low temperatures, a rather rigid environment for the bound chromophore, in which conformational heterogeneity is reduced and low-frequency motions (both harmonic vibrations and anharmonic contributions) are hindered. The system becomes anharmonic at approximately 180 K; however, above approximately 280 K, anharmonicity starts to increase much more rapidly than for the dye free in solution; this can be attributed to the onset of wobbling of the dye in its intercalation site, which is likely connected with the onset of (functionally relevant) DNA motions, involving local opening/unwinding of the double helix. As shown by parallel measurements of the melting curves, these motions precede the melting of the double helix and depend upon solvent composition much more than does the melting itself.

Heme geometry in the 10 K photoproduct from sperm whale carbonmonoxymyoglobin.

We have measured the Soret band of the photoproduct obtained by complete photolysis of sperm whale carbonmonoxymyoglobin at 10 K. The experimental spectrum has been modeled with an analytical expression that takes into account the homogeneous bandwidth, the coupling of the electronic transition with both high and low frequency vibrational modes, and the effects of static conformational heterogeneity. The comparison with deoxymyoglobin at low temperature reveals three main differences. In the photoproduct, the Soret band is shifted to red. The band is less asymmetric, and an enhanced coupling to the heme vibrational mode at 674 cm-1 is observed. These differences reflect incomplete relaxation of the active site after ligand dissociation. The smaller band asymmetry of the photoproduct can be explained by a smaller displacement of the iron atom from the mean porphyrin plane, in quantitative agreement with the X-ray structure analysis. The enhanced vibrational coupling is attributed to a subtle heme distortion from the planar geometry that is barely detectable in the X-ray structure.

Structural fluctuations of myoglobin from normal-modes, Mössbauer, Raman, and absorption spectroscopy.

A normal-mode analysis of carbon monoxymyoglobin (MbCO) and deoxymyoglobin (Mb) with 170 water molecules is performed for (54)Fe and (57)Fe. A projection is defined that extracts iron out-of-plane vibrational modes and is used to calculate spectra that can be compared with those from resonance Raman scattering. The calculated spectra and the isotopic shift (57)Fe versus (54)Fe agree with the experimental data. At low temperatures the average mean square fluctuations (MSFs) of the protein backbone atoms agree with molecular dynamics simulation. Below 180 K the MSFs of the heme iron agree with the data from Mossbauer spectroscopy. The MSFs of the iron atom relative to the heme are an order of magnitude smaller than the total MSFs of the iron atom. They agree with the data from optical absorption spectroscopy. Thus the MSFs of the iron atom as measured by Mossbauer spectroscopy can be used to probe the overall motion of the heme within the protein matrix, whereas the Gaussian thermal line broadening of the Soret band and the resonance Raman bands can be used to detect local intramolecular iron-porphyrin motions.

Low temperature optical spectroscopy of low-spin ferric hemeproteins.

We report the Soret absorption spectra (500-350 nm) of the cyanomet derivatives of human hemoglobin and horse myoglobin, in the temperature range 300-20 K and in two different solvents (65% v/v glycerol-water or 65% v/v ethylene glycol-water). In order to obtain information on stereodynamic properties of active site of the two hemeproteins, we perform an analysis of the band profiles within the framework of electron-vibrations coupling. This approach enables us to single out the various contributions to the spectral bandwidth, such as those arising from non-radiative decay of the excited electronic state (homogeneous broadening) and from the coupling of the electronic transition i) with high frequency modes (that determines the vibronic structure of the band) and ii) with a "bath" of low frequency modes (that is responsible for the temperature dependence of the experimental spectra). We discuss the relevant parameters and their temperature dependence and compare them with the ones already reported for other derivatives of the same hemeproteins in the same solvents. In particular, non-harmonic contributions to soft modes are found, for cyanomet derivatives, to be larger than those observed for liganded carbonmonoxy but smaller than those observed for unliganded deoxy derivatives. The reported data enable us to obtain information on the dependence of stereodynamic properties of the heme pocket upon iron oxidation state, dimensions of the exogenous ligand and composition of the external matrix.