beta-lactoglobulin under high pressure studied by small-angle neutron scattering.

We used small-angle neutron scattering to study the effects of the high hydrostatic pressure on the structure of beta-lactoglobulin. Experiments were carried out at pH 7 on the dimeric form of the protein in a pressure range going from 50 MPa to 300 MPa. These measurements allow the protein size and the interactions between macromolecules to be studied during the application of pressure. Increasing pressure up to 150 MPa leads to a swollen state of the protein that gives rise to an increase of the radius of gyration by about 7%. Within this pressure range, we also show that the interaction between macromolecules weakens although it remains repulsive. The measurements show an aggregation process occurring above 150 MPa. From the spectra analysis, it appears that the aggregation occurs mainly by association of the dimeric units.

Characterization of the denatured states distribution of neocarzinostatin by small-angle neutron scattering and differential scanning calorimetry.

The denatured states of a small globular protein, apo-neocarzinostatin (NCS), have been characterized using several techniques. Structural properties were investigated by optical spectroscopy techniques and small-angle neutron scattering (SANS), as a function of guanidinium chloride (GdmCl) concentration. SANS experiments show that in heavy water, the protein keeps its native size at GdmCl concentrations below 2.5 M. A sharp transition occurs at about 3.6 M GdmCl, and NCS behaves like an excluded volume chain above 5 M. The same behavior is observed in deuterated buffer by fluorescence and circular dichroism measurements. For the H(2)O buffer, the transition occurs with lower concentration of denaturant, the shift being about 0.6 M. 8-Anilino-1-naphthalenesulfonate (ANS) was used as a hydrophobic fluorescent probe for studying the early stages of protein unfolding. Protein denaturation modifies the fluorescence intensity of ANS, a maximum of intensity being detected close to 2 M GdmCl in hydrogenated buffer, which shows the existence of at least one intermediate state populated at the beginning of the unfolding pathway. Differential scanning calorimetry (DSC) was used to obtain thermodynamic values for NCS denaturation. The melting curves recorded between 20 and 90 degrees C in the presence of various GdmCl concentrations (0-3 M) cannot be explained by a simple two-state model. Altogether, the data presented in this paper suggest that before unfolding the protein explores a distribution of states which is centered around compact states at denaturant concentrations below 2 M in H(2)O, and then shifts to less structured states by increasing denaturant concentrations.

Change in backbone torsion angle distribution on protein folding.

Understanding protein folding requires the determination of the configurational space accessible to the protein at different stages in folding. Here, computer simulation analysis of small angle neutron scattering results is used to probe the change in the distribution of configurations on strong denaturation of a globular protein, phosphoglycerate kinase, in 4 M guanidine hydrochloride solution. To do this atomic-detail ensembles of the unfolded protein chain are modeled and their scattering profiles compared with the experiment. The local conformational statistics are found to strongly influence the experimental intensity at scattering vectors between 0.05 and 0.3 A(-1). Denaturation leads to a reduction in the protein atom-pair distance distribution function over the approximately 3-15 A region that is associated with a quantifiable shift in the backbone torsional angle (phi, psi) distribution toward the beta region of the Ramachandran plot.

Excluded volume in the configurational distribution of a strongly-denatured protein.

The configurational distribution of phosphoglycerate kinase (PGK) strongly-denatured in 4 M guanidine hydrochloride solution is investigated using small-angle neutron scattering (SANS) and Monte Carlo computer simulation. It is shown that the experimental scattering profile can be represented by a random flexible chain of spheres of excess scattering density with excluded volume interactions, the best agreement being achieved when partial sphere intersection is allowed. The radius of gyration of the chain increases by a factor of 4 on denaturation, whereas the average length of segments approximately 5 residues long increases by only approximately 10%, consistent with a picture in which the large expansion on denaturation originates primarily from increased long-range flexibility of the polypeptide chain. The results provide a description of the chain statistics from which the construction of starting points for simulation studies of folding of the protein can be envisaged.

Repulsive interparticle interactions in a denatured protein solution revealed by small angle neutron scattering.

In order to investigate the effect of concentration in biological processes such as protein folding, small angle neutron scattering measurements were used to determine the second virial coefficient of solutions of both native and strongly denatured phosphoglycerate kinase and the radius of gyration of the protein at zero concentration. The value of the second virial coefficient is a good probe of the non-ideality of a solution. The present results show that the unfolding of the protein leads to a drastic change in the repulsive intermolecular interactions. We conclude that these interactions are due mainly to the behaviour of the denatured polypeptide chain as an excluded volume polymer.

Picosecond dynamical changes on denaturation of yeast phosphoglycerate kinase revealed by quasielastic neutron scattering.

Quasielastic neutron scattering experiments performed on yeast phosphoglycerate kinase in the native form and denatured in 1.5 M guanidinium chloride reveal a change in the fast (picosecond time scale) diffusive internal dynamics of the protein. The momentum and energy transfer dependences of the scattering for both states are fitted by an analytical model in which, on the experimentally accessible picosecond time scale and angstrom length scale, the dynamics of a fraction of the nonexchangeable hydrogens in the protein is described as a superposition of vibrations with uniform diffusion in a sphere, the rest of the hydrogens undergoing only vibrational motion. The fraction diffusing changes, from approximately 60% in the native protein to approximately 82% in the denatured protein. The radius of the sphere also changes slightly, from approximately 1.8 A in the native protein to approximately 2.2 A in the denatured protein. Possible implications of these results for the general protein folding problem are discussed.

Small-angle neutron scattering by a strongly denatured protein: analysis using random polymer theory.

Small-angle neutron scattering profiles are presented from phosphoglycerate kinase, in the native form and strongly denatured in 4 M guanidinium chloride (GdnHCl) solution. The data are interpreted using a model in which the excess scattering density associated with the protein is represented as a finite freely jointed chain of spheres. The similarity of the model-derived scattering function to experiment increases asymptotically with the number of spheres. The improvement of the fit obtained with more than approximately 200 spheres (i.e., two residues per sphere) is insignificant. The effects of finite size of the scattering units and of scattering length variation along the polypeptide chain are examined. Improved agreement with experiment is obtained when these effects are taken into account. A method for rapid calculation of the scattering profile of a full, all-atom configuration is examined. It is found that a representation of the chain containing two scattering units per residue, placed at the backbone and side-chain scattering length centroids, reproduces the full, all-atom profile to within 2%.

Solvent-induced structural distortions of horse metmyoglobin.

Structural and dynamic constraints produced by the surrounding solvent on the aquometmyoglobin molecule were investigated by means of circular dichroism and Fourier-transform infrared spectroscopies, tritium/hydrogen exchange kinetics and small-angle neutron-scattering experiments. Formamide and ethanol were chosen as cosolvents because they are known to increase and decrease protein activity, respectively. The CD measurements in the Soret region show that no changes occur in the heme molecular structure nor in the protein near the heme. The results of proton-exchange kinetics experiments indicate that the conformational dynamics of aquometmyoglobin is only marginally affected by the cosolvents. However, the small-angle neutron-scattering spectra strongly suggest that these cosolvents induce some distortions of the tertiary conformation. According to the ultraviolet CD and Fourier-transform infrared data, the alteration of the tertiary conformation results from changes in both the number of intrachain hydrogen bonds and the structures of beta turns of type I' for formamide and of type II for either of the two cosolvents. The use of several techniques allows the present approach to demonstrates that the myoglobin structure is extremely sensitive to its environmental conditions.

How random is a highly denatured protein?

There has been renewed interest in determining the physicochemical properties of denatured states of proteins. In many denatured states there is evidence for the existence of nonrandom configurational distributions. Here we examine the small-angle neutron scattering profile of yeast phosphoglycerate kinase in the native state and in highly denaturing conditions. We show that the denatured protein scattering profile can be interpreted using a model developed for synthetic polymers in which the chain behaves as a random coil in a good solvent, i.e. with excluded volume interactions. The implications of this result for our appreciation of the protein folding process are discussed.

Low-frequency vibrations of a nucleoside analog.

This paper reports on the first coherent neutron scattering measurements ever carried out on the vibrational spectrum of a nucleoside analog. Frequencies up to 3.5 Thz belonging to some half-dozen dispersion curves have been measured in a plane normal to the [1 10] direction in a triclinic crystal of 5-iodo-2'-deoxyuridine (5-iodouridine). The number of phonon branches observed suggests that the deoxyribose sugar and pyrimidine in this molecule are the vibrating entities at low frequencies. Comparisons, notably of elastic properties, are made with previous measurements on crystalline forms of DNA and various nucleic acid base derivatives. The observed frequencies are discussed with reference to a simple force constant model.

Configurational distribution of denatured phosphoglycerate kinase.

Physiochemical characterization of the denatured states of proteins is important for a complete understanding of the factors stabilizing their folded conformations. Using a combination of small angle neutron scattering (SANS), statistical mechanical modelling and molecular mechanics calculations, we examine the configurational distribution of phosphoglycerate kinase denatured in 4 M guanidine hydrochloride solution. The denaturing of the protein produces a clear change in the form of the SANS profile and a large increase of the radius of gyration. In the statistical mechanical model, the region of contrast neutron scattering density associated with the protein is pictured as a chain of freely jointed spheres. The model is fitted to the SANS data for the denatured protein. It is found that a model with a small number of spheres cannot account for the higher resolution scattering, indicating an absence of detectable structuration; a good fit is found with 100 spheres of 8.5 A radius. Single configurations of the fitted chain of spheres are used as low-resolution bounds for model-building and molecular mechanics calculations to obtain plausible atomic-detail models of the denatured chain.

Physical properties of the Escherichia coli transcription termination factor rho. 1. Association states and geometry of the rho hexamer.

To function as a DNA-RNA helicase in rho-dependent transcript termination, six genetically identical subunits of the Escherichia coli transcription termination protein rho must first assemble into a hexameric complex. To help determine the quaternary structure of this complex, we have studied the association equilibria of the rho protomers. Sedimentation equilibrium, sedimentation velocity, diffusion, X-ray scattering, and neutron-scattering data have been combined to create a "phase diagram" of the association states of this protein as a function of protein concentration and ionic environment. The results show that rho exists predominantly as a hexamer under approximately physiological conditions and that this hexamer is in equilibrium with both lower and higher states of association that may also have physiological relevance. Small-angle X-ray scattering measurements and theoretical calculations indicate that the rho hexamer has a radius of gyration of 50 +/- 3 A. The radius of gyration measured by small-angle neutron scattering in 2H2O is 47 +/- 3 A. These scattering studies also support earlier models of rho as a planar hexagon which have been developed on the basis of electron microscopy. In the following paper in this issue [Geiselmann, J., Seifried, S. E., Yager, T. D., Liang, C., & von Hippel, P. H. (1992)], these results are combined with information on symmetry, subunit interactions, and packing geometry to obtain a model of the quaternary structure of the functional rho hexamer.

Temperature and pH dependence of immunoglobulin G conformation.

Previous indirect observations have indicated that IgG may change its conformation at low or high pH and at a temperature of about 35 degrees C. By means of small angle neutron scattering a change in the value of the gyration radius of two different native IgG's was observed above 44 degrees C. No similar change was detected when the sample was previously dissolved in an acidic buffer. The acidic pretreatment caused a significant decrease in the gyration radius (Rg) value measured at 20 degrees C which was partially recovered by increasing the temperature. These observations led to the assumption that the main conformational change observed appears either in the hinge region of the molecular or in the interdomain areas separating the constant and the variable domains of the Fab parts.

Vibrational modes of hemoglobin in red blood cells.

Equine red blood cells were washed in saline heavy water (2H2O) to exchange the hydrogen atoms of the non-hemoglobin components with deuterons. This led to novel neutron scattering measurements of protein vibrations within a cellular system and permitted a comparison with inelastic neutron scattering measurements on purified horse hemoglobin, either dry or wetted with 2H2O. As a function of wavevector transfer Q and the frequency transfer v the neutron response typified by the dynamic structure factor S(Q, v) was found to be similar for extracted and cellular hemoglobin at low and high temperatures. At 77 K, in the cells, a peak in S(Q, v) due to the protein was found near 0.7 THz, approximately half the frequency of a strong peak in the aqueous medium. Measurements at higher temperatures (170 and 230 K) indicated similar small shifts downwards in the peak frequencies of both components. At 260 K the low frequency component became predominantly quasielastic, but a significant inelastic component could still be ascribed to the aqueous scattering. Near 295 K the frequency responses of both components were similar and centered near zero. When scattering due to water is taken into account it appears that the protein neutron response in, or out of, red blood cells is little affected by hydration in the low frequency regime where Van der Waals forces are thought to be effective.

Structure of halophilic malate dehydrogenase in multimolar KCl solutions from neutron scattering and ultracentrifugation.

The structure and solvent interactions of malate dehydrogenase from Halobacterium marismortui in multimolar KCl solvents are found to be similar to those in multimolar NACl solvents reported previously (G. Zaccai, E. Wachtel and H. Eisenberg, J. Mol. Biol. 190 (1986) 97). KCl rather than NaCl is predominant in physiological medium. At salt concentrations up to about 3.0 M, the protein (a dimer of M 87000 g/mol) can be considered to occupy an invariant volume in which it is associated with about 4100 molecules of water and about 520 molecules of salt. At very low resolution, the enzyme particle appears to have a compact protein core and protruding protein parts in interaction with the water and salt components, structural features that are not observed in non-halophilic mitochondrial malate dehydrogenase. The above conclusions were drawn from the analysis of neutron scattering and ultracentrifugation data, and the complementarity of these approaches is discussed extensively.

Laser-induced multiphoton processes in living cells.

It is shown that specific light-induced subcellular alterations, usually referred to as phase "paling," result from multiphoton absorption processes. For green light (532 nm), four photons are required to induce paling in chromosomes and nucleoli; a two-photon process is observed for UV light (266 nm).