Protein-polynucleotide scattering centers as a protein structure probe.

When certain basic globular proteins are mixed with nucleic acids near a critical concentration ratio, large, low density scattering centers of about 10(9) particle weight are created. Scattering from these complexes is altered when thermally inactivated proteins are substituted for enzymes in their native, globular conformation. Scattering data from heat-treated ribonuclease and lysozyme mixed with four different synthetic homopolyribonucleotides are reported. The concentration of nucleic acid necessary to produce maximum scattering from a heat-treated protein sample is shown to be a direct indication of the amount of enzyme that remains biologically active after being heated.     

Force as a probe of membrane protein structure and function

Force measurement techniques are being used increasingly to explore the mechanical properties of proteins, as well as the structural origins of intermolecular forces. Developments in instrumentation and the increasing availability of engineered and purified membrane proteins have widely expanded the range of biological systems that can be addressed. Within the past year, force measurements have identified novel mechanisms of binding between cell-surface proteins, as well as the mechanical properties of integral membrane proteins and the intramolecular interactions that stabilize their structures.     

Elastic incoherent neutron scattering as a probe of high pressure induced changes in protein flexibility

We report here the results of elastic incoherent neutron scattering experiments on three globular proteins (trypsin, lysozyme and β-lactoglobulin) in different pressure intervals ranging from 1 bar to 5.5 kbar. A decrease of the mean square hydrogen fluctuations, 〈u²〉, has been observed upon increasing pressure. Trypsin and β-lactoglobulin behave similarly while lysozyme shows much larger changes in 〈u²〉. This can be related to different steps in the denaturing processes and to the high propensity of lysozyme to form amyloids. Elastic incoherent neutron scattering has proven to be an effective microscopic technique for the investigation of pressure induced changes in protein flexibility.     

Electrophoretic light scattering as a probe of reaction kinetics

A new method is proposed for determining chemical rate contants in dimerization reactions of globular proteins. Light scattering from a solution of charged macromolecules in and applied electric field gives a series of bands whose widths can be used to deduce the reaction rate contants. This method should be applicable to other types of peactions. First order reactions are also considered.     

Ultraviolet resonance Raman spectroscopy as a probe of protein structure in the fd virus

Resonance Raman spectroscopy can provide details of molecular structure via the enhancement of specific vibrational bands in the spectrum of the scattered light when the laser excitation is tuned to electronic absorption wavelengths of the molecule. The availability of lasers operating in the deep ultraviolet region makes it possible to apply this technique to problems of protein structure. The backbone conformation and the environments of aromatic side chains can be probed via appropriate enhancement of selected vibrational modes. In this article we investigate ultraviolet resonance Raman (UVRR) spectra from the coat protein of the filamentous bacteriophage, fd, in the intact virus and in sodium dodecyl sulfate (SDS) suspension. The results indicate that 1) the protein is completely alpha-helical in the mature virus, but loses a large fraction of its helix content in the SDS micelles. 2) The two tyrosine residues appear to behave as H-bond acceptors in the intact phage but this interaction is lost in the micelles. 3) The tryptophan residue is not solvent-exposed in either protein conformation, although in SDS it is accessible to H/D exchange with the solvent. 4) The three phenylalanine residues are involved in stacking interactions in the intact virus; these are disrupted in the SDS micelles. 5) The single proline residue appears to be in a trans conformation both in the virus and in the micelles.     

Solid-state NMR as a probe of amyloid structure

Solid state nuclear magnetic resonance (NMR) has developed into one of the most informative and direct experimental approaches to the characterization of the molecular structures of amyloid fibrils, including those associated with Alzheimer's disease. In this article, essential aspects of solid state NMR methods are described briefly and results obtained to date regarding the supramolecular organization of amyloid fibrils and the conformations of peptides within amyloid fibrils are reviewed.     

Dynamic light scattering as a probe of superhelical DNA-intercalating agent interaction

Polarized dynamic light-scattering measurements on superhelical pBR322-plasmid DNA solutions in 0.2M NaCl, 2 mM NaPi, pH 7.0, 2 mM EDTA result in a translational diffusion coefficient D = (3.77 ± 0.10) × 10^?8 cm²/s for the native molecule. Modeling the DNA, in the simplest approximation, as a 10 × 440-nm effective hydrodynamic rigid rod yields a good fit to the apparent diffusion coefficient angular-dependence data up to 70°; the model fails at higher angles, probably due to the effects of flexibility or branching of the rod. Diffusion coefficient titration experiments with a platinum complex intercalating agent (PtTS) result in a titratable superhelix density of σ = ?0.079 ± 0.008 under our experimental conditions, corresponding to about 34 superhelical turns in the native DNA. The DNA contour length predicted by our two independent results, the rod dimensions and the number of superhelical turns, is in excellent agreement with the contour length calculated from the number of base pairs, supporting the hydrodynamic approximation of an effective rodlike structure for this small DNA molecule in solution.     

Photobiotin as a sensitive probe for protein labeling

A sensitive method for the nonisotopic in vitro labeling of proteins under physiological conditions using photobiotin, a compound originally developed for labeling nucleic acids (Forster et al. (1985) Nucleic Acids Res. 13, 745), has been developed. Using sheep brain tubulin as a model protein it was shown that labeling with photobiotin resulted in detection limits below 10 pg when avidin-alkaline phosphatase was used in the final step. No significant loss of tubulin polymerization, colchicine binding, recognition by antitubulin antibodies, or changes in electrophoretic behavior were observed. In addition, photobiotinylation of antitubulin antibodies did not affect their recognition of unlabeled tubulin. The use of photobiotin labeling with avidin-alkaline phosphatase detection for electrophoretic separations of molecular weight standards, crude protein supernatants, and tubulin gave a 64 to 1024-fold increase in sensitivity over Coomassie blue staining.     

3-Nitrotyrosine as a spectroscopic probe for investigating protein protein interactions

3-Nitrotyrosine (NT) is approximately 10(3)-fold more acidic than Tyr, and its absorption properties are strongly pH-dependent. NT absorbs radiation in the wavelength range where Tyr and Trp emit fluorescence (300-450 nm), and it is essentially nonfluorescent. Therefore, NT may function as an energy acceptor in resonance energy transfer (FRET) studies for investigating ligand protein interactions. Here, the potentialities of NT were tested on the hirudin thrombin system, a well-characterized protease inhibitor pair of key pharmacological importance. We synthesized two analogs of the N-terminal domain (residues 1-47) of hirudin: Y3NT, in which Tyr3 was replaced by NT, and S2R/Y3NT, containing the substitutions Ser2 --> Arg and Tyr3 --> NT. The binding of these analogs to thrombin was investigated at pH 8 by FRET and UV/Vis-absorption spectroscopy. Upon hirudin binding, the fluorescence of thrombin was reduced by approximately 50%, due to the energy transfer occurring between the Trp residues of the enzyme (i.e., the donors) and the single NT of the inhibitor (i.e., the acceptor). The changes in the absorption spectra of the enzyme inhibitor complex indicate that the phenate moiety of NT in the free state becomes protonated to phenol in the thrombin-bound form. Our results indicate that the incorporation of NT can be effectively used to detect protein protein interactions with sensitivity in the low nanomolar range, to uncover subtle structural features at the ligand protein interface, and to obtain reliable Kd values for structure activity relationship studies. Furthermore, advances in chemical and genetic methods, useful for incorporating noncoded amino acids into proteins, highlight the broad applicability of NT in biotechnology and pharmacological screening.     

Solid-state NMR as a probe of amyloid fibril structure

Amyloid fibrils are intrinsically noncrystalline, insoluble, high-molecular-weight aggregates of peptides and proteins, with considerable biomedical and biophysical significance. Solid-state NMR techniques are uniquely capable of providing high-resolution, site-specific structural constraints for amyloid fibrils, at the level of specific interatomic distances and torsion angles. So far, a relatively small number of solid-state NMR studies of amyloid fibrils have been reported. These have addressed issues about the supramolecular organization of beta-sheets in the fibrils and the peptide conformation in the fibrils, and have concentrated on the beta-amyloid peptide of Alzheimer's disease. Many additional applications of solid-state NMR to amyloid fibrils from a variety of sources are anticipated in the near future, as these systems are ideally suited for the technique and are of widespread current interest.     

Monoclonal antibody as a probe for structure and function of an Escherichia coli outer membrane protein

Eight independently derived monoclonal antibodies directed against the LamB protein were produced and characterized. By using these antibodies as probes, we identified four distinct topological and functional regions in the LamB molecule. Four monoclonal antibodies recognize antigenic determinants of the protein exposed on the outer side of the membrane. Two of these have their binding sites located in a region involved in maltose transport. One monoclonal antibody presumably binds to a determinant which is normally hidden in the membrane and three monoclonal antibodies recognize determinants facing the periplasmic space.     

Surface plasmon resonance as a probe of protein isomerization

This article presents new concepts in affinity chromatography/mass spectrometry for the study of molecular interactions. Chromatographic assays involving estrogen receptor-beta, sorbitol dehydrogenase, human alpha-thrombin, cholera toxin B subunit, beta-galactosidase, and Griffonia simplicifolia isolectin B(4) were established in microaffinity columns and operated in frontal analysis mode. Methods and formalism are presented for the measurement of dissociation constants, using direct methods in which the mass spectrometric signature of the ligand is used to measure breakthrough time and, hence, binding strength. The direct approach is capable of measuring sub-micromolar K(d) and higher, on sub-pmol amounts of immobilized protein, as shown in the cholera toxin assay. Indirect assays that demonstrate the advantage of routine, rugged performance were developed. By tracking the effect of a test ligand on a selected probe, or indicator ligand, dissociation constants in the low nanomolar range could be reliably determined for ligands to estrogen receptor-beta. Mass spectrometry supports the resolution of complex ligand mixtures, and it is demonstrated in the sorbitol dehydrogenase assay that ligands can be rank ordered across approximately three orders of magnitude in K(d), in a single run. A new concept for rapid mixture prescreening is presented, in which an indicator ligand can be used to discriminate between mixtures that contain high levels of weak ligands and those that contain single strong ligands.     

Differential scattering of circularly polarized light as a unique probe of polynucleosome superstructures

A theoretical approach to modeling Circular Intensity Differential Scattering (CIDS) of native chromatin as multiple scattering of dipoles is discussed without the Born approximation. The model can explain the experimental data in the literature. It is shown that CIDS contains more structural information than does total light scattering and to a good approximation is independent of the length of the scattering molecules. Finally, CIDS in conjunction with traditional light scattering measurements should aid in discriminating between various alternative models of higher order chromatin structure now being proposed. Generalization of this theoretical study to other complex biomolecular structures, is also briefly discussed.     

CO as a vibrational probe of heme protein active sites

Carbon monoxide is a useful vibrational probe of heme binding sites in proteins, because FeCO backbonding is modulated by polar interactions with protein residues, and by variations in the donor strength of the trans ligand. This modulation is sensitively monitored by the CO and FeC stretching frequencies, which are readily detectable in infrared and resonance Raman spectra. The two frequencies are anticorrelated, and the nuFeC/nuCO position along the correlation line reflects the type and strength of distal polar interactions. Changes in the trans ligand donor strength shift the correlation to higher or lower positions. Illustrative applications of the nuFeC/nuCO diagram are reviewed for proteins bearing histidine and thiolate axial ligands. Steric crowding has not been found to affect the nuFeC/nuCO correlations significantly, except in the special case of cytochrome oxidase, where the heme-bound CO may interact with the nearby CuB center.     

Phenanthroline-Cu(II) cleavage as a probe of rRNA structure

Chemical cleavage is developing into a powerful tool for analysis and characterization of nucleic acids. Phenanthroline-Cu(II) cleavage has been used extensively for studies of DNA for the last two decades, but recently has been applied to structural studies of RNA as well. This approach has been used to study the structure and structural changes occurring in ribosomal RNA within the ribosomes. In this article we discuss the mechanism by which phenanthroline cleaves, the applications possible using this approach, and the results that can be obtained. Protocols for use of phenanthroline are outlined as well.     

Pyrrolo-C as a fluorescent probe for monitoring RNA secondary structure formation

Pyrrolo-C (PC), or 3-[beta-D-2-ribofuranosyl]-6-methylpyrrolo[2,3-d]pyrimidin-2(3H)-one, is a fluorescent analog of the nucleoside cytidine that retains its Watson-Crick base-pairing capacity with G. Due to its red-shifted absorbance, it can be selectively excited in the presence of natural nucleosides, making it a potential site-specific probe for RNA structure and dynamics. Similar to 2-aminopurine nucleoside, which base-pairs with uridine (or thymidine), PC's fluorescence becomes reversibly quenched upon base-pairing, most likely due to stacking interactions with neighboring bases. To test its utility as an RNA probe, we examined PC's fluorescent properties over a wide range of ionic strengths, pH, organic cosolvents, and temperatures. Incorporation of PC into a single-stranded RNA results in an approximately 60% reduction of fluorescence intensity, while duplex formation reduces the fluorescence by approximately 75% relative to the free ribonucleoside. We find that the fluorescence intensity of PC is only moderately affected by ionic strength, pH, and temperature, while it is slightly enhanced by organic cosolvents, making it a versatile probe for a broad range of buffer conditions. We demonstrate two applications for PC: fluorescent measurements of the kinetics of formation and dissociation of an RNA/DNA complex, and fluorescent monitoring of the thermal denaturation of the central segment of an RNA duplex. Taken together, our data showcase the potential of pyrrolo-C as an effective fluorescent probe to study RNA structure, dynamics, and function, complementary to the popular 2-aminopurine ribonucleoside.