Study Of Protein Conformation Research Articles

Effects of the β-Amyloid and Carboxyl-terminal Fragment of Alzheimer's Amyloid Precursor Protein on the Production of the Tumor Necrosis Factor-α and Matrix Metalloproteinase-9 by Human Monocytic THP-1

To explore the direct role of beta-amyloid (Abeta) and carboxyl-terminal fragments of amyloid precursor protein in the inflammatory processes possibly linked to neurodegeneration associated with Alzheimer's disease, the effects of the 105-amino acid carboxyl-terminal fragment (CT(105)) of amyloid precursor protein on the production of tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinase-9 (MMP-9) were examined in a human monocytic THP-1 cell line and compared with that of Abeta. CT(105) elicited a marked increase in TNF-alpha and MMP-9 production in the presence of interferon-gamma in a dose- and time-dependent manner. Similar patterns were obtained with Abeta despite its low magnitude of induction. Autocrine TNF-alpha is likely to be a main mediator of the induction of MMP-9 because the neutralizing antibody to TNF-alpha inhibits MMP-9 production. Genistein, a specific inhibitor of tyrosine kinase, dramatically diminished both TNF-alpha secretion and subsequent MMP-9 release in response to CT(105) or Abeta. Furthermore, PD98059 and SB202190, specific inhibitors of ERK or p38 MAPK respectively, efficiently suppressed CT(105)-induced effects whereas only PD98059 was effective at reducing Abeta-induced effects. Our results suggest that CT(105) in combination with interferon-gamma might serve as a more potent activator than Abeta in triggering inflammatory processes and that both tyrosine kinase and MAPK signaling pathways may represent potential therapeutic targets for the control of Alzheimer's disease progression.

Theoretical determination of the vibrational absorption and Raman spectra of 3-methylindole and 3-methylindole radicals

We report theoretical calculations of vibrational absorption and Raman spectra for the tryptophan analog 3-methylindole using density functional theory, the Becke3LYP hybrid functional, and the TZ/2P basis set. These results are compared to experimentally measured vibrational absorption and Raman spectra for 3-methylindole. The theoretical calculations represent accurate predictions of the observed vibrational frequencies and intensities, and of the Raman intensities. Currently, tryptophan radicals, either neutral or cationic, are believed to participate in electron transfer in enzymes such as cytochrome c peroxidase, ribonucleotide reductase, and DNA photolyase. In this paper we also report theoretical vibrational absorption and Raman spectra for 3-methylindole cation radical and 3-methylindole neutral radical. These predictions should provide specific spectroscopic markers for the detection of neutral or cationic tryptophan radicals in biological systems, providing a complement to the data available from electron paramagnetic resonance experiments. Raman spectroscopy of tryptophan is already in use in the study of protein conformations; theoretical predictions for the radical species provides a new tool for the detection of neutral or cation radicals of tryptophan in natural systems predisposed to the appropriate experiment.

Intrahelical arrangement in the integral membrane protein rhodopsin investigated by site-specific chemical cleavage and mass spectrometry.

Site-specific cleavage on the interhelical loop I on the cytoplasmic face of rhodopsin has been observed after activation of a Cu-phenanthroline tethered cleavage reagent attached on the cytoplasmic loop IV. The characterization of the reaction products by mass spectrometry, both of the membrane-bound protein and of the CNBr-cleaved peptides, allows the site of cleavage to be determined precisely. The specific cleavage of the peptide bond between Q64 and H65 on loop I leaves the N-terminal peptide (M1-Q64) intact, confirmed by MALDI-MS detection of the two N-linked glycosyl groups near the N-terminus of rhodopsin. The limited extension of the tether side chain requires a interresidue distance between the cleavage site, Q64, and the site of ligand attachment, C316, of less than 12 A. Upon photoactivation of the receptor, no change in the cleavage pattern is observed; however, a simulated Meta II intermediate activation state indicates a much more complex cleavage pattern. The development of this cleavage method, previously used primarily as a "chemical nuclease", in combination with mass spectrometry, may provide a powerful method on membrane protein conformation studies that can be used to complement other biophysical characterizations.

Structure and dynamics of membrane proteins as studied by infrared spectroscopy

Infrared (IR) spectroscopy is a useful technique in the study of protein conformation and dynamics. The possibilities of the technique become apparent specially when applied to large proteins in turbid suspensions, as is often the case with membrane proteins. The present review describes the applications of IR spectroscopy to the study of membrane proteins, with an emphasis on recent work and on spectra recorded in the transmission mode, rather than using reflectance techniques. Data treatment procedures are discussed, including band analysis and difference spectroscopy methods. A technique for the analysis of protein secondary and tertiary structures that combines band analysis by curve-fitting of original spectra with protein thermal denaturation is described in detail. The assignment of IR protein bands in H 2O and in D 2O, one of the more difficult points in protein IR spectroscopy, is also reviewed, including some cases of unclear assignments such as loops, β-hairpins, or 3 10-helices. The review includes monographic studies of some membrane proteins whose structure and function have been analysed in detail by IR spectroscopy. Special emphasis has been made on the role of subunit III in cytochrome c oxidase structure, and the proton pathways across this molecule, on the topology and functional cycle of sarcoplasmic reticulum Ca 2+-ATPase, and on the role of lipids in determining the structure of the nicotinic acetylcholine receptor. In addition, shorter descriptions of retinal proteins and references to other membrane proteins that have been studied less extensively are also included.

Conformational change of human lens insoluble alpha-crystallin.

Human lens alpha-crystallin becomes progressively insoluble with age and is the major crystallin component in the water-insoluble (WI) fraction. The mechanism that causes the originally water-soluble (WS) alpha-crystallin to become insoluble is unknown. A conformational change by chemical modification may be the cause, but the nature of insolubility renders it impossible to study protein conformation in the WI fraction by most spectroscopic measurements. In the present study, alpha-crystallin in the WI fraction was extracted by urea and reconstituted to a folded protein by dialysis. The refolded urea-soluble (US) alpha-crystallin was compared with WS alpha-crystallin. The US alpha-crystallin has a greater amount of polymeric species, but fewer degraded subunits than the WS alpha-crystallin as shown by SDS-PAGE and Western blot. Circular dichroism (CD) measurements indicate that they have the same secondary structure but a different tertiary structure, possibly a partial unfolding in the US alpha-crystallin. This is supported by fluorescence measurements: Trp residues are more exposed and protein has a more-hydrophobic surface in the US than in the WS alpha-crystallin. Blue fluorescence further indicates that the US alpha-crystallin has a greater amount of pigment than the WS alpha-crystallin. Together, these results indicate that the US alpha-crystallin is a chemically and conformationally modified protein.

High Pressure as a Tool to Study Protein Conformation.

The recent interest on high pressure biotechnology requires fundamental studies on the pressure behavior of biochemical constituents. In this laboratory, we use hydrostatic pressure both as a thermodynamic parameter, complementary to temperature, to determine the energetic quantities of enzyme reactions and as a tool to study protein conformation. For the second point, different new approaches are presented mainly based on spectroscopic observations which necessitate special high pressure adaptations.

Conformation of T4 lysozyme in solution. Hinge-bending motion and the substrate-induced conformational transition studied by site-directed spin labeling.

T4 lysozyme and mutants thereof crystallize in different conformations that are related to each other by a bend about a hinge in the molecule. This observation suggests that the wild type protein may undergo a hinge-bending motion in solution to allow substrate access to an otherwise closed active site cleft [Faber, H.R., & Matthews, B.W. (1990) Nature 348, 263-266]. To test this hypothesis, either single or pairs of nitroxide side chains were introduced into the protein to monitor tertiary contact interactions and inter-residue distances, respectively, in solution. A set of constraints for these structural parameters was derived from a reference state, a covalent enzyme-substrate adduct where the enzyme is locked in the closed state. In the absence of substrate, differences in both inter-residue distances and tertiary contact interactions relative to this reference state are consistent with a hinge-bending motion that opens the active site cleft. Quantitative analysis of spin-spin interactions between nitroxide pairs reveals an 8 A relative domain movement upon substrate binding. In addition, it is demonstrated that the I3P mutation, which produces a large hinge-bending angle in the crystal, has no effect on the solution conformation. Thus, the hinge motion is not the result of the mutation but is an integral part of T4 lysozyme catalysis in solution, as suggested recently [Zhang, X.J., Wozniak, J.A., & Matthews, B.W. (1995) J. Mol. Biol. 250, 527-552]. The strategy employed here, based on site-directed spin labeling, should be generally applicable to the study of protein conformation and conformational changes in solution.

Polyacrylamide Gel Electrophoresis in Discontinuous Transverse Urea-Gradient Gels

Polyacrylamide gel electrophoresis in transverse urea-gradient gels is widely used for the study of changes in conformation and quaternary structure of proteins induced by increasing concentrations of urea. However, possible uncertainties regarding the effective concentration of urea at any given point across the gel must be considered when this technique is used for quantitative purposes. We describe here a simple, practical procedure for preparing urea-gradient gels discontinuously, by stacking various layers of acrylamide solution with increasing urea concentrations. We show that the urea concentration in different lanes of discontinuous gradient gels prepared in this way can be predetermined accurately enough for quantitative purposes and provide as an example the study of the dissociation of thyroglobulin in urea. Various levels of resolution can be attained by this technique. The use of discontinuous gradient gels may extend the usefulness of urea gel electrophoresis in relation with the quantitative aspects of the study of protein conformation.

The ubiquitous cofactor NADH protects against substrate-induced inhibition of a pyridoxal enzyme.

In the usual reaction catalyzed by D-amino acid transaminase, cleavage of the alpha-H bond is followed by the reversible transfer of the alpha-NH2 to a keto acid cosubstrate in a two-step reaction mediated by the two vitamin B6 forms pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP). We report here a reaction not on the main pathway, i.e., beta-decarboxylation of D-aspartate to D-alanine, which occurs at 0.01% the rate of the major transaminase reaction. In this reaction, beta-C-C bond cleavage of the single substrate D-aspartate occurs rather than the usual alpha-bond cleavage in the transaminase reaction. The D-alanine produced from D-aspartate slowly inhibits both transaminase and decarboxylase activities, but NADH or NADPH instantaneously prevent D-aspartate turnover and D-alanine formation, thereby protecting the enzyme against inhibition. NADH has no effect on the enzyme spectrum itself in the absence of substrates, but it acts on the enzyme.D-aspartate complex with an apparent dissociation constant of 16 microM. Equivalent concentrations of NAD or thiols have no such effect. The suppression of beta-decarboxylase activity by NADH occurs concomitant with a reduction in the 415-nm absorbance due to the PLP form of the enzyme and an increase at 330 nm due to the PMP form of the enzyme. alpha-Ketoglutarate reverses the spectral changes caused by NADH and regenerates the active PLP form of the enzyme from the PMP form with an equilibrium constant of 10 microM. In addition to its known role in shuttling electrons in oxidation-reduction reactions, the niacin derivative NADH may also function by preventing aberrant damaging reactions for some enzyme-substrate intermediates. The D-aspartate-induced effect of NADH may indicate a slow transition between protein conformational studies if the reaction catalyzed is also slow.

Protein conformation in solution by three-dimensional fluorescence spectrometry

The conformations of bovine serum albumin (USA) and egg albumin (EA) in solution and their conformation changes under different conditions were studied by using three-dimensional fluorescence spectrometry (TDFS) such as three-dimensional fluorescence (TDF) spectra and three-dimensional fluorescence polarization (TDFP) spectra with tryptophan residues in protein molecules as an intrinsic fluorescent probe. The results show that the microenvironment of tryptophan residues of protein molecules in various solutions can be directly indicated and TDFS is an effective tool for studying protein conformation in solution. Meantime, some valuable results were obtained.

New approach to the study of transient protein conformations: the formation of a semiburied salt link in the folding pathway of barnase.

We use in this study a novel kinetic approach to determine the H+ titration properties of a semiburied salt link in the transition state for unfolding of barnase. The approach is based on changes in the pH dependence of the kinetics upon mutation of a target residue. This makes it relatively insensitive to the absolute value of the stability and, thereby, to artifacts caused by structural rearrangements around the site of mutation. The semiburied salt bridge studied here is between Asp93 and Arg69. Mutation of either residue significantly destabilized the protein, and the pKa value of Asp93 is severely lowered in the native state to below 1 because of the ionic interaction with Arg69. The Asp93-Arg69 salt link appears to be formed early in the folding process; the pKa value of Asp93 in the transition state (approximately 1) is similar to that in the native state, and deletion of the ionic interaction with Arg69 substantially destabilizes the folding intermediate and changes the kinetic behavior from multistate to two-state or close to two-state, depending on the conditions. The results suggest that the formation of ionic interactions within clusters of hydrophobic residues can be important for early folding events and can control kinetically the folding pathway. This is not because of the inherent stability of the salt link but because the presence of two unpaired charges is very unfavorable. The data reveal also that fractional phi values are consistent with a uniformly expanded transition state or one with closely spaced energy levels and not with parallel folding pathways.

Time-resolved tryptophan phosphorescence spectroscopy: a sensitive probe of protein folding and structure

Room-temperature tryptophan phosphorescence from proteins is a sensitive probe of the local structural environment of the luminescent amino acid. Tryptophan phosphorescence lifetimes can vary over 3-4 orders of magnitude depending on the structural rigidity of the emitter environment and the proximity of quenching interactions, and this sensitivity can be used to characterize the local structural environment of the chromophore. Tryptophan phosphorescence lifetime can be easily monitored as a function of time, and thus is capable of producing effectively real-time information on dynamic processes in proteins. We discuss the origins and characteristics of tryptophan phosphorescence, and its application to monitoring folding processes in proteins and to studying protein conformation and flexibility. We also present the extension of phosphorescence techniques to study, for the first time, triplet emission at room temperature from tryptophan residues engineered into specific positions as reporters of protein structure.

Protein fluorescence decay: discrete components or distribution of lifetimes? Really no way out of the dilemma?

A new methodology of fluorescence decay analysis by iterative reconvolution is presented. It is based on the recent finding that the statistics of single-photon time-correlated data are best described by a compound Poisson law and requires the recording of a sample of at least 20 decays. Application of multivariate statistical methods to the analysis of the recovered decay parameters results in improved accuracy and better estimation of the uncertainties of mono- and multiexponential decays. If it is, of course, not possible to distinguish unambiguously between discrete components and a continuous distribution of lifetimes, it is, however, possible to determine a higher limit of the width of such a distribution should it be present. With our methodology, the presence of a distribution of lifetimes with a width of approximately 20% of its center value inevitably leads to a failure in the deconvolution procedure, a fact of crucial importance in protein conformational studies, for example.

Pyrene excimer fluorescence as a probe of protein conformational change.

Fluorescence probes are useful in studies of protein conformation and interactions because of the availability of fluorophors with a variety of fluorescence properties, of derivatives with different labeling specificities, and because of the accessibility of excellent fluorescence instruments (Haugland, 1992; Lakowicz, 1983). Derivatives of the fluorophor can be attached to specific amino acid residues of proteins while maintaining the spectroscopic properties of the parent chromophore. Information about the protein domain to which the probe is attached can be obtained because the sensitive fluorescence properties depend on the nature of the excited state. Due to the more polar electron distribution in the excited state than the ground state, the probe can interact strongly with the immediate environment, allowing for the use of a variety of fluorescence measurements: (1) the spectral characteristics depend on the polarity of the immediate environment; (2) the relative quantum yield or intensity depends on the proximity of quenching groups; (3) changes in relative probe exposure can be obtained by solute quenching techniques; (4) as fluorescence energy donors and acceptors, probes can provide information on changes in distance in the 15–60 Å range; and (5) polarization of fluorescence measurements can give information about the flexibility of the domain to which the probes are attached.

26] Simple force field for study of peptide and protein conformational properties

Publisher Summary This chapter discusses the simple force field for use in the study of peptide and protein conformation. Both backbone and side chains have been examined and are shown to be in generally good agreement with properties derived from high-resolution protein structures. CRASS, a simple force field for use in the study of conformational properties of peptides and proteins is derived. CRASS explores the conformational properties of small peptides. To keep CRASS simple, the united atoms approximation is adopted: nonpolar hydrogens are not included explicitly; instead, the radii of the atoms to which they are attached are enlarged accordingly. A set of vander Waals radii, Ri, is determined from a survey of small molecule crystal structures. CRASS vander Waals radii and atom (and group) types are listed. The residue is simulated in the center of flexible blocked tripeptides using the CRASS force field at a temperature of 298 K. The simulated serine and threonine distributions do not agree with those observed in proteins, presumably because electrostatic interactions are ignored in CRASS. In particular, within CRASS it is possible for two atoms of like charge (for example, two oxygen) to interact favorably at short distances.

Significance of Root-Mean-Square Deviation in Comparing Three-dimensional Structures of Globular Proteins

In the study of globular protein conformations, one customarily measures the similarity in three-dimensional structure by the root-mean-square deviation (RMSD) of the C α atomic coordinates after optimal rigid body superposition. Even when the two protein structures each consist of a single chain having the same number of residues so that the matching of C α atoms is obvious, it is not clear how to interpret the RMSD. A very large value means they are dissimilar, and zero means they are identical in conformation, but at what intermediate values are they particularly similar or clearly dissimilar? While many workers in the field have chosen arbitrary cutoffs, and others have judged values of RMSD according to the observed distribution of RMSD for random structures, we propose a self-referential, non-statistical standard. We take two conformers to be intrinsically similar if their RMSD is smaller than that when one of them is mirror inverted. Because the structures considered here are not arbitrary configurations of point atoms, but are compact, globular, polypeptide chains, our definition is closely related to similarity in radius of gyration and overall chain folding patterns. Being strongly similar in our sense implies that the radii of gyration must be nearly identical, the root-mean-square deviation in interatomic distances is linearly related to RMSD, and the two chains must have the same general fold. Only when the RMSD exceeds this level can parts of the polypeptide chain undergo nontrivial rearrangements while remaining globular. This enables us to judge when a prediction of a protein's conformation is "correct except for minor perturbations", or when the ensemble of protein structures deduced from NMR experiments are "basically in mutual agreement".

Separation of Freezing- and Drying-Induced Denaturation of Lyophilized Proteins Using Stress-Specific Stabilization: II. Structural Studies Using Infrared Spectroscopy

The conformation of two labile enzymes, lactate dehydrogenase and phosphofructokinase, has been examined in the aqueous and lyophilized states, using infrared spectroscopy. In the preceding paper it was demonstrated that a stress-specific stabilization scheme, which employs a combination of a cryoprotectant (polyethylene glycol) and a compound which protects the dried protein (sugars or mannitol), can be used to optimize recovery of activity of these enzymes upon freeze-drying and rehydration. The purpose of the present study is to determine the effects of these additives on the conformation of these enzymes during lyophilization. Lyophilization in the absence of stabilizers was observed to induce significant conformational changes in both enzymes. Addition of 10 mM mannitol, lactose, or trehalose or 1% polyethylene glycol to the enzyme solutions attenuated the unfolding, but significant spectral differences for the enzymes in the dried state are still observed when compared to the aqueous conformation. Addition of any one of these stabilizers does not improve recovery of activity. However, when a combination of 1% PEG and either 10 mM mannitol, lactose, or trehalose is added, the native structure is preserved during lyophilization and essentially full enzymatic activity is recovered upon reconstitution. The ability of the stabilizers to preserve the native structure during lyophilization correlates directly with the recovery of enzymatic activity upon reconstitution. It appears that for labile proteins, preservation of the native structure during lyophilization is requisite for recovery of activity following rehydration. This study demonstrates that the infrared spectroscopic technique is a rapid and useful method for studying protein conformation in the dried state and can aid in determining the optimal conditions for stabilization of proteins during lyophilization.

In situ studies of protein conformation in supercritical fluids: trypsin in carbon dioxide.

The conformation of the monomeric enzyme trypsin has been studied in supercritical carbon dioxide. Steady-state fluorescence spectroscopy is used to follow the conformation of trypsin in situ as a function of CO2 density. Our results show for the first time that protein denaturation can occur during the fluid compression step and that the native trypsin is only slightly more stable (1.2 kcal/mol) than the unfolded form. These results demonstrate the power of fluorescence spectroscopy as a tool for studying protein conformation and dynamics in supercritical fluids.

Synthesis of the proline analogue [2,3- 3H]azetidine-2-carboxylic acid Uptake and incorporation in Arabidopsis thaliana and Escherichia coli

Azetidine-2-carboxylic acid, the 4-membered ring noranalogue or proline, is regularly used in the study or proline metabolism as well as the study of protein conformation. We prepared ??-[2,3- 3H]azetidine-2-carboxylic acid with an optimized 10% yield from commercially available 4-amino-[2,3- 3H]butyric acid. Purification was performed by fast-protein liquid chromatography. The biological activity was checked in both Arabidopsis thaliana and Escherichia coli. The obtained specific activity or 10 mCl/mmol was sufficient for most uptake and incorporation studies.

Comparison of various molecular forms of bovine trypsin: correlation of infrared spectra with x-ray crystal structures

Fourier-transform infrared spectroscopy is a valuable method for the study of protein conformation in solution primarily because of the sensitivity to conformation of the amide I band (1700-1620 cm-1) which arises from the backbone C = O stretching vibration. Combined with resolution-enhancement techniques such as derivative spectroscopy and self-deconvolution, plus the application of iterative curve-fitting techniques, this method provides a wealth of information concerning protein secondary structure. Further extraction of conformational information from the amide I band is dependent upon discerning the correlations between specific conformational types and component bands in the amide I region. In this paper, we report spectra-structure correlations derived from conformational perturbations in bovine trypsin which arise from autolytic processing, zymogen activation, and active-site inhibition. IR spectra were collected for the single-chain (beta-trypsin) and once-cleaved, double-chain (alpha-trypsin) forms as well as at various times during the course of autolysis and also for zymogen, trypsinogen, and beta-trypsin inhibited with diisopropyl fluorophosphate. Spectral differences among the various molecular forms were interpreted in light of previous biochemical studies of autolysis and the known three-dimensional structures of the zymogen, the active enzyme, and the DIP-inhibited form. Our spectroscopic results from these proteins in D2O imply that certain loop structures may absorb in the region of 1655 cm-1. Previously, amide I' infrared bands near 1655 cm-1 have been interpreted as arising solely from alpha-helices. These new data suggest caution in interpreting this band. We have also proposed that regions of protein molecules which are known from crystallographic experiments to be disordered absorb in the 1645 cm-1 region and that type II beta-turns absorb in the region of 1672-1685 cm-1. Our results also corroborate assignment of the low-frequency component of extended strands to bands below 1636 cm-1. Additionally, the results of multiple measurements have allowed us to estimate the variability present in component band areas calculated by curve fitting the resolution-enhanced IR spectra. We estimate that this approach to data analysis and interpretation is sensitive to changes of 0.01 unit or less in the relative integrated intensities of component bands in spectra whose peaks are well resolved.