Showing papers in "Biochemistry in 1993"

The carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer's disease.

Abstract: Several variants of the beta amyloid protein, differing only at their carboxy terminus (beta 1-39, beta 1-40, beta 1-42, and beta 1-43), have been identified as the major components of the cerebral amyloid deposits which are characteristic of Alzheimer's disease. Kinetic studies of aggregation by three naturally occurring beta protein variants (beta 1-39, beta 1-40, beta 1-42) and four model peptides (beta 26-39, beta 26-40, beta 26-42, beta 26-43) demonstrate that amyloid formation, like crystallization, is a nucleation-dependent phenomenon. This discovery has practical consequences for studies of the beta amyloid protein. The length of the C-terminus is a critical determinant of the rate of amyloid formation ("kinetic solubility") but has only a minor effect on the thermodynamic solubility. Amyloid formation by the kinetically soluble peptides (e.g., beta 1-39, beta 1-40, beta 26-39, beta 26-40) can be nucleated, or "seeded", by peptides which include the critical C-terminal residues (beta 1-42, beta 26-42, beta 26-43, beta 34-42). These results suggest that nucleation may be the rate-determining step of in vivo amyloidogenesis and that beta 1-42 and/or beta 1-43, rather than beta 1-40, may be the pathogenic protein(s) in AD.

Tris(phenanthroline)ruthenium(II) enantiomer interactions with DNA: Mode and specificity of binding

Abstract: Absorbance and fluorescence methods, circular dichroism, UV melting experiments, viscosity, and competition dialysis were used to study the interaction of delta and lambda tris(phenanthroline)ruthenium(II) with DNA. The results of these studies indicated that both isomers bind to DNA by a single mode. The two isomers differ, however, in their effect on the hydrodynamic properties of DNA as measured by viscosity and, therefore, probably differ in their individual binding modes. The optical properties of the fully bound compounds differ from those of the free, but the perturbations of their visible absorbance and fluorescence emission spectra are modest when compared to changes observed for other DNA binding compounds. Binding of both isomers to DNA was found to be weak (in comparison to proven intercalators), with binding constants on the order of 10(4) M-1 determined for their binding to calf thymus DNA. A small, positive enthalpy was found for the binding of each isomer to DNA, suggesting that binding is entropically driven. Both isomers increased the melting temperature of DNA, with little quantitative difference between the two. A modest base specificity was found for each isomer, with the delta isomer preferentially binding to GC base pairs, and the lambda isomer preferentially binding to AT base pairs. Competition dialysis was used to examine the preference of delta and lambda Ru for right-handed B DNA and left-handed Z DNA. Neither isomer exhibits significant selectivity for these radically different DNA secondary structures.

DNA double-crossover molecules.

Abstract: DNA molecules containing two crossover sites between helical domains have been suggested as intermediates in recombination processes involving double-strand breaks. We have modeled these double-crossover structures in an oligonucleotide system. Whereas the relative orientations of the helical domains must be specified in designing these molecules, there are two broad classes of the molecules, the parallel, DP, and antiparallel, DA, molecules. The distance between crossover points must be specified as multiples of half-turns, in order to avoid torsional stress in this system; hence, there are two further subdivisions, those double-crossover molecules separated by odd, O, and even, E, numbers of half-turns. In addition, the parallel molecules with odd numbers of half-turns between crossovers must be divided into those with an excess major or wide-groove separation, W, or those with an excess minor- or narrow-groove separation, N. We have constructed models of all five of these classes, DAE, DAO, DPE, DPOW, and DPON. DPE molecules containing 1 and 2 helical turns between crossovers have been constructed; the DAE molecule contains 1 turn between crossovers, and the DAO, DPOW, and DPON molecules contain 1.5 helical turns between crossovers. None of the parallel molecules is well-behaved; the molecules either dissociate or form multimers when visualized on native polyacrylamide gels. In contrast, antiparallel molecules form single bands when assayed in this fashion. Hydroxyl radical autofootprinting analysis of these molecules reveals protection at expected sites of crossover and of occlusion, suggesting that all the complexes contain linear helix axes that are roughly coplanar between crossovers. However, the DPOW molecule and the DPE molecule with 2 turns between crossovers show decreased protection in the portion between crossovers, suggesting that their helices may bow in response to charge repulsion. We conclude that the helices between parallel double crossovers must be shielded from each other or distorted from linearity if they are to participate in recombination. We have analyzed the possibilities of branch migration and crossover isomerization in double-crossover molecules. Parallel molecules need no sequence symmetry beyond homology to branch migrate, but the sequence symmetry requirements for antiparallel molecules restrict migration to directly repetitive segments that iterate the sequence between crossovers. Crossover isomerization appears to be a very complex process in parallel double-crossover molecules, suggesting that it may be catalyzed by topoisomerases if it occurs within the cell.

Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein

Abstract: The green-fluorescent proteins (GFP) are a unique class of proteins involved in bioluminescence of many cnidaria. The GFPs serve as energy-transfer acceptors, receiving energy from either a luciferase-oxyluciferin complex or a Ca(2+)-activated photoprotein, depending on the organism. Upon mechanical stimulation of the organism, GFP emits green light spectrally identical to its fluorescence emission. These highly fluorescent proteins are unique due to the nature of the covalently attached chromophore, which is composed of modified amino acid residues within the polypeptide. This report describes the characterization of the Aequorea victoria GFP chromophore which is released as a hexapeptide upon digestion of the protein with papain. The chromophore is formed upon cyclization of the residues Ser-dehydroTyr-Gly within the polypeptide. The chromophore structure proposed here differs from that described by Shimomura [(1979) FEBS Lett. 104, 220] in a number of ways.

Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing

Abstract: The only component of the infectious scrapie prion identified to date is a protein designated PrPSc. A posttranslational process converts the cellular PrP isoform (PrPC) into PrPSc. Denatured PrPSc was digested with endoproteases, and the resulting fragments were isolated by HPLC. By both mass spectrometry and Edman sequencing, the primary structure of PrPSc was found to be the same as that deduced from the PrP gene sequence, arguing that neither RNA editing nor protein splicing feature in the synthesis of PrPSc. Mass spectrometry also was used to search for posttranslational chemical modifications other than the glycosylinositol phospholipid anchor attached to the C-terminus and two Asn-linked oligosaccharides already known to occur on both PrPSc and PrPC. These results contend that PrPSc molecules do not differ from PrPC at the level of an amino acid substitution or a posttranslational chemical modification; however, we cannot eliminate the possibility that a small fraction of PrPSc is modified by an as yet unidentified posttranslational process or that PrPC carries a modification that is removed in the formation of PrPSc. It seems likely that PrPSc differs from PrPC in its secondary and tertiary structure, but the possibility of a tightly bound, disease-specific molecule which purifies with PrPSc must also be considered.

Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MgATP and peptide inhibitor.

Abstract: The structure of a ternary complex of the catalytic subunit of cAMP-dependent protein kinase, MgATP, and a 20-residue inhibitor peptide was determined at a resolution of 2.7 A using the difference Fourier technique starting from the model of the binary complex (Knighton et al., 1991a). The model of the ternary complex was refined using both X-PLOR and TNT to an R factor of 0.212 and 0.224, respectively. The orientation of the nucleotide and the interactions of MgATP with numerous conserved residues at the active site of the enzyme are clearly defined. The unique protein kinase nucleotide binding site consists of a five-stranded antiparallel beta-sheet with the base buried in a hydrophobic site along beta-strands 1 and 2 and fixed by hydrogen bonds to the N6 amino and N7 nitrogens. The small lobe secures the nucleotide via a glycine-rich loop and by ion pairing with Lys72 and Glu91. While the small lobe fixes the nontransferable alpha- and beta-phosphates in this inhibitor complex, the gamma-phosphate is secured by two Mg2+ ions and interacts both directly and indirectly with several residues in the large lobe--Asp184, Asn171, Lys168. Asp166 is positioned to serve as a catalytic base. The structure is correlated with previous chemical evidence, and the features that distinguish this nucleotide binding motif from other nucleotide binding proteins are delineated.

Antagonistic action of imidazolineoxyl N-oxides against endothelium-derived relaxing factor/.NO through a radical reaction.

Abstract: A labile inorganic free radical, nitric oxide (.NO), is produced by nitric oxide synthase from the substrate L-arginine in various cells and tissues. It acts as an endothelium-derived relaxing factor (EDRF) or as a neurotransmitter in vivo. We investigated the reactivity of stable radical compounds, imidazolineoxyl N-oxides such as 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), carboxy-PTIO, and carboxymethoxy-PTIO against .NO/EDRF in both chemical and biological systems. By using electron spin resonance (ESR) spectroscopy, imidazolineoxyl N-oxides were found to react with .NO in a stoichiometric manner (PTIO/.NO = 1.0) in a neutral solution (sodium phosphate buffer, pH 7.4) with rate constants of approximately 10(4) M-1 s-1, resulting in the generation of NO2-/NO3- and imidazolineoxyls such as 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl (PTI), carboxy-PTI, or carboxymethoxy-PTI. Furthermore, the effects of imidazolineoxyl N-oxides on acetylcholine- or ATP-induced relaxation of the smooth muscle of rabbit aorta were tested. The vasorelaxations were inhibited by all three imidazolineoxyl N-oxides markedly. The inhibitory effects of carboxy-PTIO was almost 2-fold stronger than those of .NO synthesis inhibitors, N omega-nitro-L-arginine and N omega-monomethyl-L-arginine. Generation of EDRF/.NO was identified by reacting the PTIO in aortic strips and quantitating the reaction product with ESR spectroscopy. Thus, it was clarified that imidazolineoxyl N-oxide antagonize EDRF/.NO via a unique radical-radical reaction with .NO.

Mode of formation and structural features of DNA-cationic liposome complexes used for transfection.

Abstract: Complexes formed between cationic liposomes and nucleic acids represent a highly efficient vehicle for delivery of DNA and RNA molecules into a large variety of eukaryotic cells. By using fluorescence, gel electrophoresis, and metal-shadowing electron microscopy techniques, the factors that affect the, yet unclear, interactions between DNA and cationic liposomes as well as the structural features of the resulting complexes have been elucidated. A model is suggested according to which cationic liposomes bind initially to DNA molecules to form clusters of aggregated vesicles along the nucleic acids. At a critical liposome density, two processes occur, namely, DNA-induced membrane fusion, indicated by lipid mixing studies, and liposome-induced DNA collapse, pointed out by the marked cooperativity of the encapsulation processes, by their modulations by DNA-condensing agents, and also by their conspicuous independence upon DNA length. The DNA collapse leads to the formation of condensed structures which can be completely encapsulated within the fused lipid bilayers in a fast, highly cooperative process since their exposed surface is substantially smaller than that of extended DNA molecules. The formation of the transfecting DNA-liposome complexes in which the nucleic acids are fully encapsulated within a positively-charged lipid bilayer is proposed, consequently, to be dominated by mutual effects exerted by the DNA and the cationic liposomes, leading to interrelated lipid fusion and DNA collapse.

cDNA cloning, chromosomal mapping, and functional characterization of the human peroxisome proliferator activated receptor.

Abstract: The human peroxisome proliferator activated receptor (hPPAR) was cloned from a human liver cDNA library. The cDNA exhibited 85% and 91% DNA and deduced amino acid sequence identity with mouse PPAR (mPPAR), respectively. The hPPAR gene was mapped on human chromosome 22 slightly telomeric to a linkage group of six genes and genetic markers that are located in the general region 22q12-q13.1. Cotransfection assays of mouse Hepa 1 cells were used to roughly compare the ability of hPPAR- and mPPAR-expressed cDNAs to trans-activate the acyl CoA oxidase (ACO) PPAR response element located 5' upstream to the minimal thymidine kinase promoter driving the expression of the chloramphenicol acetyl transferase (CAT) reporter gene. Both receptors elicited a response with the prototypical peroxisome proliferators nafenopin, clofibrate, and WY-14,643. Moreover, using cotransfection assays in which the CAT reporter plasmid contained the CYP4 A6 gene response element rather than the ACO element, it was shown that hPPAR is capable of very efficiently trans-activating a second PPAR response element. These results indicate that the PPAR is present in humans in a form that is functional and can trans-activate response elements derived from two different genes, the rat ACO and the rabbit CYP4A6.

Binding of acylated peptides and fatty acids to phospholipid vesicles : pertinence to myristoylated proteins

Abstract: We studied the binding of fatty acids and acylated peptides to phospholipid vesicles by making electrophoretic mobility and equilibrium dialysis measurements. The binding energies of the anionic form of the fatty acids and the corresponding acylated glycines were identical; the energies increased by 0.8 kcal/mol per number of carbons in the acyl chain (Ncarbon = 10, 12, 14, 16), a value identical to that for the classical entropy-driven hydrophobic effect discussed by Tanford [The Hydrophobic Effect (1980) Wiley, New York]. The unitary Gibbs free binding energy, delta Gou, of myristoylated glycine, 8 kcal/mol, is independent of the nature of the electrically neutral lipids used to form the vesicles. Similar binding energies were obtained with other myristoylated peptides (e.g., Gly-Ala, Gly-Ala-Ala). The 8 kcal/mol, which corresponds to an effective dissociation constant of 10(-4) M for myristoylated peptides with lipids, provides barely enough energy to attach a myristoylated protein in the cytoplasm to the plasma membrane. Thus, other factors that reduce (e.g., hydrophobic interaction of myristate with the covalently attached protein) or enhance (e.g., electrostatic interactions of basic residues with acidic lipids; protein-protein interactions with intrinsic receptor proteins) the interaction of myristoylated proteins with membranes are likely to be important and may cause reversible translocation of these proteins to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Slow- and tight-binding inhibitors of the 85-kDa human phospholipase A2.

Abstract: A trifluoromethyl ketone analogue of arachidonic acid in which the COOH group is replaced with COCF3 (AACOCF3) was prepared and found to be a tight- and slow-binding inhibitor of the 85-kDa cytosolic human phospholipase A2 (cPLA2). Enzyme inhibition was observed when AACOCF3 was tested in assays using either phospholipid vesicles or phospholipid/Triton X-100 mixed micelles. The fact that the inhibition developed over several minutes in both assays establishes that AACOCF3 inhibits by direct binding to the enzyme rather than by decreasing the fraction of enzyme bound to the substrate interface. From the measured values of the inhibitor association and dissociation rate constants, an upper limit of the equilibrium dissociation constant for the Ca(2+).AACOCF3.PLA2 complex of 5 x 10(-5) mole fraction was obtained. Thus, detectable inhibition of cPLA2 by AACOCF3 occurs when this compound is present in the assay at a level of one inhibitor per several thousand substrates. Arachidonic acid analogues in which the COOH group is replaced by COCH3, CH(OH)CF3, CHO, or CONH2 did not detectably inhibit the cPLA2. The arachidonyl ketones AACOCF2CF3 and AACOCF2Cl were found by 19F NMR to be less hydrated than AACOCF3 in phospholipid/Triton X-100 mixed micelles, and compared to AACOCF3 these compounds are also weaker inhibitors of cPLA2. In keeping with the fact that cPLA2 displays substrate specificity for arachidonyl-containing phospholipids, the arachidic acid analogue C19H39COCF3 is a considerably less potent inhibitor compared to AACOCF3.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of fast protein size-exclusion liquid chromatography to study the unfolding of proteins which denature through the molten globule

Abstract: Fast protein size-exclusion liquid chromatography (SEC-FPLC) was used to study solvent-induced unfolding of six proteins. Two of them (sperm whale myoglobin and hen white lysozyme) denature on the simple N (native) U (completely unfolded) scheme. The other four proteins [bovine and human alpha-lactalbumin, bovine carbonic anhydrase B (BCAB), and beta-lactamase from Staphylococcus aureus] denature through the molten globule (MG) state (i.e., on the N MG U denaturation scheme). We have shown that the permeation properties of the Superose 12 columns are practically independent of temperature, pH, and denaturants in wide concentration intervals. In the case of myoglobin and lysozyme denaturation at 4 degrees C (when the exchange between the native and unfolded states is slower than the characteristic time of chromatography), a bimodal distribution on molecular dimensions in the transition region was observed. This indicates that, under denaturant action, protein molecules can only be in one of the two states with different compactness. In other words, this shows that FPLC is one of the most direct approaches to establish the "all-or-none" mechanism of the equilibrium solvent-induced denaturation of globular proteins. The curves of guanidinium hydrochloride- (GdmHCl) or urea-induced unfolding (N U or MG U transitions) of a protein on a column (monitored either by the relative areas of two peaks or--for fast exchange--by the position of the average peak) coincide with those monitored by far-UV CD in solution. The Stokes radius values obtained with the use of FPLC for the molten globule states of BCAB (1.6 M GdmHCl in 0.1 M sodium phosphate, pH 6.8, and acid form at pH 3.6) and for the human alpha-lactalbumin molten globule (2.0 M GdmHCl in 0.1 M sodium phosphate, pH 6.8) coincide with those known from literature. Thus, it has been shown that fast protein size-exclusion liquid chromatography (FPLC) is an "inert" technique, i.e., it does not shift the equilibrium between N, MG, and U states and, therefore, can be used for qualitative and quantitative studies of protein denaturation.

Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases

Abstract: Various ATPases have been tested for their sensitivity to naturally occurring unusual macrolides and their chemically modified derivatives, which are structurally related to bafilomycin A1 (1), the first specific inhibitor of vacuolar ATPases. The structure-activity study showed that in general the concanamycins, 18-membered macrolides, are better and more specific inhibitors than the bafilomycins of this class of membrane-bound ATPases. The additional carbohydrate residue is not responsible for the improved activity. The importance of an intact hemiketal ring, which is part of an intramolecular hydrogen-bonding network, and the effects of the size of the macrolactone ring are discussed. The structurally related elaiophylin (13), a C2-symmetric macrodiolide antibiotic, proved to be inactive on vacuolar ATPases but still retained its inhibitory effect on P-type ATPases.

Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors.

Abstract: By examining inhibitor interactions with single and multiple site-specific mutants of mouse acetylcholinesterase, we have identified three distinct domains in the cholinesterase structure that are responsible for conferring selectivity for acetyl- and butyrylcholinesterase inhibitors. The first domain is the most obvious; it defines the constraints on the acyl pocket dimensions where the side chains of F295 and F297 primarily outline this region in acetylcholinesterase. Replacement of these phenylalanine side chains with the aliphatic residues found in butyrylcholinesterase allows for the catalysis of larger substrates and accommodates butyrylcholinesterase-selective alkyl phosphates such as isoOMPA. Also, elements of substrate activation characteristic of butyrylcholinesterase are evident in the F297I mutant. Substitution of tyrosines for F295 and F297 further alters the catalytic constants. The second domain is found near the lip of the active center gorge defined by two tyrosines, Y72 and Y124, and by W286; this region appears to be critical for the selectivity of bisquaternary inhibitors, such as BW284C51. The third domain defines the site of choline binding. Herein, in addition to conserved E202 and W86, a critical tyrosine, Y337, found only in the acetylcholinesterases is responsible for sterically occluding the binding site for substituted tricyclic inhibitors such as ethopropazine. Analysis of a series of substituted acridines and phenothiazines defines the groups on the ligand and amino acid side chains in this site governing binding selectivity. Each of the three domains is defined by a cluster of aromatic residues. The two domains stabilizing the quaternary ammonium moieties each contain a negative charge, which contributes to the stabilization energy of the respective complexes.

Assembly of purified GDP-tubulin into microtubules induced by taxol and taxotere: reversibility, ligand stoichiometry, and competition.

Abstract: Purified tubulin fully liganded to GDP at the exchangeable nucleotide binding site has been prepared by a new direct nucleotide exchange procedure. This normally inactive GDP-tubulin is driven to assemble into microtubules by the binding of the antitumor drug taxol or its more soluble side-chain analogue Taxotere in Mg(2+)-containing buffer, and it disassembles by cooling the solution. Therefore this ligand-induced equilibrium microtubule assembly system dispenses with the requirement of a gamma-phosphate-metal cation ligand bound at the nucleotide site for tubulin to be active. GDP-tubulin can also form characteristic pseudo-ordered aggregates of double rings. These aggregates dissociate upon warming or by addition of GTP. Back-substitution of the nucleotide gamma-phosphate permits glycerol-induced assembly without taxol and reduces the critical protein concentration required for drug-induced microtubule assembly by a factor of 2.6 +/- 0.1. The ligand-induced assembly is maximal at taxol or Taxotere concentrations equimolar with tubulin, and both drugs bind to assembled tubulin with a stoichiometry of 0.99 +/- 0.04 ligand per alpha beta dimer. Taxotere apparently competes with taxol for the same binding site, with 1.9 +/- 0.1 times larger effective affinity. Similarly, the Taxotere-induced assembly of GDP-tubulin or GTP-tubulin proceeds with a critical protein concentration 2.1 +/- 0.1 times smaller than with taxol.

The search for structure-specific nucleic acid-interactive drugs: effects of compound structure on RNA versus DNA interaction strength.

Abstract: The RNA genomes of a number of pathogenic RNA viruses, such as HIV-1, have extensive folded conformations with imperfect A-form duplexes that are essential for virus function and could serve as targets for structure-specific antiviral drugs. As an initial step in the discovery of such drugs, the interactions with RNA of a wide variety of compounds, which are known to bind to DNA in the minor groove, by classical or by threading intercalation, have been evaluated by thermal melting and viscometric analyses. The corresponding sequence RNA and DNA polymers, poly(A).poly(U) and poly(dA).poly(dT), were used as test systems for analysis of RNA binding strength and selectivity. Compounds that bind exclusively in the minor groove in AT sequences of DNA (e.g., netropsin, distamycin, and a zinc porphyrin derivative) do not have significant interactions with RNA. Compounds that bind in the minor grove in AT sequences of DNA but have other favorable interactions in GC sequences of DNA (e.q., Hoechst 33258, DAPI, and other aromatic diamidines) can have very strong RNA interactions. A group of classical intercalators and a group of intercalators with unfused aromatic ring systems contain compounds that intercalate and have strong interactions with RNA. At this time, no clear pattern of molecular structure that favors RNA over DNA interactions for intercalators has emerged. Compounds that bind to DNA by threading intercalation generally bind to RNA by the same mode, but none of the threading intercalators tested to date have shown selective interactions with RNA.

GC base sequence recognition by oligo(imidazolecarboxamide) and C-terminus-modified analogues of distamycin deduced from circular dichroism, proton nuclear magnetic resonance, and methidiumpropylethylenediaminetetraacetate-iron(II) footprinting studies

Abstract: The DNA binding properties of a series of imidazole-containing and C-terminus-modified analogues 4-7 of distamycin are described. These analogues contain one to four imidazole units, respectively. Data from the ethidium displacement assay showed that these compounds bind in the minor groove of DNA, with the relative order of binding constants of 6 (Im3) > 7 (Im4) > 5 (Im2) > 4 (Im1). The reduced binding constants of these compounds for poly(dA-dT) relative to distamycin, while they still interact strongly with poly(dG-dC), provided evidence of GC sequence acceptance. The preferences for GC-rich sequences by these compounds were established from a combination of circular dichroism (CD) titration, proton nuclear magnetic resonance (1H-NMR), and methidiumpropylethylenediaminetetraacetate-iron(II) [MPE.Fe-(II)] footprinting studies. In the CD studies, these compounds produced significantly larger DNA-induced ligand bands with poly(dG-dC) than poly(dA-dT) at comparable ligand concentrations. 1H-NMR studies of the binding of 5 to d-[CATGGCCATG]2 provided further evidence of the recognition of GC sequences by these compounds, and suggested that the ligand was located on the underlined sequence in the minor groove with the C-terminus oriented over the T residue. MPE footprinting studies on a GC-rich BamHI/SalI fragment of pBR322 provided unambiguous evidence for the GC sequence selectivity for some of these compounds. Compounds 4 and 7 produced poor footprints on the gels; however, analogues 5 and 6 gave strong footprints.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of temperature on protein structure and dynamics: X-ray crystallographic studies of the protein ribonuclease-A at nine different temperatures from 98 to 320 K.

Abstract: Structures using X-ray diffraction data collected to 1.5-A resolution have been determined for the protein ribonuclease-A at nine different temperatures ranging from 98 to 320 K. It is determined that the protein molecule expands slightly (0.4% per 100 K) with increasing temperature and that this expansion is linear. The expansion is due primarily to subtle repacking of the molecule, with exposed and mobile loop regions exhibiting the largest movements. Individual atomic Debye-Waller factors exhibit predominantly biphasic behavior, with a small positive slope at low temperatures and a larger positive slope at higher temperatures. The break in this curve occurs at a characteristic temperature of 180-200 K, perhaps indicative of fundamental changes in the dynamical structure of the surrounding protein solvent. The distribution of protein Debye-Waller factors is observed to broaden as well as shift to higher values as the temperature is increased.

Interactions of long-chain fatty acids and albumin: determination of free fatty acid levels using the fluorescent probe ADIFAB.

Abstract: Equilibrium binding of long-chain fatty acids (FA) with albumin from human serum (HSA), bovine serum (BSA), and murine serum (MSA) has been studied by measuring the equilibrium levels of free fatty acids (FFA) FFA levels were measured directly, using a new fluorescent probe composed of acrylodan-derivatized intestinal fatty acid binding protein (ADIFAB) Measurements of [FFA] were done as a function of the ratio of total FA to total albumin (v) for v values between 0 and 6, at pH 74 and 37 degrees C Under conditions observed in normal human physiology (v < or = 2), [FFA] values of the most abundant serum FA (palmitate, stearate, oleate) in equilibrium with human or bovine albumin are less than 15 nM These values are considerably smaller than the generally quoted values of [FFA] in equilibrium with albumin: more than 20-fold for palmitate and more than 50-fold for oleate FFA levels were found to increase monotonically with for all three albumins and all FA In most cases [FFA] increased, for the same chain length, with increasing degree of acyl chain unsaturation, suggesting that FA aqueous solubility may play a significant role in the equilibrium between FA association with albumin and the aqueous phase [The highest FFA levels (approximately 3000 nM), for example, were observed for linoleate (18:3) at the maximum v value (6)] Although aqueous-phase solubility of the FA may be important in understanding the interaction between FA and albumin, protein structure, as reflected in differences among the three albumins, also significantly affects the equilibrium(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and dynamic membrane properties of alpha-tocopherol and alpha-tocotrienol: implication to the molecular mechanism of their antioxidant potency.

Abstract: d-alpha-Tocopherol and d-alpha-tocotrienol are two vitamin E constituents having the same aromatic chromanol "head" but different hydrocarbon "tails". alpha-Tocotrienol has been shown to be more potent in protecting against free radical-induced oxidative stress than alpha-tocopherol. Simple models of phospholipid membrane systems were used to investigate the mechanism of the antioxidant potency of alpha-tocotrienol in terms of its effects on membrane order and reorientation dynamics. Chemiluminescence and fluorescence measurements demonstrated that alpha-tocotrienol exhibits significantly greater peroxyl radical scavenging potency than alpha-tocopherol in phosphatidylcholine liposomes, whereas both antioxidants have identical activity in hexane. This suggests that the antioxidant potency of alpha-tocotrienol requires the membrane environment. When alpha-tocopherol and alpha-tocotrienol were examined for their effects on phospholipid molecular order using conventional ESR spin labeling with 5- and 16-position-labeled doxylstearic acid, although both vitamin E constituents disordered the gel phase and stabilized the liquid-crystalline phase, no differences were observed between the effects of the two compounds. A slightly greater increase (19% vs 15%) in ordering of the liquid-crystalline state due to alpha-tocopherol, however, was discerned in noninvasive 2H NMR experiments. The difference is most noticeable near C10-C13 positions of the phospholipid chain, possibly suggesting alpha-tocotrienol is located closer to the membrane surface. Saturation-transfer ESR, furthermore, revealed that on the time scale tau c = 10(-7)-10(-3) s the rates of rotation about the long molecular axis and of the wobbling motion of the axis are modified to differing extents by the two forms of the vitamin E.(ABSTRACT TRUNCATED AT 250 WORDS)

A DNA aptamer which binds to and inhibits thrombin exhibits a new structural motif for DNA.

Abstract: The investigation of the three-dimensional structure of the DNA aptamer d(G1G2T3-T4G5G6T7G8T9G10G11T12T13G14G15) which binds to and inhibits thrombin has been carried out by NMR methods. This DNA exhibits a number of long-range NOEs between residues which are not adjacent in sequence, which allowed the determination of the novel tertiary structure adopted. This DNA adopts a highly compact, highly symmetrical structure which consists of two tetrads of guanosine base pairs and three loops. The residues of the tetrads alternate anti-syn-anti-syn. This novel structural motif for DNA may also be relevant to the structure of telomere DNA.

Engineered disulfide bonds as probes of the folding pathway of barnase: increasing the stability of proteins against the rate of denaturation.

Abstract: Disulfide bridges have been introduced into barnase to act as probes of folding. One disulfide (between residues 85 and 102) links two loops known to pack together early in the folding pathway. A second disulfide bond (between residues 43 and 80) links two elements of secondary structure known to pack together only after the rate-determining step of folding. The disulfide-bridged proteins are more stable than wild-type by 4.1 and 1.2 kcal mol-1, respectively. The kinetics of unfolding and refolding of the mutant proteins has been measured, and a comparison of the disulfide proteins and their corresponding dithiol forms has been made by use of thermodynamic cycles. These data have been used to construct folding profiles of the disulfide proteins. The disulfide bond engineered into the part of the protein that folds early confers stability upon the intermediate and transition states of folding. The protein with a disulfide bond connecting parts of the protein that fold late is not stabilized until the protein reaches its final folded form. Conversely, in the unfolding pathway, the rate of unfolding of this mutant is lowered considerably. This points to a method of decreasing the rate of denaturation of proteins that are used in medical and biotechnological applications: elements of structure that unfold in or before the rate-determining step of overall unfolding may be stabilized and so slow down the overall unfolding process. The barnase mutant linked between Cys 43 and Cys 80, for example, unfolds 20 times slower than wild-type and 170 times slower than the reduced protein.

Evidence for the presence of five distinct proteolytic components in the pituitary multicatalytic proteinase complex. Properties of two components cleaving bonds on the carboxyl side of branched chain and small neutral amino acids.

Abstract: Initial studies on the specificity of the multicatalytic proteinase complex (MPC; EC 3.4.99.46) led to the identification of three distinct proteolytic components designated as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing, all sensitive to inactivation by 3,4-dichloroisocoumarin (DCI), a general serine proteinase inhibitor. The three components cleave the peptidyl-arylamide bonds in the model synthetic substrates, Z-(D)-Ala-Leu-Arg-2-naphthylamide, Z-Gly-Gly-Leu-p-nitroanilide, and Z-Leu-Leu-Glu-2-naphthylamide, respectively. We report here evidence for the presence in the MPC of two additional distinct components, neither of them capable of cleaving the three model substrates. One of these components cleaves the Leu-Gly and the Leu-Ala bonds in the substrates Cbz-Gly-Pro-Ala-Leu-Gly-p-aminobenzoate and Cbz-Gly-Pro-Ala-Leu-Ala-p-aminobenzoate, respectively, and is activated by treatment of the MPC with DCI, N-ethylmaleimide, Mg2+, Ca2+, and low concentrations of sodium dodecyl sulfate and fatty acids. This component is apparently identical with the previously identified DCI-resistant component of the MPC that cleaves preferentially bonds on the carboxyl side of branched chain amino acids in natural peptides including neurotensin and proinsulin [Cardozo, C., Vinitsky, A., Hidalgo, M. C., Michaud, C., & Orlowski, M. (1992) Biochemistry 31, 7373-7380]. It is probably also identical with the component proposed to be the main factor responsible for the caseinolytic activity [Pereira, M. E., Nguyen, T., Wagner, B. J., Margolis, J. W., Yu, B., & Wilk, S. (1992a) J. Biol. Chem. 267, 7949-7955]. The designation "branched chain amino acid preferring" (BrAAP) is proposed for this component. The second component cleaves peptide bonds between the small neutral amino acids Ala-Gly and Gly-Gly in the substrates Cbz-Gly-Pro-Ala-Ala-Gly-p-aminobenzoate and Cbz-Gly-Pro-Ala-Gly-Gly-p-aminobenzoate, respectively. This component is sensitive to inactivation by DCI, N-ethylmaleimide, and organic mercurials, but unlike the BrAAP it is significantly activated neither by Mg2+ or Ca2+ nor by fatty acids or sodium dodecyl sulfate. The designation "small neutral amino acid preferring" (SNAAP) is proposed for this component. Both components are sensitive to inhibition by the peptidyl-aldehydes N-acetyl-Leu-Leu-norleucinal (Ac-LLnL-CHO; calpain inhibitor I) and N-acetyl-Leu-Leu-methioninal (Ac-LLM-CHO; calpain inhibitor II) but are resistant to inhibition by Z-LLF-CHO, a potent inhibitor of the chymotrypsin-like activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of hydrazine to release in intact and unreduced form both N- and O-linked oligosaccharides from glycoproteins.

Abstract: The use of hydrazine to release unreduced N- and O-linked oligosaccharides from glycoproteins has been investigated using several "standard" glycoproteins of previously defined glycosylation. It is shown that hydrazinolysis can be used to release intact N- and O-linked oligosaccharides in an unreduced form. The release of O-linked oligosaccharides occurs with a lower temperature dependence than the release of N-linked oligosaccharides, and the kinetic parameters governing release of oligosaccharides from these standard glycoproteins have been determined. These parameters allow a definition of reaction conditions under which anhydrous hydrazinolysis can be used to selectively release O-linked oligosaccharides (60 degrees C, 5 h) or release both N- and O-linked oligosaccharides (95 degrees C, 4 h) in high yield (> 85%) from all glycoproteins investigated (n = 11). Under these reaction conditions, the recovered N- and O-linked oligosaccharides are structurally intact (as judged by 600-MHz 1H-NMR, laser-desorption mass spectrometry, HPAEC-PAD, gel filtration, and glycosidase digestion), with the possible exception of certain N- and O-acyl substituents of sialic acid. This use of mild hydrazinolysis therefore allows both the simultaneous and sequential chemical release from glycoproteins of O- and N-linked oligosaccharides in their intact unreduced form.

Understanding the rates of certain enzyme-catalyzed reactions: Proton abstraction from carbon acids, acyl transfer reactions, and displacement reactions of phosphodiesters

Abstract: The structural factors responsible for the rapid rates (kcats) of enzyme-catalyzed reactions are not well understood. In this paper, we outline an analysis that we believe can provide a quantitative understanding of the kcats of three types of reactions: abstraction of the alpha-protons from carbon acids, acyl-transfer reactions, and displacement reactions of phosphodiesters. We propose that these reactions proceed via the formation of intermediates in which negative charge develops on the carbonyl or phosphoryl oxygens. Our analysis is based on Marcus formalism that separates the activation energy barrier for conversion of bound substrate to the intermediate, delta G++, into contributions from a thermodynamic barrier, delta G zero, and an intrinsic kinetic barrier, delta G++int. We propose that one (or more) general acid catalyst positioned adjacent to the carbonyl or phosphoryl oxygens of the substrate is primarily responsible for reducing both delta G zero and delta G++int from the values that characterize nonenzymatic reactions. The proton donors (1) stabilize the intermediates via the formation of short, strong hydrogen bonds (the pKas of the protonated intermediates and the general acid catalysts are matched), thereby reducing delta G zero, and (2) stabilize the transition states for formation of the intermediates by negating the developing charge on the oxygens without the requirement for significant structural reorganization, thereby reducing delta G++int. The possible reductions in delta G zero and delta G++int are sufficient to understand the rapid kcats of these reactions.

Free Radical-Mediated Lipid Peroxidation in Cells: Oxidizability Is a Function of Cell Lipid bis-Allylic Hydrogen Content

Abstract: Oxidizability of lipids in homogeneous solution varies linearly with the extent of their unsaturation. In vitro cellular, as well as in vivo, studies of oxidizability have generally relied upon chemical indicators of peroxidation such as thiobarbituric acid-reactive substances. To examine the oxidizability of lipids in cells, we have measured oxygen uptake and, using electron paramagnetic resonance spin trapping with alpha-(1-oxo-4-pyridyl)-N-tert-butylnitrone (POBN), the real time generation of lipid-derived free radicals. We have used our experimental in vitro cellular lipid modification model to examine the rate and extent of lipid peroxidation versus the degree of lipid unsaturation in L1210 murine leukemia cells. Lipid peroxidation was stimulated using the prooxidants iron, ascorbate, and the ether lipid compound 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine. We did a total cellular lipid analysis to determine the number of lipid carbon-carbon double bonds contained in L1210 cells enriched with eight fatty acids of different degrees of unsaturation. We found in cellular lipids that (i) lipid chain length had no apparent effect on the rate or extent of radical formation; (ii) the maximum amount of lipid radical generated increases with the total number of bis-allylic positions in the cellular lipids; and, most importantly, (iii) the rate of cellular lipid peroxidation increases exponentially with the number of bis-allylic positions. Our quantitative results clearly demonstrate, for the first time, that the number of bis-allylic positions contained in the cellular lipids of intact cells determines their susceptibility, i.e., oxidizability, to free radical-mediated peroxidative events.

Betaine can eliminate the base pair composition dependence of DNA melting

Abstract: We show that the amino acid analogue betaine shares with small tetraalkylammonium ions [Melchior, W. B., Jr., & von Hippel, P. H. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 298-302] the ability to reduce or even eliminate the base pair composition dependence of DNA thermal melting transitions. The "isostabilizing" concentration of betaine (at which AT and GC base pairs are equally stable) is approximately 5.2 M. Betaine exerts its isostabilizing effect without appreciably altering the conformation of double-stranded DNA from the B form. The presence of > 5 M betaine also does not greatly change the behavior of DNA as a polyelectrolyte; this lack of effect on electrostatic interactions is expected because betaine exists as a zwitterion near neutral pH. Study of DNA melting transitions in high concentrations of betaine thus allows the experimental separation of compositional and polyelectrolyte effects on DNA melting. As a consequence, betaine solutions can also be used to investigate DNA-protein interactions under isostabilizing (or close to isostabilizing) conditions, which has not been possible using isostabilizing salts. This potential is illustrated by examining the highly salt concentration-dependent interaction of ribonuclease A with DNA in concentrated betaine solutions.

Differential scanning calorimetric study of the effect of cholesterol on the thermotropic phase behavior of a homologous series of linear saturated phosphatidylcholines.

Abstract: We have studied the effects of cholesterol on the thermotropic phase behavior of aqueous dispersions of a homologous series of linear saturated phosphatidylcholines, using high-sensitivity differential scanning calorimetry and an experimental protocol which ensures that broad, low-enthalpy phase transitions are accurately monitored. We find that the incorporation of small amounts of cholesterol progressively decreases the temperature and the enthalpy, but not the cooperativity, of the pretransition of all phosphatidylcholines exhibiting such a pretransition and that the pretransition is completely abolished at cholesterol concentrations above 5 mol % in all cases. The incorporation of increasing quantities of cholesterol also alters the main or chain-melting phase transition of these phospholipid bilayers in both hydrocarbon chain length-dependent and hydrocarbon chain length-independent ways. At cholesterol concentrations of from 1 to 20-25 mol %, the DSC endotherms of all phosphatidylcholines studied consist of a superimposed sharp and broad component, the former ascribed to the melting of cholesterol-poor and the latter to the melting of the cholesterol-rich phosphatidylcholine domains. The temperature and cooperativity of the sharp component are reduced only slightly and in a chain length-independent manner with increasing cholesterol concentration, an effect we ascribe to the colligative effect of the presence of small quantities of cholesterol at the domain boundaries. Moreover, the enthalpy of the sharp component decreases and becomes zero at 20-25 mol % cholesterol for all of the phosphatidylcholines examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Probing the combining site of an anti-carbohydrate antibody by saturation-mutagenesis: role of the heavy-chain CDR3 residues.

Abstract: The carbohydrate-binding site in Fab fragments of an antibody specific for Salmonella serogroup B O-polysaccharide has been probed by site-directed mutagenesis using an Escherichia coli expression system. Of the six hypervariable loops, the CDR3 of the heavy chain was selected for exhaustive study because of its significant contribution to binding-site topography. A total of 90 mutants were produced and screened by an affinity electrophoresis/Western blotting method. Those of particular interest were further characterized by enzyme immunoassay, and on this basis seven of the mutant Fabs were selected for thermodynamic characterization by titration microcalorimetry. With regard to residues that hydrogen bond to ligand through backbone interactions, Gly102H could not be substituted, while several side chains could be introduced at Gly100H and Tyr103H with relatively little effect on antigen binding. There was, however, a preference for nonpolar side chains at position 103H. Substitution of His101H with carboxylate and amide side chains gave mutants with binding affinities approaching that of the wild type; complete side-chain removal by mutation to Gly was tolerated with a 10-fold reduction in binding constant. Analysis of binding by titration microcalorimetry revealed some dramatic thermodynamic changes hidden by the similarity of the binding constants. Similar effects were observed with residue changes in an Arg-Asp salt-bridge at the base of the loop. These results indicate that alterations to higher affinity anti-carbohydrate antibodies are characterized by an enthalpy-entropy compensation factor which allows for fundamental changes in the nature of the binding interactions but impedes engineering for increases in affinity.