One-dimensional diffusion is used by transcription factors and restriction endonucleases to locate specific sites on double-stranded DNA. The backbone of RNA, like that of DNA, could allow for the facilitated diffusion of proteins. Yet, the facilitated diffusion of a protein along RNA (or any single-stranded nucleic acid) has not been demonstrated so far. Single-stranded DNA is an excellent substrate analog for ribonuclease A (RNase A), and this analogy is the basis for the work described in the chapter. First, this chapter reports the use of DNA oligonucleotides and fluorescence polarization to probe the binding of adenine to the B1 subsite of RNase A. Then, it describes the use of DNA/RNA chimeric oligonucleotides to distinguish between three-dimensional and one-dimensional diffusion mechanisms for catalysis by RNase A. The results provide a biophysical rationale, as well as direct evidence for the diffusion of a protein along a single-stranded nucleic acid. Bovine pancreatic RNase A is a distributive endoribonuclease that catalyzes the cleavage of the P-O5′ bond of RNA on the 3′ side of pyrimidine residues. RNase A binds to polymeric substrates and can use one-dimensional diffusion along a poly(dA) tract to accelerate the location of a uridine substrate. Use of this mechanism depends on the concentration of NaCl, as expected if the enzyme is binding to the nucleic acid by nonspecific interactions with phosphoryl groups. Binding of the enzymic active site to adenosine residues is 20-fold weaker than uridine residues, which could enhance the ability of the enzyme to slide along the poly(dA) tract.
A self-assembling 42-residue peptide, known as “Aβi-42,” is the major component of the senile plaque amyloid deposits associated with Alzheimer's disease. It is generated by proteolytic processing of the amyloid precursor protein. Fmoc-aminoacyl fluorides have been shown to exhibit advantages in synthesis of this peptide over standard coupling reagents, the Aβi-42 synthesis procedure has been modified to employ solutions of aminoacyl fluorides in dry N,N-dimethylformamlde (DMF) to see if improvement in yield and purity of the crude product could be obtained. The fluoride method allowed coupling for reduced times (10 min), except Fmoc-Arg(Pbf)-OH and Fmoc-His(Trt)-OH, which are activated with O-(7-azabenzotriazol-1-yl)-1,3,3-tetramethyluronium hexafluorophosphate/ dilsopropylethylamlne (HATU/DIEA) and coupled for one hour, at 22°C. However, to ensure complete removal of the Nα-protecting groups at each step, Fmoc deprotection was maintained at elevated (40°C) temperatures. The deprotection temperature was also raised to 55°C in a further effort to improve the yield of the crude product in a subsequent synthesis. The protocols employing Fmoc–aminoacyl fluoride aminoacylations resulted in an improved purity and yield of crude product (by analytical RP HPLC) compared to the optimized BOP/HOBt coupling tactics.
Publisher Summary Glycosylation-inhibiting factor (GIF) inhibits N-glycosylation of IgE-binding factors. The unglycosylated IgE-binding factor then selectively suppresses IgE synthesis. Further, GIF appears to be a subunit of antigenspecific suppressor T cell factors that facilitate the generation of antigen-specific suppressor T cells. Recent studies indicate that post-translational modification of GIF in suppressor T cells is required for the generation of the biological activity. However, the relationship between GIF bioactivity and the conformational transition of the protein is not known. To understand the mechanisms of GIF functions, this chapter studies the crystal structure of recombinant human GIF. It is apparent that GIF has a novel tertiary structure. The chapter discusses the crystallographic analysis of GIF. The overall structure of GIF trimer is found to be a three-fold-related barrel structure, which is composed of three six-stranded β-sheets on the inside and six α-helices on the outside. Each subunit consists of two β-α-α motifs related by a pseudo-twofold axis. The trimer structure is formed by intermonomer hydrogen bonds and hydrophobic interfaces among β-sheets. There is a 5-A diameter “hole” through the middle of the barrel. The barrel structure of GIF in part resembles “trefoil” cytokines, such as interleukin-1 and fibroblast growth factor.
It is believed that a substrate bound to the protease would form interactions similar to peptidomimetic inhibitors. However, as a substrate would be processed rapidly, no structure of a protease/substrate complex is available. As a first step in studying such interactions, a mutant of feline immunodeficiency virus protease (FIV PR) is expressed in which the catalytic Asp30 was mutated into an Asn, leading to inactive protease—designated as FIV PR(D30N) in the chapter. A complex between this mutant and a substrate should therefore be stable. To investigate the extent of perturbation of the active site of FIV PR caused by this mutation, the crystal structure of FIV PR(D30N) is determined at 2.0 Å resolution in a complex with LP-149, a statine-based inhibitor. This structure is then compared with the structure of the wild type enzyme FIV PR(wt) complexed with the same inhibitor. The study described reveals that the mode of binding of LP-149 to FIV PR(D30N) is similar to the mode of binding of LP-149 to FIV PR(wt), making the mutant a valuable model to study the interactions of substrates with FIV PR, and retroviral proteases in general. The results described show that the crystal structure of the FIV PR(D30N)/LP-149 complex is very similar to that of the FIV PR(wt)/LP-149 complex. The position and orientation of the inhibitor molecule LP-149 in both proteins is nearly identical. Some hydrogen bond distances between the inhibitor and the active site pockets of FIV PR(D30N) have been found to be slightly different from those observed in the FIV PR(wt)/LP-149 complex. These differences are however, too small to affect the binding mode of the inhibitor in the mutated active site.
This chapter describes the basic statistical thermodynamic formalism required to interpret hydrogen exchange protection factors. The agreement between predicted and experimental hydrogen exchange protection factors for staphylococcal nuclease (SNase) and other proteins suggests that the probability distribution of partially folded states generated with the COREX algorithm mimics the general features of the ensemble of conformations under native conditions and that this approach can be used to examine general aspects of the equilibrium ensemble of partially folded states. It has also been shown that this approach accounts for the cooperativity of folding/unfolding transitions and successfully predicts the apparent two-state behavior observed in temperature and denaturant induced folding/unfolding reactions. The folding/unfolding equilibrium of proteins as observed by global macroscopic observables can be usually described by simple models, including two, three, or at most a few conformational states. The situation is different when the equilibrium is studied by physical observables that monitor the behavior of individual residues. It is possible for some residues to exhibit high stability constants or protection factors and simultaneously m values close to zero. This behavior should be contrasted with that expected for the logarithm of a two-state equilibrium constant that should be linear with denaturant concentration.
Chemical or enzymatic methods can be employed to release intact oligosaccharides. Among various electrophoretic and chromatographic methods that give reliable profile results of the released oligosaccharides, reversed-phase (rp) high-performance liquid chromatography (HPLC) offers an advantage. With bovine fetuin as a model system in the chapter, the use of 1-phenyl-3-methyl-5-pyrazolone (PMP) labeling in the routine rp-HPLC profile analysis of glycoproteins has been evaluated. The advantage in this case is, as the system usually uses volatile or salt-free buffer, the recovered samples are suitable for further analysis without additional manipulation. Unfortunately, oligosaccharides lack a hydrophobic domain as well as a chromophore for sensitive detection. To circumvent this disadvantage, it has been demonstrated that oligosaccharides labeled with 1-phenyl-3-methyl-5-pyrazolone (PMP) are suitable for rp-HPLC analysis with conventional UV detection. As a result, two analysis kits are now available commercially to perform this method routinely. A kit performs enzymatic release of N-linked oligosaccharides followed by PMP labeling, and a second kit provides chromatographic conditions for separation of these labeled oligosaccharides. With the use of fetuin as a model system, a study is initiated to examine whether the PMP-labeling and rp-HPLC approach is also suitable for analyzing chemically released oligosaccharides. The recovered PMP-oligosaccharide samples from rp-HPLC are also analyzed by MALDI TOF and LC ESI MS.
Techniques of using size-exclusion chromatography (SEC) with on-line light-scattering, uv absorbance, and refractive index detectors to characterize the polypeptide molecular weights of simple proteins or glycoproteins or to determine the stoichiometry of protein complexes are described. Two unique advantages of this approach over conventional SEC are that the molecular weight measurement is independent of elution position and can exclude the contributions from carbohydrates. When a protein or complex contains no carbohydrates, a two-detector method, i.e., light scattering combined with refractive index, can be used to calculate the molecular weight. When a protein contains carbohydrates, a three-detector method is used to calculate the molecular weight of polypeptide alone. Finally, a self-consistent three-detector method is used to determine the stoichiometry of a protein complex containing carbohydrates. Example applications for all these methodologies are described.
The most successful drugs are compounds of relative mass < 500 Da. Studies show that such small compounds, while capable of being bound tightly to pocket-like sites that accommodate natural ligands of similar structure and size, usually cannot achieve binding with sufficiently high affinity to the molecular surfaces of proteins recognized by other proteins. As a result, it remains to be determined how drugs that disrupt protein–protein interactions can be developed. In such conditions, it appears worthwhile to create model systems that allow aspects of this problem to be analyzed. In this chapter, a monoclonal antibody has been used that binds to human interleukin-1β (IL-1β) by recognizing amino acid residues that are also recognized by the IL-1β receptor (IL-1R). A monoclonal antibody that recognizes the receptor-binding residues of a cytokine can be considered a surrogate for the cytokine's natural receptor. Such a reagent might be valuable in assessing the structural basis of cytokine-receptor affinity, and could furnish a starting point from which to attempt the design of smaller competitive agents. To be an appropriate subject for downsizing, an antibody must achieve critical interactions with at least a part of the receptor-binding surface of IL-1β. The selection of an antibody is excluded that blocks access of IL-1β to the receptor, merely by steric overlap of its molecular bulk with the space occupied by bound receptor. Such an antibody, on downsizing, would lead to a compound that fails to compete with IL-1β binding. This chapter describes the selection and characterization of an antibody, and its Fab fragment that provides a suitable starting point for this endeavor.
In recent years, there has been a recurring interest and demand for the quantitation of free amino acids in physiological fluids, for example, urine, blood serum, tissue cultures, as well as in hydrolysates. Amino acid analysis is a well established technique for the quantitation of free amino acids found in either hydrolysates or physiological fluids. Because of such increased interest in analysis of physiological samples, several studies establishes analyzer methods that would allow choosing between the standard protocol for protein and peptide hydrolysates and a separate protocol for an expanded number of amino acids. This is to include the most important free amino acids found in physiological samples. A separation protocol has been developed that allows for the separation and quantitation of up to 30 amino acids using the 420H system. While this separation protocol does not allow for the separation of all possible free amino acids found in physiological samples, it enables to identify and quantify those most commonly requested. The major difference in separation conditions among the standard versus the physiological method is the addition of an extra step at six minutes into the gradient. This step decreases the steepness of the slope of the gradient development, thus allowing for the separation of citrulline, taurine, and arginine.
Publisher Summary Ethylenediaminetetraacetic acid (EDTA), which complexes divalent ions, is a poor stabilizing agent to prevent ex vivo generation of C4a, whereas Futhan (nafamostat mesilate)—a powerful serine protease inhibitor—is an excellent stabilizing agent in this regard. On the other hand, heparin, when combined with EDTA, significantly reduces ex vivo generation of C4a, which may indicate an involvement of coagulation enzymes in C4 cleavage. It has been hypothesized that other enzymes of the coagulation or fibrinolytic system are responsible for at least a part of the ex vivo C4a generation that is observed in EDTA plasma. Even though many coagulation factors require phosphohpids or Ca 2+ for activation, in the chapter, the hypothesis that some coagulation enzymes may be able to cleave C3 and/or C4 in a Ca 2+ - and phospholipid-free environment has been tested. In the study described in this chapter, EDTA-plasma shows little ex vivo generation of C3a at 4°C within 48 hours, whereas C4a levels increased significantly. This effect is even more pronounced in whole blood that has undergone the same treatment. Heparin does not appear to be an effective stabilizing agent by itself, but works well when combined with EDTA. However, the best results are obtained with the serine protease inhibitor Futhan, indicating that enzymes of the complement activation and/or coagulation pathway possess residual activity after the divalent ions in plasma has been chelated by EDTA.
Glial cell line-derived neurotrophic factor (GDNF) is one of the more recently identified neurotrophic factors, first purified from the conditioned medium of a rat glial cell line (B49). Because of its ability in supporting the growth of midbrain dopaminergic neurons in vitro, GDNF has been implicated to have therapeutic potential in the treatment of Parkinson's disease. Mature GDNF is a single polypeptide with 134 amino acid residues, containing seven cysteines and functions as a glycosylated, disulfide linked dimer. Examination of the protein's primary structure suggests that GDNF is a distant member of the transforming growth factor-fi (TGF-fi) superfamily of growth factors. The recombinant protein has been expressed in Escherichia coli and is currently being developed as a candidate for human therapeutic use. Because of its complex structure, the protein was refractory to extensive proteolytic degradation under native conditions. This chapter discusses the issue of disulfide assignment by subjecting GDNF to partial reduction using the chemical reagent tris-(2-carboxyethyl)phosphine (TCEP) in 0.17 M acetic acid at pH 2.5 and the structural characterization of the single reduced disulfide bond is reviewed.
Methods of amino acid analysis (AAA) based on precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) quantifies hydrolyzed samples with a high degree of accuracy. Rugged, reproducible chromatographic systems for resolving some common mixtures of AQC-derivatized amino acids, such as those produced by the hydrolysis of collagen or free amino acids present in cell culture fluids, have been developed. The AQC-based method also derivatize effectively in the presence of salts and lipids. Considering such strengths, the excellent stability of the derivatives, and the unique fluorescence properties that allow for direct injection of the reaction mixture without cleanup, the AQC methodology represents an ideal choice for the analysis of complex samples. Recent studies on separation optimization show that accurate control of mobile phase pH is essential to successfully resolve a number of important nonhydrolysate amino acids. With effective control of a complex gradient profile, the system could resolve a mixture of amino acids, including Asn, Gin, cysteine derivatives carboxymethyl cysteine and pyridylethyl cysteine, and the hydroxylated amino acids hydroxyproline (Hyp) and hydroxylysine (Hyl) as well as the hydrolysate amino acids. However, the required precision in the control of eluent pH unnecessarily complicated transfer of the method among laboratories. The method also lacked the ability to separate Orn from the hydrolysate amino acids. This chapter describes the utility of quaternary high-performance liquid chromatography (HPLC) gradient systems for facilitating methods development and simplifying routine eluent preparation with excellent pH control. Retention time reproducibility is also enhanced with a new HPLC system, especially in the shallow region of the gradient profile. Analysis of collagen hydrolysates and cell culture supernatants are shown as representative applications.
A miniaturized protein and peptide microsequencer consisting of either a fused silica capillary reactor or a microreactor made of Teflon is described. The performance of the miniaturized sequencer was evaluated by sequencing 33 and 27 picomoles of myoglobin that were covalently attached to Sequelon-DITC. The products generated by the sequencer were analyzed using capillary electrophoresis with thermo-optical absorbance detection. This CE system provides reproducible migration time (<0.4% of RSD) and detection limits of less than 4 fmol.
Recently, it has been shown that Cys (X)2 Cys (X)4 His (X)4 Cys array (CCHC) zinc finger peptides are susceptible to chemical attack by a wide variety of oxidizing agents. The metal-chelated sulfur thiolates in the CCHC zinc fingers of HIV-1 p7 are known to react with a variety of chemical groups, including maleimides, nitrosos, disulfoxides, thiocarbamoyl-disulfides, and other substituted disulfides as well as oxidizing agents, such as Cu+2, Fe+3, and Hg+2 ions. The reaction mechanism for the thiuram disulfide class of oxidizing agents and maleimide class of alkylating agents are examined and presented in this chapter. Thiuram disulfides are examined in detail, as a member of this class of compounds, tetraethylthiuram disulfide (Antabuse) is an FDA-approved drug for alcohol abuse therapy and has very low in vivo toxicity. These compounds have functional groups that can modify zinc fingers in nucleocapsid (NC) protein and have antiviral activity but are not necessarily specific for the virus. To initiate studies leading to the design of reagents with greater specificity for the viral NC protein, it is necessary to determine the mechanism of action for model compounds and in particular to determine the initial site of attack on the NC protein. While the studies of NC protein alone demonstrated little if any cross-linking, the results with HIV-1 virus showed extensive oligomerization. The mature virion contains a compact ribonucleoprotein complex formed by the genomic RNA and ca. 2,500 copies of the NC protein. Therefore, the high concentration of NC in the viral particle the formation of intermolecular disulfide bonds over intramolecular ones is expected to be favored following virus treatment with thiuram disulfides.
Publisher Summary The task of sequencing major histocompatibility complex (MHC) peptides is difficult as MHC class I proteins can bind and present 10,000–15,000 different cellularly derived peptides present at the sub-pico–femtomole level. The development of methods to sequence MHC class I and class II peptides has been the matter of interest. Specifically, two-dimensional microcapillary HPLC-MS/MS has been utilized to separate and sequence such peptides. This chapter describes the use of a new orthogonal two-dimensional chromatography-MS/MS approach employing reversed-phase high performance liquid chromatography (HPLC) followed by on-line membrane preconcentration capillary electrophoresis-MS/MS (mPC-CE-MS/MS) to separate and sequence MHC class I peptides. For this purpose, an orthogonal two-dimensional chromatography has been employed that consists of HPLC fractionation and on-line mPC-CE-MS. The use of mPC-CE-MS allows loading and on-line sample cleanup for >100 μL solutions containing analyte peptides and utilizes an impregnated membrane adsorptive phase contained in a cartridge placed at the inlet of the CE capillary. It has been shown that the mPC-CE cartridge has no adverse effects on overall CE-MS performance. Ultimately, this approach has been used to structurally characterize peptides derived from EL-4/K b immunoprecipitated MHC class I molecules and determine the sequence derived from MS/MS analysis of K b .
In the reduced state the iron center of each of the four heme group in recombinant hemoglobin, rHb1.1, reversibly binds molecular oxygen. Upon binding oxygen the hemoglobin can autoxidize forming metHb, which is incapable of binding oxygen. The metHb can be reduced back to the ferrous state using ascorbate and reduced oxygen conditions. In the presence of oxygen, however, ascorbate reacts with molecular oxygen to form dehydroascorbate and superoxide anion. The dehydroascorbate can undergo hydrolytic ring rupture to form 2,3-diketogulonic acid that in turn can react further with oxygen forming additional byproducts, which may modify proteins. One of the more prevalent protein modifications detected has been carboxymethylation of lysine groups to form Nɛ-(carboxymethyl)lysine. Although it is known that the superoxide reacts with and oxidizes the hemoglobin, little is known concerning the modification of the hemoglobin by dehydroascorbate and its byproducts. Subsequent interest lies in determining how rHb1.1 is modified in the presence of dehydroascorbate as well as in the presence of ascorbate and oxygen. The location of the modification has been determined using trypsin mapping. Following dehydroascorbate modification of deoxy-rHb, the main β-globin peak and the lagging shoulder of the p-globin are purified from reverse phase HPLC and mapped with trypsin. The map of the unmodified P-globin has been shown in this chapter and is not different from that of P-globin not exposed to ascorbate.
Juvenile Hormone Esterase (JHE) plays an essential role in the development of insects since it is partially responsible for clearing juvenile hormone (JH), one of the hormones that is responsible for insect metamorphosis. JHE is a 60 kDa enzyme that selectively hydrolyzes the alpha/beta unsaturated ester of JH, Because of its pivotal role in insect development, we have targeted JHE for use as a biopesticide. In this study we have constructed a homology-based molecular model of JHE from the agricultural crop pest, Heliothis virescens. JHE is a member of the alpha/beta hydrolase fold family of enzymes and was built according to two structures in the same family: acetylcholinesterase from Torpedo californica and lipase from Geotrichum candidum. Analysis of the active site region reveals extensive conservation between JHE and its templates. A surprise was the presence of a conserved Ser near the catalytic triad, Docking of JH III into the active site has provided insight into protein-substrate interactions that are corroborated by experimental observation. The model is being used as a predictive basis to design biopesticides. In this regard, we have identified a site on the protein surface that is suggestive of a recognition site for the putative JHE receptor. (C) 1999 Wiley-Liss, Inc.
Publisher Summary This chapter describes the degree to which precise spatial complementarity among core residues is required to maintain native-like protein properties. Sites within the carboxy-terminal domain core of phage T4 lysozyme has been substituted singly and as a group with methionine to produce a simplified core sequence. The properties of such mutant lysozymes are briefly described. In addition, the chapter describes a method to isolate mutant protein from inclusion bodies and a sensitive enzymatic assay to detect small differences in mutant protein activities. The carboxy-terminal domain of T4 lysozyme is composed of seven helices and includes the largest contiguous set of buried residues in the protein. Side-chains are considered a part of the core if they have less than 10% solvent accessible surface. The carboxy-terminal domain also contains a single, completely buried methionine (Met 102), and two others (Met 106 and Met 120), the side-chains of which are about 80% buried. The fact that at least seven core residues can be replaced as a group with methionine in phage T4 lysozyme without introducing molten globule-like characteristics shows that strict side-chain complementarity is not required to maintain native-like protein properties. The crystallographic thermal factors of the side-chains of some of the seven methionines in the mutant structure are slightly higher than the wild-type amino acids they replace. The thermal factors of the three methionines that are present within the carboxy-terminal domains of both structures are, if anything, better ordered in the mutant molecule. In all cases, the electron density for the introduced methionines is well defined. There is no suggestion that substitution of seven methionines leads to disorder within the protein core or in other regions of the structure.
