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Reported here is a native chemical ligation strategy for the total chemical synthesis of the B1 domain of protein L. A synthetic construct of this 76 amino acid protein domain was prepared by the chemoselective ligation of two unprotected polypeptide fragments, one containing an N-terminal cysteine residue and one containing a C-terminal thioester moiety. The polypeptide fragments utilized in the ligation reaction were readily prepared by stepwise solid phase peptide synthesis (SPPS) methods for Boc-chemistry. The milligram quantities of protein required for conventional biophysical studies were readily accessible using the synthetic protocol described here. The folding properties of the synthetic protein L construct were also determined and found to be very similar to those of a similar wild-type protein L constructs prepared by recombinant-DNA methods. This work facilitates future unnatural amino acid mutagenesis experiments on this model protein system to further dissect the molecular basis of its folding and stability.

Quantum mechanical/molecular mechanical (QM/MM) calculations and experimental kinetic studies have been performed on 4-oxalocrotonate tautomerase (4OT) for two different substrates, 2-hydroxymuconate (2HM) and 2-oxo-4-hexenedioate (2o4hex). Potential (deltaE) and free energy (deltaG) paths for both steps of the reaction using both substrates were calculated to determine the free energy barriers and compared to the experimental values obtained from the kinetic studies via the transition state theory. In the first step, a proton from the hydroxyl oxygen on the second carbon of 2HM, or from the third carbon of 2o4hex, is abstracted by Pro-1. In the second step, the proton is transferred to the fifth carbon of the substrate to form the product, 2-oxo-3-hexenedioate (2o3hex). For both substrates we obtain a calculated deltaG of approximately 13 kcal/mol, in agreement with experimental determinations. The calculated free energy barrier difference deltaG2o4hex - deltaG2HM (deltadeltaG) is 0.87 kcal/mol. We obtained an experimental deltadeltaG of 0.85 kcal/mol. These results suggest that 2HM is turned over faster than 2o4hex by 4OT. However, these energy differences are so small that both 2HM and 2o4hex need to be taken into account in considering the mechanism of catalysis of 4OT.

With the recent development of chemical and biological methods to introduce backbone modifications into the polypeptide chains of proteins, there have been a growing number of site-directed mutagenesis experiments focused on understanding the role of the polypeptide backbone in protein folding and function. The substitution of a main chain amide bond with an ester bond is now a popular mutation to investigate the role of the polypeptide backbone in ligand, binding, enzyme catalysis, and protein folding. Here we review the results of studies on some 25 ester-bond containing analogues from nine different protein systems. The structural, thermodynamic, and functional consequences of introducing backbone amide- to ester-bond mutations into these protein systems are discussed.

The 45-amino acid polypeptide chain of the homodimeric transcriptional repressor, CopG, was chemically synthesized by stepwise solid phase peptide synthesis (SPPS) using a protocol based on Boc-chemistry. The product obtained from the synthesis was readily purified by reversed-phase HPLC to give a good overall yield (21% by weight). Moreover, the synthetic CopG constructs prepared in this work folded into three-dimensional structures similar to the wild-type protein prepared using conventional recombinant methods as judged by far UV-CD spectroscopy. A fluorescent CopG analog, (Y39W)CopG, was also designed and chemically synthesized to facilitate biophysical studies of CopG's protein folding and assembly reaction. The guanidinium chloride-induced equilibrium unfolding properties of the wild-type CopG and (Y39W)CopG constructs in this work were characterized and used to develop a model for CopG's equilibrium unfolding reaction. Our results indicate that CopG's folding and assembly reaction is well modeled by a two-state process involving folded dimer and unfolded monomer. Using this model, DeltaG(f) and m-values of -13.42 +/- 0.04 kcal/mole dimer and 1.92 +/- 0.01 kcal/(mole M) were calculated for CopG.

Structure−activity relationships were investigated on a novel series of sulfonyldihydropyridine-containing KATP openers. Ring sizes, absolute stereochemistry, and aromatic substitution were evaluated for KATP activity in guinea pig bladder cells using a fluorescence-based membrane potential assay and in a pig bladder strip assay. The inhibition of spontaneous bladder contractions in vitro was also examined for a select group of compounds. All compounds studied showed greater potency to inhibit spontaneous bladder contractions relative to their potencies to inhibit contractions elicited by electrical stimulation. In an anesthetized pig model of myogenic bladder overactivity, compound 14 and (−)-cromakalim 1 were found to inhibit spontaneous bladder contractions in vivo at plasma concentrations lower than those that affected hemodynamic parameters. Compound 14 showed approximately 5-fold greater selectivity than 1 in vivo and supports the concept that bladder-selective KATP channel openers may have utility ...

The application of SUPREX (stability of unpurified proteins from rates of H/D exchange) to the thermodynamic analysis of protein-DNA complexes is described. A series of five model protein-DNA complexes involving two known DNA binding proteins, Arc repressor and CopG, were analyzed in order to determine the accuracy, precision, and generality of the SUPREX technique for quantifying the strength of protein-DNA interactions. For protein-DNA complexes that reversibly unfold in a two-state manner, we demonstrate that reasonably precise Kd values in agreement with those determined by conventional techniques can be determined by SUPREX. In the case of protein-DNA complexes that are not well modeled by a two-state unfolding mechanism, we find that relative binding affinities can be determined in the SUPREX experiment.

Recently, we developed a new method for measuring the thermodynamic stability of proteins. The method, termed Stability of Unpurified Proteins from Rates of H/D Exchange (SUPREX), utilizes matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and exploits the H/D exchange properties of proteins to determine folding free energies (i.e. ΔG°f values) for proteins. Here we report on the SUPREX analysis of seven new model proteins. The results of these analyses and the results of previously reported SUPREX analysis on seven additional proteins are used to assess the accuracy and precision of the SUPREX technique for measuring ΔG°f values. We find that the accuracy of the SUPREX technique for measuring the ΔG°f values of proteins is on the order of 20% and precision (relative standard deviation) of the technique is on the order 10%. These measures of accuracy and precision are comparable to those of conventional methods.

Direct matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of human serum yielded ion signals from only a fraction of the total number of peptides and proteins expected to be in the sample. We increased the number of peptide and protein ion signals observed in the MALDI-TOF mass spectra analysis of human serum by using a prefractionation protocol based on liquid phase isoelectric focusing electrophoresis. This pre-fractionation technique facilitated the MALDI-TOF MS detection of as many as 262 different peptide and protein ion signals from human serum. The results obtained from three replicate fractionation experiments on the same serum sample indicated that 148 different peptide and protein ion signals were reproducibly detected using our isoelectric focusing and MALDI-TOF MS protocol.

A hydrophilic surface suitable for solid-state peptide synthesis was developed on a solid support called a lantern. The split-and-mix combinatorial technique was used to prepare about 500 surfaces in a very short time. Surfaces were analyzed according to values for gel formation, percentage weight grafted, grafted copolymer composition, and number of functional groups per lantern. These values were correlated to the purity of a peptide synthesized on these surfaces. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 3371–3378, 2003

Here we describe the total chemical synthesis and biophysical characterization of two backbone-modified, ester bond-containing analogues of the homohexameric enzyme 4-oxalocrotonate tautomerase (4OT). The amide-to-ester bond mutations in the two analogues in this study, (OI2)4OT and (OI7)4OT, were designed to effectively delete specific backbone-backbone hydrogen bonds in the beta-sheet region of the native 4OT hexamer. The (OI2)4OT and (OI7)4OT analogues each contained one ester bond per monomer that effectively deleted 12 backbone-backbone hydrogen bonds per hexamer. The structural properties of each analogue were characterized by size-exclusion chromatography (SEC), far-UV CD spectroscopy, and catalytic activity measurements, and they were found to be very similar to the structural properties of the wild-type enzyme. The results of equilibrium unfolding studies revealed that the (OI2)4OT and (OI7)4OT analogues were stabilized by 47.7 +/- 2.5 and 45.0 +/- 2.5 kcal/mol, respectively, under standard state conditions (1 M hexamer) as compared to a value of 69.6 +/- 3.3 kcal/mol for the wild-type control. Our results suggest that the two different, but structurally similar, backbone-backbone hydrogen bonds deleted in (OI2)4OT and (OI7)4OT make nearly equivalent contributions to the thermodynamic stability of the 4OT hexamer.

A new H/D exchange- and MALDI mass spectrometry-based technique, termed SUPREX, was used to characterize the thermodynamic properties of a series of model protein-peptide complexes of the Abelson tyrosine kinase SH3 domain (abl-SH3) and the S-Protein (S-Pro). The SUPREX technique was employed to evaluate the folding free energies (DeltaG(f) values) of each model protein in the absence and in the presence of a series of different peptide ligands. Ultimately, these SUPREX-derived DeltaG(f) values were used to calculate dissociation constants (K(d) values) for each of the nine protein-peptide complexes in this study. As part of this work, we describe a new data collection and analysis method that allows the accurate and precise determination of protein folding m-values in the SUPREX experiment. The m-values that we determined for the abl-SH3 domain and the S-Pro system were in good agreement with those determined by conventional techniques. Our results also indicate that the SUPREX-derived K(d) values for the protein-peptide complexes in this work were in reasonably good agreement with those determined by conventional techniques.

Solid-phase synthesis is greatly dependent on the solid support. Here, we report the use of a new hydrophilic grafted surface on SynPhase lanterns in solid-phase organic chemistry. A convenient and facile solid-phase synthesis of disubstituted 1,4-benzodiazepine-2-ones on polyamide SynPhase lanterns is described. The key step of the synthesis involved a reduction-cyclization of a nitroaryl methyl ester with a mixture of tin(II) chloride dihydrate and ammonium acetate in water and ethanol at elevated temperature to give the desired target compounds. A library of 21 disubstituted 1,4-benzodiazepine-2-ones was prepared.

4-Oxalocrotonate tautomerase (4-OT) is a multimeric, bacterial enzyme comprised of 6 identical 62-amino acid subunits, which associate under native conditions to form a homo-hexameric structure stabilized entirely by noncovalent interactions. We have previously shown that the GuHCl-induced equilibrium unfolding of 4-OT at pH 8.5 is well modeled as a two-state process involving only hexamer and unfolded monomer; and we have obtained spectroscopic evidence that intermediate state(s) is (are) populated in the equilibrium unfolding reaction at pHs 6.0 and 7.4 [Silinski, P., Allingham, M. J., and Fitzgerald, M. C. (2001) Biochemistry 40, 4493-4502]. Here, we report on the pH-induced equilibrium unfolding of 4-OT using size-exclusion chromatography (SEC), far-UV-circular dichroism (CD) spectroscopy, and catalytic activity measurements over the pH range from 1.5 to 10.1. Our results indicate that the native hexamer of 4-OT is the predominant species in solution at pHsor =6.2, that a partially folded dimeric state of 4-OT is stabilized in solution at pH 4.8, and that the enzyme is largely denatured in strongly acidic solutions (pHor =3.1). GuHCl-induced equilibrium unfolding studies on 4-OT at pH 4.8 indicate that the folded 4-OT dimer populated at this pH is stabilized by 11.7 kcal.mol(-1). The results of biophysical studies on a fluorescent analogue of the enzyme, 4-OT(F50Y), and the results of UV photo-cross-linking studies on a synthetically derived 4-OT analogue, 4-OT(P1Bpa), suggest the polypeptide chains in the 4-OT dimer are nativelike in structure with the exception of their C-termini.

A series of indole containing oxazolines has been discovered as a result of structural modifications of the lead compound A-105972. The compounds exert their anticancer activity through inhibition of tubulin polymerization by binding at the colchicine site. A-289099 was identified as an orally active antimitotic agent active against various cancer cell lines including those that express the MDR phenotype. The anticancer activity, pharmacokinetics, and an efficient and enantioselective synthesis of A-289099 are described.

RNA-seq and denaturation studies demonstrated that allergens are among the most highly expressed and stable proteins from Dermatophagoides pteronyssinus. The combination of high levels of transcription and stability for the major allergens Der p 1, Der p 2, and Der p 23 is rare.

Reported here is the use of stable isotope labeling with amino acids in cell culture (SILAC) and pulse proteolysis (PP) for detection and quantitation of protein–ligand binding interactions on the proteomic scale. The incorporation of SILAC into PP enables the PP technique to be used for the unbiased detection and quantitation of protein–ligand binding interactions in complex biological mixtures (e.g., cell lysates) without the need for prefractionation. The SILAC-PP technique is demonstrated in two proof-of-principle experiments using proteins in a yeast cell lysate and two test ligands including a well-characterized drug, cyclosporine A (CsA), and a non-hydrolyzable adenosine triphosphate (ATP) analogue, adenylyl imidodiphosphate (AMP-PNP). The well-known tight-binding interaction between CsA and cyclophilin A was successfully detected and quantified in replicate analyses, and a total of 33 proteins from a yeast cell lysate were found to have AMP-PNP-induced stability changes. In control experiments, the method’s false positive rate of protein target discovery was found to be in the range of 2.1% to 3.6%. SILAC-PP and the previously reported stability of protein from rates of oxidation (SPROX) technique both report on the same thermodynamic properties of proteins and protein–ligand complexes. However, they employ different probes and mass spectrometry-based readouts. This creates the opportunity to cross-validate SPROX results with SILAC-PP results, and vice-versa. As part of this work, the SILAC-PP results obtained here were cross-validated with previously reported SPROX results on the same model systems to help differentiate true positives from false positives in the two experiments.

Over the past 15 years, a series of energetics-based techniques have been developed for the thermodynamic analysis of protein folding and stability. These techniques include Stability of Unpurified Proteins from Rates of amide H/D Exchange (SUPREX), pulse proteolysis, Stability of Proteins from Rates of Oxidation (SPROX), slow histidine H/D exchange, lysine amidination, and quantitative cysteine reactivity (QCR). The above techniques, which are the subject of this review, all utilize chemical or enzymatic modification reactions to probe the chemical denaturant– or temperature-induced equilibrium unfolding properties of proteins and protein-ligand complexes. They employ various mass spectrometry-, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)-, and optical spectroscopy–based readouts that are particularly advantageous for high-throughput and in some cases multiplexed analyses. This has created the opportunity to use protein folding and stability measurements in new applications such as in high-throughput screening projects to identify novel protein ligands and in mode-of-action studies to identify protein targets of a particular ligand.

Described here is a quantitative mass spectrometry-based proteomics method for the large-scale thermodynamic analysis of protein-ligand binding interactions. The methodology utilizes a chemical modification strategy termed, Stability of Proteins from Rates of Oxidation (SPROX), in combination with a Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) approach to compare the equilibrium folding/unfolding properties of proteins in the absence and presence of target ligands. The method, which is general with respect to ligand, measures the ligand-induced changes in protein stability associated with protein-ligand binding. The methodology is demonstrated in a proof-of-principle study in which the well-characterized protein-drug interaction between cyclosporine A (CsA) and cyclophilin A was successfully analyzed in the context of a yeast cell lysate. A control experiment was also performed to assess the method's false positive rate of ligand discovery, which was found to be on the order of 0.4 - 3.5%. The new method was utilized to characterize the adenosine triphosphate (ATP)-interactome in Saccharomyces cerevisiae using the nonhydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP), and the proteins in a yeast cell lysate. The new methodology enabled the interrogation of 526 yeast proteins for interactions with ATP using 2035 peptide probes. Ultimately, 325 peptide hits from 139 different proteins were identified. Approximately 70% of the hit proteins identified in this work were not previously annotated as ATP binding proteins. However, nearly two-thirds of the newly discovered ATP interacting proteins have known interactions with other nucleotides and co-factors (e.g. NAD and GTP), DNA, and RNA based on GO-term analyses. The current work is the first proteome-wide profile of the yeast ATP-interactome, and it is the largest proteome-wide profile of any ATP-interactome generated, to date, using an energetics-based method. The data is available via ProteomeXchange with identifiers PXD000858, DOI 10.6019/PXD000858, and PXD000860.

4-Oxalocrotonate tautomerase (4-OT) is a bacterial enzyme that is comprised of 6 identical 62 amino acid subunits. The 4-OT enzyme is an attractive model system in which to study the interrelationship between protein folding, subunit assembly, and catalytic function. Here we report on the GuHCl-induced equilibrium unfolding properties of wild-type 4-OT using catalytic activity measurements and using far-UV circular dichroism (CD) spectroscopy. We demonstrate that the unfolding of wild-type 4-OT in 50 mM phosphate buffers containing 6 M GuHCl is reversible at pHs 6.0, 7.4, and 8.5; and we find that there is both an enzyme concentration dependence and a pH dependence to the equilibrium unfolding properties of 4-OT. Our data suggests that the GuHCl-induced unfolding of 4-OT in 50 mM phosphate buffer at pH 8.5 can be modeled as a two-state process involving folded hexamer and unfolded monomer. On the basis of this model, we determined a free-energy value for the unfolding of 4-OT at pH 8.5 to be 68.7 +/- 3.2 kcal/mol under standard state conditions (1 M hexamer). In 50 mM phosphate buffers at pHs 6.0 and 7.4, only the catalytic activity denaturation curves are consistent with a two-state folding mechanism. At the lower pHs the far-UV-CD transitions are not well described by a two-state model. Our results at pHs 6.0 and 7.4 suggest that intermediate state(s) are populated in the equilibrium unfolding reaction at these lower pHs and that these intermediate state(s) have some helical content but no measurable catalytic activity.

In proteomic research, it is often necessary to screen a large number of polypeptides for the presence of stable structure. Described here is a technique (referred to as SUPREX, stability of unpurified proteins from rates of H/D exchange) for measuring the stability of proteins in a rapid, high-throughput fashion. The method uses hydrogen exchange to estimate the stability of microgram quantities of unpurified protein extracts by using matrix-assisted laser desorption/ionization MS. The stabilities of maltose binding protein and monomeric λ repressor variants determined by SUPREX agree well with stability data obtained from conventional CD denaturation of purified protein. The method also can detect the change in stability caused by the binding of maltose to maltose binding protein. The results demonstrate the precision of the method over a wide range of stabilities.

Noncoding RNAs have been implicated in the regulation of expression of numerous genes; however, the mechanism is not fully understood. We identified bidirectional, long noncoding RNAs upstream of the TNF gene using five different methods. They arose in a region where the repressors LRRFIP1, EZH2, and SUZ12 were demonstrated to bind, suggesting a role in repression. The noncoding RNAs were polyadenylated, capped, and chromatin associated. Knockdown of the noncoding RNAs was associated with derepression of TNF mRNA and diminished binding of LRRFIP1 to both RNA targets and chromatin. Overexpression of the noncoding RNAs led to diminished expression of TNF and recruitment of repressor proteins to the locus. One repressor protein, LRRFIP1, bound directly to the noncoding RNAs. These data place the noncoding RNAs upstream of TNF gene as central to the transcriptional regulation. They appear to serve as a platform for the assembly of a repressive complex.

A method to use matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for real-time monitoring of organic reactions on polymeric supports used in solid-phase synthesis is described. The strategy utilizes a synthetic construct that allows for the rapid and convenient direct MALDI analysis of the attached substrates as well as their subsequent chemical cleavage as desired. We have used this strategy to monitor nucleophilic substitutions, palladium-catalyzed coupling reactions, and solid phase peptide synthesis reactions using both Boc- and Fmoc-based chemistries. © 1997 Elsevier Science Ltd.

The synthesis of defined arrays of polypeptide analogues in conjunction with a simple self-encoded chemical readout system provides a powerful method for the systematic investigation of the relationship between polypeptide molecular structure and function. A novel solid-phase synthesis procedure was used to prepare arrays of polypeptide analogues in which a specific modification was systematically incorporated into a unique position in a peptide sequence. The synthesis was carried out in such a way that the resulting arrays contained a defined family of modified peptides, with each peptide molecule containing only a single specific modification. The array of polypeptide analogues was self-encoded in a positional fashion by incorporating a selectively cleavable bond into the analogue structure. Following cleavage of the polypeptide analogue array, analysis of the resulting peptide fragments by MALDI mass spectrometry defined, in a single step, the presence and identity of each peptide analogue in the mixtu...

Proteins have evolved to carry out very specific functions within the cell by interacting with a diverse set of biomolecules. Understanding how a protein's higher order structure relates to its function is important for defining the molecular basis of these interactions. In recent years, mass spectrometry has become an important tool for dissecting protein structure and function. Using electrospray ionization (ESI)- and matrix-assisted laser desorption/ionization (MALDI)-based approaches, it has been possible to monitor protein folding, characterize noncovalent protein complexes, and assess the contribution of individual amino acid residues to a protein's function. Here, it is our goal to summarize these approaches and highlight recent, biologically relevant applications where mass spectrometry has provided unique insight into the mysteries of protein structure and function.

C2-Symmetrical tetrahydroxyazepanes were synthesized as inhibitors for glycosidases. Tetrahydroxyazepane 1 is a non-specific inhibitor of various glycosidases, while compounds 2, 3 and 4 specifically inhibit β-N-acetylglucosaminidase, β-glucosidase, and a-fucosidase, respectively, with Ki in the micromolar range. Compound 1 is not an inhibitor of HIV/FIV proteases, but its 3,6-difluorobenzyl derivatives are moderate inhibitors of both enzymes.

Background: Modifying the covalent structure of a protein is an effective empirical route to probing three-dimensional structure and biological function. Here we describe a combinatorial protein chemistry strategy for studying structure-activity relationships in proteins. Our approach (termed 'protein signature analysis') involves functional selection from an array of self-encoded protein analogs prepared by total synthesis, coupled to a simple chemical readout that unambiguously identifies the modified proteins in the resulting active and inactive populations. Results: Protein signature analysis was used to study the interaction of the amino-terminal SH3 domain from the cellular adaptor protein c-Crk with its cognate proline-rich peptide, C3G. Using a functional selection assay, the qualitative effects of scanning a series of synthetic analog units through the amino-acid sequence of the SH3 domain were evaluated. The analog units were designed to alter both amino-acid sidechains and the polypeptide backbone within the protein. These chemical studies revealed that the sidechain of Asp150 in the SH3 domain is essential for ligand binding and that changes in the structure of the polypeptide backbone can also result in loss of binding activity. Conclusions: These chemical studies have provided new insight into how ligand binding is related to the covalent structure of the SH3 domain. Protein signature analysis is a powerful and conceptually novel way of studying the molecular and chemical basis of protein function; it combines the advantages of systematic modification of a protein's chemical structure with the practical convenience of combinatorial synthesis.

In this work we demonstrate that analytes, covalently linked to a polymeric support through a photolabile linker, can be directly analyzed by matrix-assisted laser desorption/ionization (MALDI). Mass spectral analysis is performed in a single step requiring no pretreatment of the sample to induce cleavage from the support. Our results show that the UV laser light in the MALDI experiment can be used to simultaneously promote an analyte's photolytic cleavage from a solid support and its gas phase ionization for subsequent mass spectral analysis. In this manner, MALDI facilitates the dissociation and identification of resin-bound analytes in a single analytical procedure without the need for any prior chemical treatment.

In the past several years, significant progress has been made in the application of matrix-assisted laser desorption-ionization (MALDI) mass spectrometry to the analysis of large biopolymers, including nucleic acids. By isolating analyte molecules in an appropriate matrix and irradiating the sample with a high-intensity, pulsed laser beam, MALDI can generate intact, gas-phase ions of these analytes. Primarily used with time-of-flight mass spectrometers, this relatively new, soft ionization technique has allowed for the routine analysis of oligonucleotides up to 60 or so nucleotides in length. Recent results have also shown that base specific, matrix-dependent fragmentation is an important factor in the MALDI analysis of oligonucleotides. Further extension of the technique to longer oligonucleotides will rely on both the continued search for new matrix materials and an increased understanding of the desorption and ionization process in MALDI.

The fragmentation of small oligodeoxynucleotides using matrix-assisted laser desorption/ionization (MAL DI) mass spectrometry with 355-nm radiation from the matrix 2,5-dihydroxybenzoic acid is studied. Negative ion mass spectra of the homopolymer oligodeoxynucleotides d(A){sub n}, d(C){sub n}, and d(G){sub n} (n = 4, 6, 8, and 10) show substantial cleavage at N-glycosidic and phosphodiester bonds, in contrast to d(T){sub n} which shows little fragmentation. The number and intensity of the fragment peaks increase significantly with the length of the parent molecules. A series of asymmetric oligodeoxynucleotides were synthesized to study this fragmentation in greater detail. In dT{sub 4}GT{sub 10}, dT{sub 4}G{sub 4}T{sub 7}, and dT{sub 7}G{sub 4}T{sub 4}, the primary fragmentation pathway is loss of a base followed by backbone cleavage at the 3{prime} C-O bond of the corresponding deoxyribose. Similar fragmentation patterns were observed with dT{sub 4}N{sub 4}T{sub 7} samples where N = C or A. A statistical cleavage model described the observed patterns of fragmentation well; the model yields fractions of backbone cleavage at A, C, and G of 0.13, 0.26, and 0.27, respectively. In contrast to the fragmentation observed using 2,5-dihydroxybenzoic acid as a matrix, little or no fragmentation is observed with the matrix 3-hydroxypicolinic acid. Fragmentation of oligodeoxynucleotidesmore » in UV/MALDI is thus both nucleobase and matrix dependent. 35 refs., 9 figs., 5 tabs.« less

beta-amyloid peptide (A beta) is the major protein component of senile plaques and cerebrovascular amyloid deposits in Alzheimer's patients. Several researchers have demonstrated that A beta is neurotoxic in in vitro and in vivo systems. Peptide aggregation state and/or conformation might play a significant role in determining the toxicity of the peptide. The size and flexibility of fibrils formed from the synthetic peptide beta (1-39), corresponding to the first 39 residues of A beta, were determined. Samples were prepared either directly from lyophilized peptide or diluted from a 10 mg/ml stock solution in 0.1% trifluoroacetic acid (TFA). All samples had a final peptide concentration of 0.5 mg/ml, a final pH of 7.4, and a final NaCl concentration of 0.14 M. The molecular weight and linear density of the fibrils increased with increasing pre-incubation time in TFA, based on static light scattering measurements. Analysis of the angular dependence of the intensity of scattered light indicated that the fibrils were semi-flexible chains and that the fibril flexibility decreased with increasing pre-incubation time in TFA. There was a concomitant change in phase behavior from precipitation to gelation with the decrease in fibril flexibility.

SummaryThe redox-regulated chaperone Hsp33 protects organisms against oxidative stress that leads to protein unfolding. Activation of Hsp33 is triggered by the oxidative unfolding of its own redox-sensor domain, making Hsp33 a member of a recently discovered class of chaperones that require partial unfolding for full chaperone activity. Here we address the long-standing question of how chaperones recognize client proteins. We show that Hsp33 uses its own intrinsically disordered regions to discriminate between unfolded and partially structured folding intermediates. Binding to secondary structure elements in client proteins stabilizes Hsp33's intrinsically disordered regions, and this stabilization appears to mediate Hsp33's high affinity for structured folding intermediates. Return to nonstress conditions reduces Hsp33's disulfide bonds, which then significantly destabilizes the bound client proteins and in doing so converts them into less-structured, folding-competent client proteins of ATP-dependent foldases. We propose a model in which energy-independent chaperones use internal order-to-disorder transitions to control substrate binding and release.

Described here is a mass spectrometry-based covalent labeling protocol that utilizes the amine reactive reagent, s-methyl thioacetimidate (SMTA), to study the chemical denaturant-induced equilibrium unfolding/refolding properties of proteins and protein-ligand complexes in solution. The protocol, which involves evaluating the rate at which globally protected amine groups in a protein are modified with SMTA as a function of chemical denaturant concentration, is developed and applied to the analysis of eight protein samples including six purified protein samples (ubiquitin, BCAII, RNaseA, 4OT, and lysozyme with, and without GlcNAc), a five-protein mixture comprised of ubiquitin, BCAII, RNaseA, Cytochrome C, and lysozyme, and a yeast cell lysate. In ideal cases the folding free energies of proteins and the dissociation constants of protein-ligand complexes can be accurately evaluated using the protocol. A direct MALDI-TOF readout is demonstrated for analysis of purified protein samples. Bottom-up proteomic strategies involving gel-based and/or LC-MS-based shotgun proteomic platforms are also demonstrated for the analyses of complex protein samples. Analysis of proteins in a yeast cell lysate suggests the SMTA-labeling protocol expands the peptide and protein coverage in chemical modification- and shotgun proteomics-based strategies for making thermodynamic measurements of protein folding and stability on the proteomic scale.

In order to examine the importance of pH in the matrix-assisted laser desorption/ionization (MAL-DI) analysis of proteins and oligonucleotides, 37 highly substituted pyrimidine, pyridine, and benzene derivatives containing basic amino groups were screened as potential matrices. Several of these compounds were useful for the preparation and analysis of samples under neutral and basic pH conditions. The matrix, 2-amino-4-methyl-5-nitropyridine, gave good results with small proteins (up to [approximately] 12,000 Da). The best results with oligonucleotides were obtained with 2-amino-5-nitropyridine, which was a useful matrix for the analysis of mixed-base oligonucleotides smaller than 20 nucleotides in length and homopolymers of thymidine. In general, oligonucleotide analysis using basic matrices was found to be subject to similar constraints of size and base composition that hold when acidic matrices are used. A major advantage of these newly discovered matrix materials over the more commonly used acidic matrices is that they extend the utility of MALDI to the analysis of acid-sensitive species. 19 refs., 6 figs., 2 tabs.

The cell autonomous response to viral infection is carefully regulated to induce type I interferons (IFNs), which in turn induce the establishment of an antiviral state. Leucine-rich repeat (in Flightless I) interacting protein-1 (LRRFIP1) and LRRFIP2 are 2 related proteins that have been identified as interacting with MyD88 and Flightless I homolog, a leucine-rich repeat protein. LRRFIP2 positively regulates NFκB and macrophage cytokine production after lipopolysaccharide, but less is known about LRRFIP1. We hypothesized that LRRFIP1 could be more important in antiviral responses, as overexpression led to type I IFN production in a pilot study. The induction of type I IFNs occurred even in the absence of virus, but was enhanced by the presence of virus. Conversely, knockdown of LRRFIP1 compromised IFN expression. We found that LRRFIP1 was rapidly recruited to influenza-containing early endosomes in a p38-dependent fashion. This was specific for virus-containing endosomes as there was almost no colocalization of LRRFIP1 with early endosomes in the absence of virus. Further, LRRFIP1 was recruited to RNA-containing vesicles. Taken together, these data suggest that LRRFIP1 participates in cell responses to virus at early time points and is important for type I IFN induction.

Immunosuppressive drugs are used to prevent rejection of transplanted organs and treat autoimmune diseases. Clinically approved immunosuppressive drugs possess undesirable side effects, including acute neurological toxicity, chronic nephrotoxicity, and osteoporosis. As a result, considerable efforts have been devoted to the identification of immunosuppressive natural products that lack cytotoxicity and undesirable side effects on bone structure. Subglutinols A (1 a) and B (1 b) are diterpene pyrones isolated from Fusarium subglutinans. Compounds 1 a and 1 b are equipotent in the mixed lymphocyte reaction assay and thymocyte proliferation assay (IC(50) = 0.1 microM). Owing to the lack of toxicity, 1 a and 1 b are expected to be promising new immunosuppressive drugs. Herein, we detail our efforts that have culminated in a stereoselective synthesis of 1 a and 1 b from the (S)-(+)-5-methyl-Wieland-Miescher ketone and determined their absolute stereochemistries. We also present initial biological data to show the great potential of 1 a as an immunosuppressive drug with dose-dependent osteogenic activity.

Molecular chaperones are a highly diverse group of proteins that recognize and bind unfolded proteins to facilitate protein folding and prevent nonspecific protein aggregation. The mechanisms by which chaperones bind their protein substrates have been studied for decades. However, there are few reports about the affinity of molecular chaperones for their unfolded protein substrates. Thus, little is known about the relative binding affinities of different chaperones and about the relative binding affinities of chaperones for different unfolded protein substrates. Here we describe the application of SUPREX (stability of unpurified proteins from rates of H-D exchange), an H-D exchange and MALDI-based technique, in studying the binding interaction between the molecular chaperone Hsp33 and four different unfolded protein substrates, including citrate synthase, lactate dehydrogenase, malate dehydrogenase, and aldolase. The results of our studies suggest that the cooperativity of the Hsp33 folding-unfolding reaction increases upon binding with denatured protein substrates. This is consistent with the burial of significant hydrophobic surface area in Hsp33 when it interacts with its substrate proteins. The SUPREX-derived K(d) values for Hsp33 complexes with four different substrates were all found to be within the range of 3-300 nM.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been used to mass analyze a variety of synthetic oligodeoxyribonucleotides ranging in size from 6 to 30 nucleotides. Molecular ions were observed almost exclusively, with little or no fragmentation and generally minor contributions from multiply charged species. Mixtures of poly d(T)12–30 with low picomole amounts of each component gave well resolved peaks. These results are encouraging for the concept of DNA sequence analysis by mass spectrometry.

A new approach has been developed for the rapid fragmentation and fractionation of DNA into a size suitable for shotgun cloning and sequencing. The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. Atypical reaction conditions which alter the specificity of this enzyme (CviJI**) yield a quasi-random distribution of DNA fragments from the small molecule pUC19 (2686 base pairs). To quantitatively evaluate the randomness of this fragmentation strategy, a CviJI** digest of pUC19 was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lacZ minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI** restricts PyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation. Advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 micrograms instead of 2-5 micrograms), fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed), and higher cloning efficiencies are obtained (CviJI** digested and column fractionated DNA transforms 3-16 times more efficiently than sonicated, end-repaired, and agarose fractionated DNA).

A discrete, hexanuclear zirconium metallocycle has been synthesized, isolated, and characterized by single-crystal X-ray diffraction. It is sustained in a chair-like conformation by glycolic acids. Formation and growth of the complex has been observed using in situ time-resolved dynamic light scattering measurements.

The double-stranded RNA (dsRNA) transport protein SID-1 enables systemic RNA interference (RNAi) in Caenorhabditis elegans, whereby silencing initiated by local exposure to dsRNA spreads throughout the animal and to its progeny. Previously, we showed that providing dsRNA in the growth medium of Drosophila S2 cells that express C. elegans SID-1 efficiently triggers RNAi. In these experiments long dsRNA proved to be significantly more effective than short dsRNA in silencing the target gene. Here, we show that equivalent masses of long or short dsRNA accumulate in these cells, indicating that size-dependent silencing is not due to size-selective transport through SID-1. Furthermore, using pulse-chase dsRNA uptake experiments, we show that short dsRNA accumulates more rapidly than long dsRNA. We found that import rates are dependent on dsRNA concentration, consistent with energy-independent, diffusion-limited transport through the SID-1 channel. Comparison of silencing efficiencies between Drosophila S2 cells heterologously expressing SID-1 and primary-cultured C. elegans cells shows similar dsRNA concentration and size dependencies, suggesting that C. elegans regulatory proteins do not measurably enhance or restrict dsRNA transport through SID-1. Finally, we find that coexpressing mutant SID-1 with wild-type SID-1 in S2 cells interferes with SID-1 function, indicating that SID-1 may function as a multimer.

Knowledge about the structural and biophysical properties of proteins when they are free in solution and/or in complexes with other molecules is essential for understanding the biological processes that proteins regulate. Such knowledge is also important to drug discovery efforts, particularly those focused on the development of therapeutic agents with protein targets. In the last decade a variety of different covalent labeling techniques have been used in combination with mass spectrometry to probe the solution-phase structures and biophysical properties of proteins and protein-ligand complexes. Highlighted here are five different mass spectrometry-based covalent labeling strategies including: continuous hydrogen/deuterium (H/D) exchange labeling, hydroxyl radical-mediated footprinting, SUPREX (stability of unpurified proteins from rates of H/D exchange), PLIMSTEX (protein-ligand interaction by mass spectrometry, titration, and H/D exchange), and SPROX (stability of proteins from rates of oxidation). The basic experimental protocols used in each of the above-cited methods are summarized along with the kind of biophysical information they generate. Also discussed are the relative strengths and weaknesses of the different methods for probing the wide range of conformational states that proteins and protein-ligand complexes can adopt when they are in solution.

Primary hyperoxaluria type I is a severe kidney stone disease caused by mutations in the protein alanine:glyoxylate aminotransferase (AGT). Many patients have mutations in AGT that are not deleterious alone but act synergistically with a common minor allele polymorphic variant to impair protein folding, dimerization, or localization. Although studies suggest that the minor allele variant itself is destabilized, no direct stability studies have been carried out. In this report, we analyze AGT function and stability using three approaches. First, we describe a yeast complementation growth assay for AGT, in which we show that human AGT can substitute for function of yeast Agx1 and that mutations associated with disease in humans show reduced growth in yeast. The reduced growth of minor allele mutants reflects reduced protein levels, indicating that these proteins are less stable than wild-type AGT in yeast. We further examine stability of AGT alleles in vitro using two direct methods, a mass spectrometry-based technique (stability of unpurified proteins from rates of H/D exchange) and differential scanning fluorimetry. We also examine the effect of known ligands pyridoxal 5′-phosphate and aminooxyacetic acid on stability. Our work establishes that the minor allele is destabilized and that pyridoxal 5′-phosphate and aminooxyacetic acid binding significantly stabilizes both alleles. To our knowledge, this is the first work that directly measures relative stabilities of AGT variants and ligand complexes. Because previous studies suggest that stabilizing compounds (i.e. pharmacological chaperones) may be effective for treatment of primary hyperoxaluria, we propose that the methods described here can be used in high throughput screens for compounds that stabilize AGT mutants.

IL-23, a heterodimer of IL-12 p40 and IL-23 p19, is critical for an effective immune response to many infections and has been implicated in several autoimmune diseases, however, little is known about the regulation of IL-23 gene expression in monocytes. We found that poly I:C, LPS, flagellin, and zymogen activated significant IL-23 production in primary human monocytes. Using chromatin immunoprecipitation, we found that a distal upstream region of the IL-23 p19 promoter at −601 to −521 underwent extensive histone modifications in response to stimuli. This distal region of the promoter is not highly conserved between species and has not been previously implicated in the regulation of IL-23 expression. Knockdown of CBP markedly decreased IL-23 p19 responses to poly I:C but had a less dramatic effect on LPS responses, confirming different chromatin responses to these two stimuli. Our data suggest that one of the mechanisms regulating IL-23 expression is the regulation of histone modifications at this distal upstream region of the promoter.

An H/D exchange- and MALDI mass spectrometry-based screening assay was applied to search for novel ligands that bind to cyclophilin A, a potential therapeutic and diagnostic target in lung cancer. The assay is based on stability of unpurified proteins from rates of H/D exchange (SUPREX), which exploits the H/D exchange properties of amide protons to measure the increase in a protein's thermodynamic stability upon ligand binding in solution. The current study evaluates the throughput and efficiency with which 880 potential ligands from the Prestwick Chemical Library (Illkirch, France) could be screened for binding to cyclophilin A. Screening was performed at a rate of 3 min/ligand using a conventional MALDI mass spectrometer. False positive and false negative rates, based on a set of control data, were as low as 0% and 9%, respectively. Based on the 880-member library screening, a false positive rate of 0% was observed when a two-tier selection strategy was implemented. Although novel ligands for cyclophilin A were not discovered, cyclosporin A, a known ligand to CypA and a blind control in the library, was identified as a hit. We also describe a new strategy to eliminate some of the complications related to back exchange that can arise in screening applications of SUPREX.

Described here is a new technique, termed SPROX (stability of proteins from rates of oxidation), that can be used to measure the thermodynamic stability of proteins and protein-ligand complexes. SPROX utilizes hydrogen peroxide in the presence of increasing concentrations of a chemical denaturant to oxidize proteins. The extent of oxidation at a given oxidation time is determined as a function of the denaturant concentration using either electrospray or matrix-assisted laser desorption/ionization mass spectrometry. Ultimately, the denaturant concentration dependence of the oxidation reaction rate is used to evaluate a folding free energy (DeltaG(f)) and m value (deltaDeltaG(f)/delta[Den]) for the protein's folding/unfolding reaction. Measurements of such SPROX-derived DeltaG(f) and m values on proteins in the presence and absence of ligands can also be used to evaluate protein-ligand affinities (e.g., DeltaDeltaG(f) and Kd values). Presented here are SPROX results obtained on four model protein systems including ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). SPROX-derived DeltaG(f) and m values on these proteins are compared to values obtained using more established techniques (e.g., CD spectroscopy and SUPREX). The dissociation constants of several known protein-ligand complexes involving these proteins were also determined using SPROX and compared to previously reported values. The complexes included the CypA-cyclosporin A complex and the BCAII-4-carboxybenzenesulfonamide complex. The accuracy and precision of SPROX-derived thermodynamic parameters for the model proteins and protein-ligand complexes in this study are discussed as well as the caveats of the technique.

Ferric binding protein, FbpA, is a member of the transferrin superfamily whose function is to move an essential nutrient, iron, across the periplasm and into the cytosol through formation of a ternary complex containing Fe (3+) and a synergistic anion, X. Here we utilize SUPREX ( stability of unpurified proteins from rates of H/D exchange) to determine the identification and distribution of the synergistic anion in FeFbpA-X species in periplasmic preparations from Gram-negative bacteria. SUPREX is a mass spectrometry-based technique uniquely suited for thermodynamic analyses of protein-ligand complexes in complex biological mixtures such as periplasmic preparations. Model binary mixtures of FeFbpA-Cit and FeFbpA-PO 4 were initially characterized by SUPREX due to the likely presence of citrate and phosphate ions in the periplasm. Ex vivo SUPREX analyses were performed on FeFbpA-X species overexpressed in an Escherichia coli cell line and on endogenous FeFbpA-X species in Neisseria gonorrheae. Detected in the E. coli periplasmic extract were two distinct populations of FbpA, including one in which the protein was unliganded (i.e., apoFbpA) and one in which the protein was bound to iron and the synergistic anion, phosphate (i.e., FeFbpA-PO 4). FeFbpA-PO 4 was the only population of FbpA molecules detected in the N. gonorrheae periplasmic extract. This work provides the first determination of the identity of the in vivo anion bound to FeFbpA-X in the periplasm and substantiates the hypothesis that the synergistic anion plays a structural and functional role in FbpA-mediated transport of iron across the periplasm and into the cytosol.

The obligate human pathogens Haemophilus influenzae, Neisseria gonorrhoeae, and N. meningitidis utilize a highly conserved, three-protein ATP-binding cassette transporter (FbpABC) to shuttle free Fe3+ from the periplasm and across the cytoplasmic membrane. The periplasmic binding protein, ferric binding protein (FbpA), is capable of transporting other trivalent cations, including Ga3+, which, unlike Fe3+, is not redox-active. Because of a similar size and charge as Fe3+, Ga3+ is widely used as a non-redox-active Fe3+ substitute for studying metal complexation in proteins and bacterial populations. The investigations reported here elucidate the similarities and differences in FbpA sequestration of Ga3+ and Fe3+, focusing on metal selectivity and the resulting transport function. The thermodynamic binding constant for Ga3+ complexed with FbpA at pH 6.5, in 50 mM 4-morpholineethanesulfonic acid, 200 mM KCl, 5 mM KH2PO4 was determined by UV-difference spectroscopy as $$ \log \,K_{{\text{eff}}}^\prime = 13.7 \pm 0.6. $$ This represents a 105-fold weaker binding relative to Fe3+ at identical conditions. The unfolding/refolding behavior of Ga3+ and Fe3+ holo-FbpA were also studied using a matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy technique, stability of unpurified proteins from rates of H/D exchange (SUPREX). This analysis indicates significant differences between Fe3+ and Ga3+ sequestration with regard to protein folding behavior. A series of kinetic experiments established the lability of the Ga3+FbpA–PO4 assembly, and the similarities/differences of stepwise loading of Fe3+ into apo- or Ga3+-loaded FbpA. These biophysical characterization data are used to interpret FbpA-mediated Ga3+ transport and toxicity in cell culture studies.

Publisher Summary This chapter discusses the applications of the “stability of unpurified proteins from rates of hydrogen/deuterium exchange” (SUPREX) technique to the thermodynamic analysis of protein folding and ligand binding. The requisite experimental protocols for SUPREX data acquisition and analysis are briefly described. SUPREX is a useful tool for measuring the protein folding free energies, binding free energies, and binding affinities. SUPREX is distinguished from other hydrogen/deuterium (H/D) exchange- and mass-spectrometry-based approaches for the analysis of protein stability and ligand binding in its use of chemical denaturant. Unique experimental advantages of SUPREX over existing methods for making the same types of thermodynamic measurements include the technique's high-throughput capabilities and the capacity for studying multicomponent mixtures. These advantages make SUPREX a promising technique that could open the door to the study of proteins under conditions that more closely resemble the conditions inside a cell—for example, the technique could be used to study protein folding under the conditions of molecular crowding or in the presence of molecular chaperones. There is also the possibility of examining the thermodynamic properties of large multiprotein complexes by SUPREX.