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1.
Branched peptides as therapeutics 总被引:1,自引:0,他引:1
The concept of 'magic bullet', initially ascribed to immunoglobulins by Paul Ehrlich at the beginning of the 20th century and strengthened by the hybridoma technology of Kohler and Milstein in the mid 70s, can nowadays be attributed to different target-specific molecules, such as peptides. This attribution is increasingly valid in light of the explosion of new technologies for peptide library construction and screening, not to mention improvements in peptide synthesis and conjugation and in-vivo peptide stability, which make peptide molecules specific bullets for targeting pathological markers and pathogens. Today, hundreds of peptides are being developed and dozens are in clinical trials for a variety of diseases, demonstrating that the general reluctance towards peptide drugs that existed a decade ago has now been overcome. In spite of this progress, the development of new peptide drugs has largely been limited by their short half-life. Branched peptides such as Multiple Antigen Peptides (MAPs) were invented in the 80s by Tam [Tam, J.P., (1998) Proc. Natl. Acad. Sci. USA, 85, 5409] and have been extensively tested to reproduce single epitopes to stimulate the immune system for new vaccine discovery. In our lab we discovered that MAP molecules acquire strong resistance to proteases and peptidases. This resistance renders MAPs very stable and thus suitable for drug development. Here we report our experience with several MAP molecules in different biotechnological applications ranging from antimicrobial and anti toxin peptides to peptides for tumor targeting. 相似文献
2.
3.
Zhou HX 《The Journal of chemical physics》2008,128(19):195104
Master equations are widely used for modeling protein folding. Here an approximate solution to such master equations is presented. The approach used may be viewed as a discrete variational transition-state theory. The folding rate constant kf is approximated by the outgoing reaction flux J, when the unfolded set of macrostates assumes an equilibrium distribution. Correspondingly the unfolding rate constant ku is calculated as Jpu(1-pu), where pu is the equilibrium fraction of the unfolded state. The dividing surface between the unfolded and folded states is chosen to minimize the reaction flux J. This minimum-reaction-flux surface plays the role of the transition-state ensemble and identifies rate-limiting steps. Test against exact results of master-equation models of Zwanzig [Proc. Natl. Acad. Sci. USA 92, 9801 (1995)] and Munoz et al. [Proc. Natl. Acad. Sci. USA 95, 5872 (1998)] shows that the minimum-reaction-flux solution works well. Macrostates separated by the minimum-reaction-flux surface show a gap in p(fold) values. The approach presented here significantly simplifies the solution of master-equation models and, at the same time, directly yields insight into folding mechanisms. 相似文献
4.
Reversible stretching of the alanine-rich peptide 3K (Proc. Natl. Acad. Sci. USA 1989, 86, 5286-5290) and its analogue MW (Nature 1992, 359, 653-655) is examined using molecular dynamics simulations in explicit water. In both cases, sampling of the extension pathway is obtained on the 10 ns time scale by applying an adaptive biasing force. The free energy profile reveals a single minimum associated with a contiguous alpha-helix. Short 3(10)-helical motifs are observed in folded as well as extended conformations, in accordance with their proposed role as folding intermediates. The native 3(10)-helical content of both peptides is found, however, to be no higher than a few percent. Difficulties in both the definition and the detection of secondary structure motifs, most notably in relation to bifurcated hydrogen bonds, are proposed to account for the discrepancy between 3(10)-helical propensities reported by several authors, based on experimental and computational results. 相似文献
5.
David Zanuy Francisco J. Sayago Guillem Revilla-López Gema Ballano Lilach Agemy Venkata Ramana Kotamraju Ana I. Jiménez Carlos Cativiela Ruth Nussinov April M. Sawvel Galen Stucky Erkki Ruoslahti Carlos Alemán 《Journal of computer-aided molecular design》2013,27(1):31-43
We present a chemical strategy to engineer analogs of the tumor-homing peptide CREKA (Cys-Arg-Glu-Lys-Ala), which binds to fibrin and fibrin-associated clotted plasma proteins in tumor vessels (Simberg et al. in Proc Natl Acad Sci USA 104:932–936, 2007) with improved ability to inhibit tumor growth. Computer modeling using a combination of simulated annealing and molecular dynamics were carried out to design targeted replacements aimed at enhancing the stability of the bioactive conformation of CREKA. Because this conformation presents a pocket-like shape with the charged groups of Arg, Glu and Lys pointing outward, non-proteinogenic amino acids α-methyl and N-methyl derivatives of Arg, Glu and Lys were selected, rationally designed and incorporated into CREKA analogs. The stabilization of the bioactive conformation predicted by the modeling for the different CREKA analogs matched the tumor fluorescence results, with tumor accumulation increasing with stabilization. Here we report the modeling, synthetic procedures, and new biological assays used to test the efficacy and utility of the analogs. Combined, our results show how studies based on multi-disciplinary collaboration can converge and lead to useful biomedical advances. 相似文献
6.
We discuss two possible approaches for extracting kinetic information from single-molecule experiments. The first approach is based on computing correlation functions from measured fluorescence signals, and the second on studying the statistics of on and off times of the same fluorescence signal. We show that in both cases it is possible to extract kinetic information about the nature of intramolecular fluctuations of the single molecule. We show that for single-molecule kinetics the intramolecular fluctuations produce stochastic memory effects which lead to new dynamic features that do not exist in traditional chemical kinetics. In particular, we investigate a new type of chemical oscillations in correlation functions observed experimentally by Edman and Rigler (Proc. Natl. Acad. Sci. USA 2000, 97, 8266). 相似文献
7.
Ma G Huang F Pu X Jia L Jiang T Li L Liu Y 《Chemistry (Weinheim an der Bergstrasse, Germany)》2011,17(41):11657-11666
Platinum phenanthroline complexes inhibit amyloid-β (Aβ) aggregation and reduce Aβ-caused neurotoxicity [Proc. Natl. Acad. Sci., 2008, 105, 6813-6818]. In this study, we investigated the interactions of Aβ(1-16) with [PtCl(2)(phen)] (phen=1,10-phenanthroline) using HPLC, ESI-MS, and NMR spectroscopy , and characterized the identity of products using tandem mass spectrometry. Results indicated that the phenanthroline ligand could induce noncovalent interactions between Aβ peptide and platinum complexes, leading to rapid Aβ platination. Multiple products were generated in the reaction, in which His6/His14 chelation was preferentially formed. Coordination of Asp7, His13, and Lys16 was also detected in other products. The majority of products were monoplatinated adducts with binding of the {Pt(phen)} scaffold, which impeded intermolecular interactions between Aβ peptides. Moreover, noncovalent interactions were confirmed by the interaction between Aβ peptide and [Pt(phen)(2)]Cl(2). The synergistic roles of the phen ligand and platinum(II) atom in the inhibition of Aβ aggregation are discussed. 相似文献
8.
Schilling B Row RH Gibson BW Guo X Young MM 《Journal of the American Society for Mass Spectrometry》2003,14(8):834-850
In a previous report (Young et al., Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5802-5806), we provided a proof-of-principle for fold recognition of proteins using a homobifunctional amine-specific chemical crosslinking reagent in combination with mass spectrometry analysis and homology modeling. In this current work, we propose a systematic nomenclature to describe the types of peptides that are generated after proteolysis of crosslinked proteins, their fragmentation by tandem mass spectrometry, and an automated algorithm for MS/MS spectral assignment called "MS2Assign." Several examples are provided from crosslinked peptides and proteins including HIV-integrase, cytochrome c, ribonuclease A, myoglobin, cytidine 5-monophosphate N-acetylneuraminic acid synthetase, and the peptide thymopentin. Tandem mass spectra were obtained from various crosslinked peptides using post source decay MALDI-TOF and collision induced dissociation on a quadrupole-TOF instrument, along with their automated interpretation using MS2Assign. A variety of possible outcomes are described and categorized according to the number of modified lysines and/or peptide chains involved, as well as the presence of singly modified (dead-end) lysine residues. In addition, the proteolysis and chromatographic conditions necessary for optimized crosslinked peptide recovery are presented. 相似文献
9.
Dong SL Löweneck M Schrader TE Schreier WJ Zinth W Moroder L Renner C 《Chemistry (Weinheim an der Bergstrasse, Germany)》2006,12(4):1114-1120
Beta-hairpins constitute the smallest beta-type structures in peptides and proteins. The development of highly stable, yet monomeric beta-hairpins based on the tryptophan zipper motif was therefore a remarkable success [A. G. Cochran, N. J. Skelton, M. A. Starovasnik, Proc. Natl. Acad. Sci USA 2001, 98, 5578-5583]. We have been able to design, synthesize and characterize a hairpin based on this motif which incorporates an azobenzene-based photoswitch, that allows for time-resolved folding studies of beta-structures with unprecedented time resolution. At room temperature the trans-azo isomer exhibits a mostly disordered structure; however, light-induced isomerization to the cis-azo form leads to a predominantly extended and parallel conformation of the two peptide parts, which are linked by the novel photoswitch, [3-(3-aminomethyl)phenylazo]phenylacetic acid (AMPP). While in the original sequence the dipeptide Asn-Gly forms a type I' beta-turn which connects the two strands of the hairpin, this role is adopted by the AMPP chromophore in our photoresponsive beta-hairpin that can apparently act as a beta I'-turn mimetic. The beta-hairpin structure was determined and confirmed by NMR spectroscopy, but the folding process can be monitored by pronounced changes in the CD, IR and fluorescence spectra. Finally, incorporation of the structurally and functionally important beta-hairpin motif into proteins by chemical ligation might allow for the photocontrol of protein structures and/or functions. 相似文献
10.
Tian Y Camacho R Thomsson D Reus M Holzwarth AR Scheblykin IG 《Journal of the American Chemical Society》2011,133(43):17192-17199
Chlorosomes are the largest and most efficient natural light-harvesting systems and contain supramolecular assemblies of bacteriochlorophylls that are organized without proteins. Despite a recent structure determination for chlorosomes from Chlorobaculum tepidum (Ganapathy Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 8525), the issue of a possible large structural disorder is still discussed controversially. We have studied individual chlorosomes prepared under very carefully controlled growth condition by a novel 2-dimensional polarization single molecule imaging technique giving polarization information for both fluorescence excitation and emission simultaneously. Contrary to the existing literature data, the polarization degree or modulation depth (M) for both excitation (absorption) and emission (fluorescence) showed extremely narrow distributions. The fluorescence was always highly polarized with M ≈ 0.77, independent of the excitation wavelength. Moreover, the fluorescence spectra of individual chlorosomes were identical within the error limits. These results lead us to conclude that all chlorosomes possess the same type of internal organization in terms of the arrangement of the bacteriochlorophyll c transition dipole moments and their total excitonic transition dipole possess a cylindrical symmetry in agreement with the previously suggested concentric multitubular chlorophyll aggregate organization (Ganapathy Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 8525). 相似文献
11.
End-to-end contact formation rates of several peptides were recently measured by tryptophan triplet quenching (Lapidus et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7220). Motivated by these experiments, we study loop-closure kinetics for two peptides of different lengths, Cys-(Ala-Gly-Gln)n-Trp (n = 1, 2), in multiple all-atom explicit-solvent molecular dynamics simulations with different initial conditions and force fields. In 150 simulations of approximately 20 ns each, we collect data covering 1.0 and 0.8 micros for the penta-peptide simulated with the AMBER and CHARMM force fields, respectively, and about 0.5 micros each with the two force fields for the octa-peptide. These extensive simulations allow us to analyze the dynamics of peptides in the unfolded state with atomic resolution, thus probing early events in protein folding, and to compare molecular dynamics simulations directly with experiment. The calculated lifetimes of the tryptophan triplet state are in the range of 50-100 ns, in agreement with experimental measurements. However, end-to-end contacts form more rapidly, with characteristic times less than 10 ns. The contact formation rates for the two force fields are similar despite differences in the respective ensembles of peptide conformations. 相似文献
12.
Recent studies published in Oncogene and Proc. Natl. Acad. Sci. USA ascribe a role for selenium, acting through wild type p53, in protecting skin cells in culture from ultraviolet radiation-induced death. While selenium clearly protects cells against ultraviolet radiation-induced death, data that we present and discuss in this letter shows that wild type p53 is not required for such protection. Moreover the non-physiologically high levels of selenium used in some studies leads us to question the relevance of such effects for selenium-induced photoprotection. 相似文献
13.
A method for the rapid limited enzymatic cleavage of PVDF membrane-immobilized proteins is described. This method allows the fast characterization of PVDF blotted proteins by peptide mass fingerprinting (Henzel, W. J., Billeci, T. M., Stults, J. T., Wong, S. C., Grimley, C., Wantanabe, C., Proc. Natl. Acad. Sci. USA 1993, 90, 5011-5015), LC-MS/MS, or N-terminal sequencing and has been demonstrated on a range of proteins using a full complement of proteolytic enzymes. This technique allows the generation of proteolytic fragments between 5 and 60 min (depending on the enzyme employed), which is significantly faster than previously reported on-membrane digestion methods. To date, this on-membrane rapid digestion protocol has aided in the identification and confirmation of mutation sites in over 200 recombinant proteins. 相似文献
14.
Internal sequence analysis of proteins separated on polyacrylamide gels at the submicrogram level: improved methods, applications and gene cloning strategies 总被引:19,自引:0,他引:19
The fields of protein chemistry and molecular biology are currently merging for study of biologically relevant events and conditions. To obtain partial sequences of microamounts of protein, efficient integration of high resolution separation and sequencing technologies is required. We report here on improved methods that allow extensive internal sequencing of 10 to 20 picomoles protein recovered from one- or two-dimensional gels. Each step of the standard protocol of Aebersold et al. (Proc. Natl. Acad. Sci. USA 1987, 84, 6970-6974) and the required instrumentation were examined and specifically adapted for use with submicrogram amounts of protein. Optimizations of in situ microdigests and liquid chromatography were needed for improved peptide recovery. Subsequent automated sequencing required subpicomole analysis. New methods for S-alkylation of gel-separated proteins and accurate identification of tryptophan-containing peptides were introduced to insure overall higher efficiencies. The acquired internal sequences facilitated cloning of the genes and several strategies are discussed. Applying our method, several proteins of unknown structure were sequenced and successfully identified or cloned. Internal sequences of submicrogram protein amounts, recovered from a single two-dimensional gel of Escherichia coli total protein (120 micrograms), allowed unambiguous identification of the spots but pre-gel enrichment will be required for analysis of most (90-95%) other spots. Integration of comprehensive two-dimensional gel protein databases with methods and strategies outlined here could potentially be an abundant source of DNA probes and markers useful for guidance of the human genome sequencing project and for analysis of the emerging vast amounts of data. 相似文献
15.
We use classical molecular dynamics combined with the recently developed metadynamics method [Laio, A.; Parrinello, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 20] to study the process of bulk melting in hexagonal ice. Our simulations show that bulk melting is mediated by the formation of topological defects which preserve the coordination of the tetrahedral network. Such defects cluster to form a defective region involving about 50 molecules with a surprisingly long lifetime. The subsequent formation of coordination defects triggers the transition to the liquid state. 相似文献
16.
R Tóth-Boconádi S G Taneva L Keszthelyi 《Journal of photochemistry and photobiology. B, Biology》2001,65(2-3):122-126
The study of mutant D96N played an important role in understanding proton translocation by light driven bacteriorhodopsin. Our measurement of photoelectric current for single and double flash illumination revealed new details of the photocycle of this mutant. With double flash excitation we found an intermediate absorbing near the wavelength of the ground state of bacteriorhodopsin (bR) but pumping in the opposite direction. This intermediate has the same lifetime as the species described by Zimányi et al. [Proc. Natl. Acad. Sci. USA 96 (1999) 4414-4419] and was assigned to early recovery of a fraction of the ground state after excitation. Because the electric response does not reconcile with that of the ground state, we tentatively assign it to the L intermediate or to an intermediate similar in absorption to bR (bR'). 相似文献
17.
Hauser K Krejtschi C Huang R Wu L Keiderling TA 《Journal of the American Chemical Society》2008,130(10):2984-2992
Two antiparallel beta-strands connected by a turn make beta-hairpins an ideal model system to analyze the interactions and dynamics of beta-sheets. Site-specific conformational dynamics were studied by temperature-jump IR spectroscopy and isotopic labeling in a model based on the tryptophan zipper peptide, Trpzip2, developed by Cochran et al. (Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5578). The modified Trpzip2C peptides have nearly identical equilibrium spectral behavior as Trpzip2 showing that they also form well-characterized beta-hairpin conformations in aqueous solution. Selective introduction of 13C=O groups on opposite strands lead to distinguishable cross-strand coupling of the labeled residues as monitored in the amide I' band. These frequency patterns reflect theoretical predictions, and the coupled 13C=O band loses intensity with increase in temperature and unfolding of the hairpin. Thermal relaxation kinetics were analyzed for unlabeled and cross-strand isotopically labeled variants. T-jumps of approximately 10 degrees C induce relaxation times of a few microseconds that decrease with increase of the peptide temperature. Differences in kinetic behavior for the loss of beta-strand and gain of disordered structure can be used to distinguish localized structure dynamics by comparison of nonlabeled and labeled amide I' components. Analysis of the data supports multistate dynamic and equilibrium behavior, but because of this process it is not possible to clearly define a folding and unfolding rate. Nonetheless, site-specific relaxation kinetics could be seen to be consistent with a hydrophobic collapse hypothesis for hairpin folding. 相似文献
18.
Three recent studies of the high-pressure transformations of methane hydrate [Chem. Phys. Lett. 325 (2000) 490; Proc. Natl. Acad. Sci. 97 (2000) 13484; Nature 410 (2001) 661] have reported apparently different behaviours. Detailed comparison of our X-ray and neutron diffraction data with those obtained in earlier work shows that there is in fact consistent behaviour on isothermal compression at room temperature with a transition from the clathrate I structure hydrate to a hexagonal hydrate with an unknown structure at 0.9 GPa. 相似文献
19.
Strittmatter EF Ferguson PL Tang K Smith RD 《Journal of the American Society for Mass Spectrometry》2003,14(9):980-991
We describe the application of capillary liquid chromatography (LC) time-of-flight (TOF) mass spectrometric instrumentation for the rapid characterization of microbial proteomes. Previously (Lipton et al., Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11049) the peptides from a series of growth conditions of Deinococcus radiodurans have been characterized using capillary LC MS/MS and accurate mass measurements which are captured as an accurate mass and time (AMT) tag database. Using this AMT tag database, detected peptides can be assigned using measurements obtained on a TOF due to the additional use of elution time data as a constraint. When peptide matches are obtained using AMT tags (i.e., using both constraints) unique matches of a mass spectral peak occurs 88% of the time. Not only are AMT tag matches unique in most cases, the coverage of the proteome is high; approximately 3500 unique peptide AMT tags are found on average per capillary LC run. From the results of the AMT tag database search, approximately 900 ORFs detected using LC-TOFMS, with approximately 500 ORFs covered by at least two AMT tags. These results indicate that AMT database searches with modest mass and elution time criteria can provide proteomic information for approximately one thousand proteins in a single run of <3 h. The advantage of this method over using MS/MS based techniques is the large number of identifications that occur in a single experiment as well as the basis for improved quantitation. For MS/MS experiments, the number of peptide identifications is severely restricted because of the time required to dissociate the peptides individually. These results demonstrate the utility of the AMT tag approach using capillary LC-TOF MS instruments, and also show that AMT tags developed using other instrumentation can be effectively utilized. 相似文献
20.
J W Crabb W R Harris C M Johnson T G Sotiroudis C C Kuhn L M Heilmeyer 《Electrophoresis》1990,11(2):133-140
A simple, rapid sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method is presented for isolating the alpha, alpha' and beta subunits of rabbit muscle phosphorylase kinase. The SDS-PAGE procedure can yield milligram amounts of alpha and beta from a single preparative gel and also allows isolation of the alpha' isozyme free of alpha. Notably the method provides the purified subunits in a form amenable to structural analysis. Edman degradation of alpha and alpha' reveal identical NH2-terminal structures. Amino acid analysis of the electrophoretically purified alpha and beta subunits are in good agreement with their deduced primary structures. The amino acid sequence of 488 residues in alpha and 713 residues in beta were determined by gas phase Edman degradation. The data support the recently deduced primary structures of alpha (Zander et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 9381-9385). 相似文献