Protein-bound Peptides Research Articles

Efficient conformational ensemble generation of protein-bound peptides

Conformation generation of protein-bound peptides is critical for the determination of protein–peptide complex structures. Despite significant progress in conformer generation of small molecules, few methods have been developed for modeling protein-bound peptide conformations. Here, we have developed a fast de novo peptide modeling algorithm, referred to as MODPEP, for conformational sampling of protein-bound peptides. Given a sequence, MODPEP builds the peptide 3D structure from scratch by assembling amino acids or helix fragments based on constructed rotamer and helix libraries. The MODPEP algorithm was tested on a diverse set of 910 experimentally determined protein-bound peptides with 3–30 amino acids from the PDB and obtained an average accuracy of 1.90 Å when 200 conformations were sampled for each peptide. On average, MODPEP obtained a success rate of 74.3% for all the 910 peptides and ≥ 90% for short peptides with 3–10 amino acids in reproducing experimental protein-bound structures. Comparative evaluations of MODPEP with three other conformer generation methods, PEP-FOLD3, RDKit, and Balloon, have also been performed in both accuracy and success rate. MODPEP is fast and can generate 100 conformations for less than one second. The fast MODPEP will be beneficial for large-scale de novo modeling and docking of peptides. The MODPEP program and libraries are available for download at http://huanglab.phys.hust.edu.cn/.

Improving Binding Affinity and Stability of Peptide Ligands by Substituting Glycines with D‐Amino Acids

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer-related protease urokinase-type plasminogen activator (uPA), we observed a glycine residue that has a positive ϕ dihedral angle when bound to the target. We hypothesized that replacing it with a D-amino acid, which favors positive ϕ angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D-serine in the bicyclic peptide indeed improved inhibitory activity (1.75-fold) and stability (fourfold). X-ray-structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ϕ angles are found in many protein-bound peptides. These results suggest that the glycine-to-D-amino acid mutagenesis strategy could be broadly applied.

A novel peptidomics approach to detect markers of Alzheimer’s disease in cerebrospinal fluid

Sensitive and specific diagnosis and monitoring of disease progression are of prime importance to develop new therapies for Alzheimer’s disease patients. Although the diagnostic accuracy, verified by pathological examination is high, it is currently not possible to diagnose Alzheimer’s disease with a high degree of certainty until relatively late in the disease process. Here, we have undertaken a peptidome analysis of postmortem cerebrospinal fluid of neuropathologically confirmed Alzheimer’s disease patients and non-demented controls using a combination of methods and technologies. This includes novel sample preparation based on the enrichment of endogenous, proteolytically derived peptides as well as peptides non-covalently bound to abundant proteins. We observed differences in peptide profiles associated with Alzheimer’s disease in the endogenous peptide fraction and in the protein-bound peptide fraction. The discriminating peptides in the unbound peptide fraction were identified as VGF nerve growth factor inducible precursor, and complement C4 precursor, whereas the discriminating peptides in the protein-bound fraction were identified as VGF nerve growth factor inducible precursor, and alpha-2-HS-glycoprotein.

Transferred NOESY NMR Studies of Biotin Mimetic Peptide (FSHPQNT) Bound to Streptavidin: A Structural Model for Studies of Peptide–Protein Interactions

Protein-protein interactions control signaling, specific adhesion, and many other biological functions. The three-dimensional structures of the interfaces and bound ligand can be approached with transferred nuclear Overhauser effect spectroscopy NMR, which can be applied to much larger proteins than conventional NMR and requires less concentrated protein. However, it is not clear how accurately the structures of protein-bound peptides can be determined by transferred nuclear Overhauser effect spectroscopy. We studied the structure of a biotin mimetic peptide (FSHPQNT) bound to streptavidin, because the X-ray structure of the complex is available to 1.74 Å resolution, and we found that conditions could be adjusted so that the off-rates were fast enough for transferred nuclear Overhauser effect spectroscopy NMR. The off-rate was determined with (19)F NMR, using a para-fluoro-phenylalanine analog of the peptide. A new criterion for a lower limit on kinetic off-rate was found, which allowed accurate structure determination at a slower off-rate. Non-specific binding of the peptide to streptavidin was not significant, because biotin blocked the peptide transferred nuclear Overhauser effect spectroscopy. Protein mediation for the long-range peptide transferred nuclear Overhauser effect spectroscopy cross-peaks was corrected by a transferred nuclear Overhauser effect spectroscopy/ROESY averaging procedure. The protein-bound structure of the peptide was determined by transferred nuclear Overhauser effect spectroscopy constrained and simulated annealing. The structure deduced from the NMR was close to the X-ray structure.

Studying protein–protein interactions using peptide arrays

Screening of arrays and libraries of compounds is well-established as a high-throughput method for detecting and analyzing interactions in both biological and chemical systems. Arrays and libraries can be composed from various types of molecules, ranging from small organic compounds to DNA, proteins and peptides. The applications of libraries for detecting and characterizing biological interactions are wide and diverse, including for example epitope mapping, carbohydrate arrays, enzyme binding and protein-protein interactions. Here, we will focus on the use of peptide arrays to study protein-protein interactions. Characterization of protein-protein interactions is crucial for understanding cell functionality. Using peptides, it is possible to map the precise binding sites in such complexes. Peptide array libraries usually contain partly overlapping peptides derived from the sequence of one protein from the complex of interest. The peptides are attached to a solid support using various techniques such as SPOT-synthesis and photolithography. Then, the array is incubated with the partner protein from the complex of interest. Finally, the detection of the protein-bound peptides is carried out by using immunodetection assays. Peptide array screening is semi-quantitative, and quantitative studies with selected peptides in solution are required to validate and complement the screening results. These studies can improve our fundamental understanding of cellular processes by characterizing amino acid patterns of protein-protein interactions, which may even develop into prediction algorithms. The binding peptides can then serve as a basis for the design of drugs that inhibit or activate the target protein-protein interactions. In the current review, we will introduce the recent work on this subject performed in our and in other laboratories. We will discuss the applications, advantages and disadvantages of using peptide arrays as a tool to study protein-protein interactions.

Genome mining inAmycolatopsis balhimycinafor ferredoxins capable of supporting cytochrome P450 enzymes involved in glycopeptide antibiotic biosynthesis

Ferredoxins are required to supply electrons to the cytochrome P450 enzymes involved in cross-linking reactions during the biosynthesis of the glycopeptide antibiotics balhimycin and vancomycin. However, the biosynthetic gene clusters for these antibiotics contain no ferredoxin- or ferredoxin reductase-like genes. In a search for potential ferredoxin partners for these P450s, here, we report an in silico analysis of the draft genome sequence of the balhimycin producer Amycolatopsis balhimycina, which revealed 11 putative Fe-S-containing ferredoxin genes. We show that two members (balFd-V and balFd-VII), produced as native-like holo-[3Fe-4S] ferredoxins in Escherichia coli, could supply electrons to the P450 OxyB (CYP165B) from both A. balhimycina and the vancomycin producer Amycolatopsis orientalis, and support in vitro turnover of peptidyl carrier protein-bound peptide substrates into monocyclic cross-linked products. These results show that ferredoxins encoded in the antibiotic-producing strain can act in a degenerate manner in supporting the catalytic functions of glycopeptide biosynthetic P450 enzymes from the same as well as heterologous gene clusters.

Oncopeptidomics: A Useful Approach for Cancer Diagnosis?

In this issue of Clinical Chemistry , Mary Lopez and colleagues (1) describe novel methods for isolation of protein-bound peptides from serum and their characterization by mass spectrometry. Lopez et al. used selected peptide combinations to develop a new profiling method for ovarian cancer diagnosis. To put this advance into perspective, I will briefly summarize relevant previous literature on diagnostic applications of serum proteomic and peptidomic profiling by mass spectrometry. Approximately 5 years ago, a new approach for diagnosing ovarian cancer, by use of SELDI-TOF mass spectrometry, was proposed by the coauthors of the article under discussion (2). It was then hypothesized that proteins or protein fragments released by tumor cells or their microenvironment may enter the general circulation. By the use of a SELDI chip, proteins or peptides could be extracted from crude serum and used for diagnostic purposes with the aid of mass spectrometry and a mathematical algorithm. Similar methods have subsequently been used to diagnose numerous other malignancies, such as breast, prostate, bladder, pancreatic, head and neck, lung, liver, and nasopharyngeal cancers, as well as gliomas and melanomas, with impressive diagnostic sensitivities and specificities. This method has enjoyed ample coverage in scientific journals, the media, and international conferences (3). This author and others have criticized this diagnostic approach for methodological shortcomings and bioinformatic artifacts (4)(5)(6)(7)(8)(9). During the last 5 years, healthy debates have been conducted in journals (including this one) and at conferences (10)(11). An independent validation study would be the best test for this technology. As yet, however, no published validation is available to confirm that these methodologies are working. The proponents of the serum proteomic profiling methods for cancer diagnostics have now turned their attention to another possibility, proposing to interrogate the …

A Novel, High-Throughput Workflow for Discovery and Identification of Serum Carrier Protein-Bound Peptide Biomarker Candidates in Ovarian Cancer Samples

Most cases of ovarian cancer are detected at later stages when the 5-year survival is approximately 15%, but 5-year survival approaches 90% when the cancer is detected early (stage I). To use mass spectrometry (MS) of serum proteins for early detection, a seamless workflow is needed that provides an opportunity for rapid profiling along with direct identification of the underpinning ions. We used carrier protein-bound affinity enrichment of serum samples directly coupled with MALDI orthagonal TOF MS profiling to rapidly search for potential ion signatures that contained discriminatory power. These ions were subsequently directly subjected to tandem MS for sequence identification. We discovered several biomarker panels that enabled differentiation of stage I ovarian cancer from unaffected (age-matched) patients with no evidence of ovarian cancer, with positive results in >93% of samples from patients with disease-negative results and in 97% of disease-free controls. The carrier protein-based approach identified additional protein fragments, many from low-abundance proteins or proteins not previously seen in serum. This workflow system using a highly reproducible, high-resolution MALDI-TOF platform enables rapid enrichment and profiling of large numbers of clinical samples for discovery of ion signatures and integration of direct sequencing and identification of the ions without need for additional offline, time-consuming purification strategies.

Efficiency of peptide presentation by dendritic cells compared with other cell types: implications for cross-priming

Dendritic cells (DCs) play a key role in the induction of cellular immune responses by harvesting antigens from peripheral tissue for cross-priming CD8(+) T cells. It has been demonstrated that apoptotic bodies, whole- or degraded-cell-associated or soluble antigens as well as heat shock protein-bound peptides can be taken up, processed and cross-presented by DCs. Since cells are continuously releasing peptides from their surface MHC molecules, DCs in the tissues are exposed to such peptides and might process and present them to T cells as an additional pathway for cross-priming. To investigate this possibility, we compared and characterized the presentation of exogenous peptides by DCs and other cell types employing novel recombinant antibodies with TCR-like specificities for specific peptide-MHC complexes (pMHCs). These analyses reveal that loading of immature and mature DCs with peptide is far less efficient than it is for monocytes, T and B lymphocytes, B-lymphoblastoid, melanoma and TAP-deficient T2 cells. This inefficiency of peptide transfer to the MHC molecules of DCs makes it unlikely that these cells recycle peptides released from the MHC molecules of other cells and may explain why cross-presentation of such peptides has not yet been observed.

Serum Half-Life and Biological Activity of Mutants of Human Insulin-Like Growth Factor I which Do Not Bind to Serum Binding Proteins

We have characterized the biological properties of two mutants of human insulin-like growth factor I (IGF-I) which, as we have shown previously, have normal affinity for the type I IGF receptor, but drastically reduced affinity for the acid-stable components of human serum binding proteins. [Phe-1,Val1,Asn2,Gln3,His4,Ser8,His9,Glu12 ,Tyr15,Leu16]IGF I (B-chain mutant) and [Gln3,Ala4,Tyr15,Leu16]IGF I have 1000 and 500 times lower affinity than IGF-I for the native 150K binding protein in adult rat serum. Like IGF-I, these two peptides migrate as monomers during size exclusion chromatography on TSK 125. [125I]IGF-I, [125I]B-chain mutant, and [125I] [Gln3,Ala4,Tyr15,Leu16]IGF-I have in vivo serum half-lives of 100, 27.5, and 26.9 min, respectively, after iv injection. These data suggest that serum binding protein-bound peptide is cleared from the serum more slowly than free peptide. The tissue distributions of [125I]IGF-I and [125I]B-chain mutant are similar 10 min after dosing, with more than 80% of the tissue-sequestered intact radioactive peptides in the kidney. Despite decreased serum half-lives, the B-chain mutant and [Gln3,Ala4,Tyr15,Leu16]IGF-I are, respectively, 4 times and twice as active as IGF-I in stimulating the incorporation of [14C]glucose into glycogen in rat diaphragm in vivo. This effect of IGF-I is thought to be mediated by the type 1 IGF receptor in muscle, since the same doses of peptide that stimulated glycogen synthesis more than 30-fold did not stimulate the incorporation of [14C]glucose into total lipid in adipose tissue, an effect known to be mediated by the insulin receptor. These data support the hypothesis that serum- or tissue-derived binding proteins impair the ability of IGF-I to exert its effects through the type 1 IGF receptor in vivo.

Pulsatility of immunoreactive somatomedin-C in chronically cannulated rats.

Pituitary GH secretion is pulsatile in man and the rat, but evidence of pulsatility in the GH-dependent somatomedins (insulin-like growth factors) has not been described. In this study serum immunoreactive somatomedin-C periodicity was examined in 10 chronically cannulated unstressed rats. Blood samples were taken at 15-min intervals over 6 h, and serum rat GH and somatomedin-C measured by RIA. For somatomedin-C assay samples were first extracted into acid-ethanol to dissociate protein-bound peptide. Serum GH levels indicated episodic secretion, with a frequency of 2.85 +/- 0.24 h; some secretory episodes were polyphasic. The mean frequency of all GH spikes reaching 400 ng/ml or greater was 1.99 +/- 0.87 h. Somatomedin-C levels showed fluctuations over an average 2-fold concentration range, 0.60 +/- 0.20 to 1.21 +/- 0.29 U/ml (mean, 0.86 +/- 0.18 U/ml), with peaks occurring 1-1.5 h after most GH secretory peaks. The somatomedin-C peak frequency was 1.93 +/- 0.47 h. Summed GH values from 0-5 h were significantly correlated with summed somatomedin-C values from 1-6 h (r = 0.861, P = 0.0007), suggesting a 1-h lag between GH pulses and the following rise in somatomedin-C. Somatomedin-binding protein showed no regular fluctuations. This study indicates that serum somatomedin-C levels in unstressed rats show periodicity which may be directly related to pulsatile GH secretion.

Synthesis of a linear gramicidin by a combination of biosynthetic and organic methods.

Linear gramicidin is a pentadecapeptide with the sequence N-formyl-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L- Val-D-Val-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-D-Leu-L-Tr p-et h- anolamine. Ribosome-free extracts from Bacillus brecis (ATCC 81 85), on Sephadex G-200 filtration, yielded fractions free of aminoacyl-tRNA ligases which catalyzed ATP-PPi exchanges and covalent trichloroacetic acid stable binding of all amino acids incorporated in linear gramicidin. From these fractions C13CCOOH-stable protein-bound peptides were prepared and, after alkali (pH 11) or peroxidative libera- tion, separated by thin-layer chromatography. Labeled valine, glycine, alanine, leucine, and tryptophan used alternatively with phenylalanine, i.e., all the amino acids present in linear gramicidin, were incorporated into one of the peptides. The amino acid stoichiometry found in this peptide by double labeling as described in the text was Gly :Ala :Leu :Val :Phe, 1 :2 :4 :4 :4. Moreover, in agreement with the structure of T he linear gramicidins are antibiotics produced by the same strains of Bacillus brecis (ATCC 8185 or Dubos strain ATCC 10068) that produce tyrocidine (Hotchkiss, 1944). Figure 1 shows the structures of these pentadecapeptides, which differ only in the alternative incorporation of trypto- phan, tyrosine, and phenylalanine as aromatic amino acids similar to the differences in the tyrocidines. The amino-ter- minal valine is formylated, and the carboxyl-terminal trypto- phan is peptidically linked to ethanolamine. If glycine is con-

Protein-bound peptides in human serum.

Protein-bound peptides in human serum.

Gel filtration as a method for purification of protein-bound peptides exemplified by oxytocin and vasopressin.

Gel Filtration as a Method for Purification of Protein-bound Peptides Exemplified by Oxytocin and Vasopressin