系统比较氨基酸描述子在多肽定量构效关系中的应用

氨基酸描述子是采用多元统计方法从大量氨基酸性质参数中提取得到的少数关键信息成分,目前已被广泛应用于多肽生物活性预测及蛋白质功能判别等领域。鉴于近年来氨基酸描述子种类的极速扩增,收集了目前国内外发表的27种氨基酸描述子,并将其用于8组经典多肽集的结构表征及定量构效关系研究。通过系统比较这些描述子对同一肽集与不同肽集的统计建模结果,我们认为物理化学描述子的建模效果优于拓扑描述子,拓扑描述子的建模效果优于三维结构描述子,且已有诸多氨基酸描述子已经达到性能限度,如未考虑肽链内部各氨基酸残基的交互影响,肽配基与相应靶标蛋白的相互结合,因此不再建议按照传统思路进一步提出新型描述子种类。     

Mass spectrometric analysis of protein histidine phosphorylation

Summary. Protein histidine phosphorylation is now recognized as an important form of post-translational modification. The acid-lability of phosphohistidine has meant that this phosphorylation has not been as well studied as serine/threonine or tyrosine phosphorylation. We show that phosphohistidine and phosphohistidine-containing phosphopeptides derived from proteolytic digestion of phosphohistone H4 are detectable by ESI-MS. We also demonstrate reverse-phase HPLC separation of these phosphopeptides and their detection by MALDI-TOF-MS.     

High affinity nucleocapsid protein binding to the muPsi RNA packaging signal of Rous sarcoma virus

The genomes of all retroviruses contain sequences near their 5' ends that interact with the nucleocapsid domains (NC) of assembling Gag proteins and direct their packaging into virus particles. Retroviral packaging signals often occur in non-contiguous segments spanning several hundred nucleotides of the RNA genome, confounding structural and mechanistic studies of genome packaging. Recently, a relatively short, 82 nucleotide region of the Rous sarcoma virus (RSV) genome, called muPsi, was shown to be sufficient to direct efficient packaging of heterologous RNAs into RSV-like particles. We have developed a method for the preparation and purification of large quantities of recombinant RSV NC protein, and have studied its interactions with native and mutant forms of the muPsi encapsidation element. NC does not bind with significant affinity to truncated forms of muPsi, consistent with earlier packaging and mutagenesis studies. Surprisingly, NC binds to the native muPsi RNA with affinity that is approximately 100 times greater than that observed for other previously characterized retroviral NC-RNA complexes (extrapolated dissociation constant K(d)=1.9 nM). Tight binding with 1:1 NC-muPsi stoichiometry is dependent on a conserved UGCG tetraloop in one of three predicted stem loops, and an AUG initiation codon controvertibly implicated in genome packaging and translational control. Loop nucleotides of other stem loops do not contribute to NC binding. Our findings indicate that the structural determinants of RSV genome recognition and NC-RNA binding differ considerably from those observed for other retroviruses.     

On the molecular basis of the high affinity binding of basic amino acids to LAOBP,a periplasmic binding protein from Salmonella typhimurium

The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high‐affinity binding of ligands to proteins is still limited; such is the case for l ‐lysine–l ‐arginine–l ‐ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l ‐arginine, l ‐lysine, and l ‐ornithine with nanomolar affinity and to l ‐histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l ‐histidine and l ‐arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of ~?300 cal mol^?1 K^?1, most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000‐fold higher affinity of LAOBP for l ‐arginine as compared with l ‐histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy‐driven micromolar affinity toward l ‐arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization. Copyright © 2015 John Wiley & Sons, Ltd.     

Nucleobase‐caged peptide nucleic acids: PNA/PNA duplex destabilization and light‐triggered PNA/PNA recognition

The 2‐(o‐nitrophenyl)‐propyl (NPP) group is used as caging group to mask the nucleobases adenine and cytosine in N‐(2‐aminoethyl)glycine peptide nucleic acids (aeg‐PNA). The adeninyl and cytosinyl nucleo amino acid building blocks Fmoc‐a^NPP‐aeg‐OH and Fmoc‐c^NPP‐aeg‐OH were synthesized and incorporated into PNA sequences by Fmoc solid phase synthesis relying on high stability of the NPP nucleobase protecting group toward Fmoc‐cleavage, coupling, capping, and resin cleavage conditions. Removal of the nucleobase caging group was achieved by UV‐LED irradiation at 365 nm. The nucleobase caging groups provided sterical crowding effecting the Watson–Crick base pairing, and thereby, the PNA double strand stabilities. Duplex formation can completely be suppressed for complementary PNA containing caging groups in both strands. PNA/PNA recognition can be completely restored by UV light‐triggered release of the photolabile protecting group. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Affinity chromatography of plasma proteins (guanidinobenzoatase): use of mimetic matrices and mimetic soluble ligands to prevent the binding of albumin on target affinity matrices

Serum albumin is the most abundant protein in plasma and it has a high capacity to bind many small compounds and macromolecules. In this way, albumin may promote important interferences during affinity chromatography of plasma proteins. Guanidinobenzoatase (GB) is a very relevant plasma protease that seems to be related to tumoral processes. This enzyme may be adsorbed on tailor-made agmatine-amide-agarose (CH-A) supports (e.g., the ones having 2 μmol of guanidino groups per ml of agarose attached to the support, through a 6 C aliphatic chain). Such tailor-made supports containing a very low concentration of ionized groups are hardly able to adsorb any protein by anion-exchange. However, they are able to strongly adsorb albumin. In order to solve this problem new mimetic affinity matrices have been designed: (i) by using the same ligand immobilized through a different chemical linkage [guanidino groups attached via secondary amino bonds, (AEA)] or (ii) by using slightly different ligands (e.g., 1,8-octanediamine containing a primary amino group instead of a guanidino one) also attached to the support via amido bonds (CH-DAO). Albumin adsorbs on the target and on the two mimetic matrices while GB is mainly adsorbed on the target one. Moreover, the adsorption of albumin on the affinity matrix (CH-A) is very strongly inhibited by the presence of low concentrations of soluble ligands (e.g., 1,8-octanediamine containing two ionized primary amino groups). On the contrary, the adsorption of GB on CH-A is hardly inhibited by the presence of such mimetic soluble ligand. In this way, the former offering of crude GB samples to AEA plus the use of mimetic inhibitors during adsorption of the extract on CH-A completely prevent the undesirable adsorption of albumin. In a such way, an extremely selective adsorption of GB can be performed. Such an improved chromatography procedure allows a very easy affinity purification and detection of GB.     

Stability and peptide binding affinity of an SH3 domain from the Caenorhabditis elegans signaling protein Sem-5.

We have determined the thermodynamic stability and peptide binding affinity of the carboxy-terminal Src homology 3 (SH3) domain from the Caenorhabditis elegans signal-transduction protein Sem-5. Despite its small size (62 residues) and lack of disulfide bonds, this domain is highly stable to thermal denaturation--at pH 7.3, the protein has a Tm of 73.1 degrees C. Interestingly, the protein is not maximally stable at neutral pH, but reaches a maximum at around pH 4.7 (Tm approximately equal to 80 degrees C). Increasing ionic strength also stabilizes the protein, suggesting that 1 or more carboxylate ions are involved in a destabilizing electrostatic interaction. By guanidine hydrochloride denaturation, the protein is calculated to have a free energy of unfolding of 4.1 kcal/mol at 25 degrees C. We have also characterized binding of the domain to 2 different length proline-rich peptides from the guanine nucleotide exchange factor, Sos, one of Sem-5's likely physiological ligands in cytoplasmic signal transduction. Upon binding, these peptides cause about a 2-fold increase in fluorescence intensity. Both bind with only modest affinities (Kd approximately equal to 30 microM), lower than some previous estimates for SH3 domains. By fluorescence, the domain also appears to associate with the homopolymer poly-L-proline in a similar fashion.     

Staurosporine-based binding assay for testing the affinity of compounds to protein kinases

Staurosporine is a broad-spectrum inhibitor of both tyrosine and serine/threonine protein kinases. Excitation of staurosporine and its analogues at 296 nm results in major emission bands centered at 378 and 396 nm. The intensity of the emission bands is enhanced on binding to the adenosine triphosphate (ATP) site of many protein kinases. This property was used to develop a competitive displacement assay for evaluating the binding affinity of small molecules to protein kinases. The assay was validated in both cuvette and plate formats for several phosphorylated and non-phosphorylated protein kinases. The throughput of the assay is high enough to be used in drug discovery for screening as well as lead optimization.     

Identification of products of protein phosphorylation in T37-transformed cells and comparison with normal cells

Proteins from crown gall tissue labelled in vivo with [³²P]orthophosphate were analysed by SDS-polyacrylamide gel electrophoresis. The major phosphorylated proteins were of 50.6 and 48.3 kDa, with minor bands at 80.1, 73.9, 68, 40.4, 30, 21.5, 20.2 and 15.2 kDa. Partial hydrolysates of total ³²P-labelled proteins were analysed in a number of ways. A two-dimensional separation on paper by electrophoresis in pyridine/acetic acid at pH 3.5 followed by chromatography in isobutyric acid/0.5 M ammonia revealed radioactive spots coincident with phosphoserine and phosphothreonine markers and only partially coincident with the phosphotyrosine marker. Two-dimensional electrophoresis at pH 1.9 followed by pH 3.5, however, unequivocally showed the presence of phosphotyrosine after elution of the phosphotyrosine marker. Phosphoserine, phosphothreonine and phosphotyrosine were present in the ratio 89.4:8.5:2.1. This is a much higher level of phosphotyrosine than normally found in animal cells. The three phosphoamino acids were confirmed by chromatography with authentic samples in four solvent systems on cellulose or silica TLC, and by dansylation followed by silica TLC. The radioactive compound running almost coincident with phosphotyrosine on two-way electrophoresis, pH 3.5, followed by chromatography in isobutyric acid/0.5 M ammonia was identified tentatively as uridine 5′-monophosphate on the basis of electrophoretic and chromatographic behaviour. Further experiments to compare normal (growing and non-growing) tobacco callus and T37-transformed cells did not give markedly different ratios of the three phosphoamino acids, although the rapidly-growing normal tobacco (i.e., plus cytokinin) appeared to have a greater abundance of the two minor phosphoamino acids (approx. 2-times). The lack of effect of transformation is in contrast to animal cells where transformation results in a 10-fold increase in the virally affected cells.     

NMR-based modeling and binding studies of a ternary complex between chicken liver bile acid binding protein and bile acids

Chicken liver bile acid binding protein (cL-BABP) is involved in bile acid transport in the liver cytosol. A detailed study of the mechanism of binding and selectivity of bile acids binding proteins towards the physiological pool of bile salts is a key issue for the complete understanding of the role of these proteins and their involvement in cholesterol homeostasis. In the present study, we modeled the ternary complex of cL-BABP with two molecules of bile salts using the data driven docking program HADDOCK on the basis of NMR and mass spectrometry data. Docking resulted in good 3D models, satisfying the majority of experimental restraints. The docking procedure represents a necessary step to help in the structure determination and in functional analysis of such systems, in view of the high complexity of the 3D structure determination of a ternary complex with two identical ligands. HADDOCK models show that residues involved in binding are mainly located in the C-terminal end of the protein, with two loops, CD and EF, playing a major role in ligand binding. A spine, comprising polarresidues pointing toward the protein interior and involved in motion communication, has a prominent role in ligand interaction. The modeling approach has been complemented with NMR interaction and competition studies of cL-BABP with chenodeoxycholic and cholic acids. A higher affinity for chenodeoxycholic acid was observed and a Kd upper limit estimate was obtained. The binding is highly cooperative and no site selectivity was detected for the different bile salts, thus indicating that site selectivity and cooperativity are not correlated. Differences in physiological pathways and bile salt pools in different species is discussed in light of the binding results thus enlarging the body of knowledge of BABPs biological functions.     

Copper binding to prion octarepeat peptides, a combined metal chelate affinity and immunochemical approaches

Based on the hypothetical proposal of Sulkowski [E. Sulkowski, FEBS Lett. 307 (2) (1992) 129] for the implication of transition metal ions in the structural changes/oligomerisation of normal cellular prion protein (PrPc) resulting in the pathological isoform (PrPsc), we focused our study on the octarepat domain of this protein which has been supposed to be the metal binding site. We have studied the copper binding to synthetic prion octarepeat peptides (PHGGGWGQ)n (n=1, 3, 6) using metal chelate and size-exclusion modes of chromatographies. This copper binding induces oligomerisation resulting in multiple aggregates. Moreover, heterogeneity of metal bound octarepeat oligomers by ESI-MS has been demonstrated. In addition, anti prion antibodies specific to the octarepeat region were used to discriminate between metal free and copper, nickel and zinc bound hexamer octarepeat peptide. Differential recognition of Cu(II) and Zn(II) bound complexes has been observed which signify differences in exposed epitopes of aggregated peptides.     

Novel Rath peptide for intracellular delivery of protein and nucleic acids

In the present study, a novel cell penetrating peptide (CPP) named as Rath, has been identified from the avian infectious bursal disease virus. It has the potential to penetrate and translocate cargo molecules into cells independent of temperature. Additionally, it can deliver oligonucleotide in 30 min and antibodies within an hour intracellular to chicken embryonic fibroblast primary cells. As an ideal delivery vehicle, it has the ability to protect the cargo molecules in the presence of serum, nucleases and has minimal or no cytotoxicity at even higher peptide concentrations studied. The biophysical characterizations showed that Rath has a dominant β structure with a small α helix and has remarkable binding ability with protein and DNA. Thus, the characterization of unique Rath peptide to deliver protein or nucleic acid into the cells with non-covalent interaction could be used as an effective delivery method for various cell based assays.     

Development of non-phosphorylated cyclic thioether peptide binding to the Grb2-SH2 domain

One of the critical intracellular signaling pathways involves specific interactions between growth factor receptors and the adaptor protein Grb2. These interactions normally involve specific tyrosine phosphorylated regions in receptors and other cognate proteins. Following the lead of our recent findings that a phage library based non-phosphorylated disulfide linked 11-mer peptide inhibited such interactions, we report here the synthesis of novel redox-stable cyclic peptide analogs. These include thioether cyclized and backbone cyclized structures. The thioether analog was prepared under mild conditions from an N-terminally chloroacetylated and C-terminally cysteine extended peptide precursor. The thioether peptide showed equipotent binding affinity for the Grb2-SH2 domain (IC50 = 10–15 M) when compared to the disulfide cyclized lead-peptide. The bioactive thioether linked peptide was demonstrated to offer advantages to the disulfide cyclized peptides under physiological conditions.     

Molecular dynamics simulations of peptides containing an unnatural amino acid: dimerization, folding, and protein binding

We have performed molecular dynamics (MD) simulations to study the dimerization, folding, and binding to a protein of peptides containing an unnatural amino acid. NMR studies have shown that the substitution of one residue in a tripeptide beta-strand by the unnatural amino acid Hao (5-HO2CCONH-2-MeO-C6H3-CO-NHNH2) modifies the conformational flexibility of the beta-strand and the hydrogen-bonding properties of its two edges: The number of hydrogen-bond donors and acceptors increases at one edge, whereas at the other, they are sterically hindered. In simulations in chloroform, the Hao-containing peptide 9 (i-PrCO-Phe-Hao-Val-NHBu) forms a beta-sheet-like hydrogen-bonded dimer, in good agreement with the available experimental data. Addition of methanol to the solution induces instability of this beta-sheet, as confirmed by the experiments. MD simulations also reproduce the folding of the synthetic peptide 1a (i-PrCO-Hao-Ut-Phe-Ile-Leu-NHMe) into a beta-hairpin-like structure in chloroform. Finally, the Hao-containing peptide, Ac-Ala-Hao-Ala-NHMe, is shown to form a stable complex with the Ras analogue, Rap1 A, in water at room temperature. Together with the available experimental data, these simulation studies indicate that Hao-containing peptides may serve as inhibitors of beta-sheet interactions between proteins.     

Proteins involved in binding and cellular uptake of nucleic acids

The study of mechanisms of nucleic acid transport across the cell membrane is valuable both for understanding the biological function of extracellular nucleic acids and the practical use of nucleic acids in gene therapy. It has been clearly demonstrated that cell surface proteins are necessary for transport of nucleic acids into cells. A large amount of data has now been accumulated about the proteins that participate in nucleic acid transport. The methods for revealing and identification of these proteins, possible mechanisms of protein-mediated transport of nucleic acids, and cellular functions of these proteins are described.     

The binding of phosphatidylcholine to the phosphatidylcholine transfer protein: affinity and role in folding

Bovine liver phosphatidylcholine transfer protein (PC-TP) has been expressed in Escherichia coli and purified to homogeneity from the cytosol fraction at a yield of 0.45 mg PC-TP per 10 mg total cytosolic protein. In addition, active PC-TP was obtained from inclusion bodies. An essential factor in the activation of PC-TP was phosphatidylcholine (PC) present in the folding buffer. PC-TP from the cytosol contains phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) with a preference for the di-monounsaturated species over the saturated species as determined by fast atom bombardment mass spectrometry (FAB-MS). By incubation with microsomal membranes the endogenous PE and PG were replaced by PC. Relative to the microsomal PC species composition, PC-TP bound preferentially C16:0/C20:4-PC and C16:0/C18:2-PC (twofold enriched) whereas the major microsomal species C18:0/C18:1-PC and C18:0/C18:2-PC were distinctly less bound. PC-TP is structurally homologous to the lipid-binding domain of the steroidogenic acute regulatory protein (Nat. Struct. Biol. 7 (2000) 408). Replacement of Lys(55) present in one of the beta-strands forming the lipid-binding site, with an isoleucine residue yielded an inactive protein. This suggests that Lys(55) be involved in the binding of the PC molecule.     

Conformational and dynamics changes induced by bile acids binding to chicken liver bile acid binding protein

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family.     

Turn scanning by site-directed mutagenesis: application to the protein folding problem using the intestinal fatty acid binding protein

We have systematically mutated residues located in turns between beta-strands of the intestinal fatty acid binding protein (IFABP), and a glycine in a half turn, to valine and have examined the stability, refolding rate constants and ligand dissociation constants for each mutant protein. IFABP is an almost all beta-sheet protein exhibiting a topology comprised of two five-stranded sheets surrounding a large cavity into which the fatty acid ligand binds. A glycine residue is located in seven of the eight turns between the antiparallel beta-strands and another in a half turn of a strand connecting the front and back sheets. Mutations in any of the three turns connecting the last four C-terminal strands slow the folding and decrease stability with the mutation between the last two strands slowing folding dramatically. These data suggest that interactions between the last four C-terminal strands are highly cooperative, perhaps triggered by an initial hydrophobic collapse. We suggest that this trigger is collapse of the highly hydrophobic cluster of amino acids in the D and E strands, a region previously shown to also affect the last stage of the folding process (Kim et al., 1997). Changing the glycine in the strand between the front and back sheets also results in a unstable, slow folding protein perhaps disrupting the D-E strand interactions. For most of the other turn mutations there was no apparent correlation between stability and refolding rate constants. In some turns, the interaction between strands, rather than the turn type, appears to be critical for folding while in others, turn formation itself appears to be a rate limiting step. Although there is no simple correlation between turn formation and folding kinetics, we propose that turn scanning by mutagenesis will be a useful tool for issues related to protein folding.     

Synthesis, conformational analysis and CB1 binding affinity of hairpin-like anandamide pseudopeptide mimetics.

We have designed, synthesized and evaluated the CB(1) binding affinity of a number of new conformationally restricted lipopeptides (1-17). All of them present some of the AEA key structural elements incorporated in a hairpinlike peptide framework. Among them, compounds 1-3 and 8 showed CB(1) affinities in competitive binding assays with K(i) values in the micromolar range (K(i) of AEA = 0.8 microM in the same assay). The remaining pseudopeptides showed little binding to the CB(1) receptor (with K(i) values >or= 50 microM). Conformational analysis on two representative compounds, performed by a combination of NMR studies, restrained molecular dynamics and QM calculations, allowed us to shed light on the structure-activity relationships (SAR), pointing to a correlation between the predominance of the hairpin-like structural motif and the CB(1) binding affinity. In a more general context, the present study may also prove useful in gaining additional insight into the biological relevance of the various AEA conformations.