Stabilization centers and protein stability

The well-balanced stability of protein structures allows large-scale fluctuations, which are indispensable in many biochemical functions, ensures the long-term persistence of the equilibrium structure and it regulates the degradation of proteins to provide amino acids for biosynthesis. This balance is studied in the present work with two sets of proteins by analyzing stabilization centers, defined as certain clusters of residues involved in cooperative long-range interactions. One data set contains 56 proteins, which belong to 16 families of homologous proteins, derived from organisms of various physiological temperatures. The other set is composed of 31 major histocompatibility complex (MHC)–peptide complexes, which represent peptide transporters complexed with peptide ligands that apparently contribute to the stabilization of the MHC proteins themselves. We show here that stabilization centers, which had been identified as special clusters of residues that protect the protein structure, evolved to serve also as regulators of function – related degradation of useless protein as part of protein housekeeping. Received: 25 August 2000 / Accepted: 6 September 2000 / Published online: 21 December 2000     

A mini-review on the enzyme-mediated manipulation of proteins/peptides

A number of enzymes are available in the toolbox facilitating the site-selective labeling, ligation, cyclization of proteins or peptides. In this review, some of the most important enzymes were discussed.     

Display Selection of a Hybrid Foldamer–Peptide Macrocycle

Expanding the chemical diversity of peptide macrocycle libraries for display selection is desirable to improve their potential to bind biomolecular targets. We now have implemented a considerable expansion through a large aromatic helical foldamer inclusion. A foldamer was first identified that undergoes flexizyme-mediated tRNA acylation and that is capable of initiating ribosomal translation with yields sufficiently high to perform an mRNA display selection of macrocyclic foldamer–peptide hybrids. A hybrid macrocyclic nanomolar binder to the C-lobe of the E6AP HECT domain was selected that showed a highly converged peptide sequence. A crystal structure and molecular dynamics simulations revealed that both the peptide and foldamer are helical in an intriguing reciprocal stapling fashion. The strong residue convergence could be rationalized based on their involvement in specific interactions with the target protein. The foldamer stabilizes the peptide helix through stapling and through contacts with key residues. These results altogether represent a significant extension of the chemical space amenable to display selection and highlight possible benefits of inserting an aromatic foldamer into a peptide macrocycle for the purpose of protein recognition.     

Protein Mimicry: A New Dimension for Peptides as Lead Compounds?

A short peptide as mimic for the hemopoietic growth factor erythropoietin (containing 165 amino acids) could be identified with the aid of peptide libraries on phage surfaces (phage display). The crystal structure of a peptide dimer complexed with two erythropoietin receptors (shown on the right) provides an insight into the molecular basis of this protein mimicry.     

确定蛋白质-短肽复合物结构的新方法

大部分蛋白质 -蛋白质复合物的三维结构在接触表面都显示出很好的几何匹配 .由于蛋白质的表面几何形状和其它的一些物理化学性质在分子的专一性相互作用中起了主要作用 ,所以 ,接触表面几何形状的互补常常被认为是蛋白质分子识别的基础 .一般来说 ,蛋白质接触表面的几何匹配只涉及 5到 1 0几个紧密堆积的氨基酸残基 ,因此 ,蛋白质与蛋白质配体之间的识别计算可以通过蛋白质与突变周围的或与蛋白质表面紧密接触的配体肽段的识别计算来实现 . Stoddard等 [1] 已经利用从 MBP上选取的八肽成功地计算出接近晶体结构的 MBP-受体复合物 .许多研…     

用顺序注射系统控制微流控芯片中的Edman降解

用顺序注射系统控制微流控芯片中的Edman降解反应, 提高了Edman降解的自动化程度, 得到蛋白质或多肽N-端氨基酸残基结构的准确信息. 对固体吸附材料的选择、顺序注射程序的设计和优化及影响Edman降解反应的因素进行了讨论. 该控制技术在蛋白质组学的研究中有一定的应用前景.     

Recent advances in enzyme-mediated peptide ligation

Artificial synthesis and site-specific modification of peptides and proteins have evolved into an indispensable tool for protein engineers and chemical biologists. Chemical and enzymatic approaches to peptide ligation are important alternatives of recombinant DNA technology for protein synthesis and modification. In the past decades, several natural peptide ligases have been discovered. Additionally, protein engineering for improving the ligation efficiencies of the natural peptide ligase and reversing the functionality of protease have provided more powerful peptide ligases. Herein, we briefly summarized the advances of enzyme-mediated peptide ligation and their application in protein synthesis and modification.     

多肽纳米管

环状多肽是构成多肽纳米管的主要子结构,它的结构形式和骨架分子构象直接影响多肽纳米管的特性。作为有特殊电子和光学性质的多肽纳米管,它在化学、生物、材料和医学等方面有潜在的应用。本文就自组装多肽纳米管的结构和应用作了介绍。     

Chemical Synthesis of Proteins with Base-Labile Posttranslational Modifications Enabled by a Boc-SPPS Based General Strategy Towards Peptide C-Terminal Salicylaldehyde Esters

Chemical synthesis of proteins bearing base-labile post-translational modifications (PTMs) is a challenging task. For instance, O-acetylation and S-palmitoylation PTMs cannot survive Fmoc removal conditions during Fmoc-solid phase peptide synthesis (SPPS). In this work, we developed a new Boc-SPPS-based strategy for the synthesis of peptide C-terminal salicylaldehyde (SAL) esters, which are the key reaction partner in Ser/Thr ligation and Cys/Pen ligation. The strategy utilized the semicarbazone-modified aminomethyl (AM) resin, which could support the Boc-SPPS and release the peptide SAL ester upon treatment with TFA/H₂O and pyruvic acid. The non-oxidative aldehyde regeneration was fully compatible with all the canonical amino acids. Armed with this strategy, we finished the syntheses of the O-acetylated protein histone H3(S10ac, T22ac) and the hydrophobic S-palmitoylated peptide derived from caveolin-1.     

Determination of Protein Concentration from CD Spectrum

Abstract

Protein concentration can be determined from its circular dichroism(CD) spectrum and from programs that calculate protein secondary structure content from CD. The method established was successfully applied to lysozyme and a de novo designed peptide with a standard error of about 5%, which is comparable to concentrations determined from an estimated extinction coefficient. This procedure is widely applicable in protein and peptide samples.     

Peptide affinity chromatography media that bind N fusion proteins under chaotropic conditions

To design a generic purification platform and to combine the advantages of fusion protein technology and matrix-assisted refolding, a peptide affinity medium was developed that binds inclusion body-derived N^pro fusion proteins under chaotropic conditions. Proteins were expressed in Escherichia coli using an expression system comprising the autoprotease N^pro from Pestivirus, or its engineered mutant called EDDIE, with C-terminally linked target proteins. Upon refolding, the autoprotease became active and cleaved off its fusion partner, forming an authentic N-terminus. Peptide ligands binding to the autoprotease at 4 M urea were screened from a combinatorial peptide library. A group of positive peptides were identified and further refined by mutational analysis. The best binders represent a common motif comprising positively charged and aromatic amino acids, which can be distributed in a random disposition. Mutational analysis showed that exchange of a single amino acid within the peptide ligand caused a total loss of binding activity. Functional affinity media comprising hexa- or octapeptides were synthesized using a 15-atom spacer with terminal sulfhydryl function and site-directed immobilization of peptides derivatized with iodoacetic anhydride. The peptide size was further reduced to dipeptides comprising only one positively charged and one aromatic amino acid. Based on this, affinity media were prepared by immobilization of a poly amino acid comprising lysine or arginine, and tryptophan, phenylalanine, or tyrosine, respectively, in certain ratios. Binding capacities were in the range of 7–15 mg protein mL⁻¹ of medium, as could be shown for several EDDIE fusion proteins. An efficient protocol for autoproteolytic cleavage using an on-column refolding method was implemented.     

Facile synthesis of C-terminal peptide hydrazide and thioester of NY-ESO-1 (A39-A68) from an Fmoc-hydrazine 2-chlorotrityl chloride resin

The NY-ESO-1 (A39-A68) peptide hydrazide was prepared through 9-fluorenyl-methoxycarbonyl solid-phase peptide synthesis (Fmoc SPPS) from a new 9-fluorenyl-methoxycarbonyl hydrazine 2-chlorotrityl chloride (Fmoc-hydrazine 2CTC) resin. The new resin was ideal for long-term storage and usage in Fmoc SPPS. Besides, the title peptide hydrazide could be transformed nearly quantitatively into the corresponding peptide thioester, which was both isolable and usable directly in native chemical ligation (NCL).     

Branched Multimeric Peptides as Affinity Reagents for the Detection of α-Klotho Protein

α-Klotho, an aging-related protein found in the kidney, parathyroid gland, and choroid plexus, acts as an essential co-receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. Decreased levels of α-Klotho are a hallmark of age-associated diseases. Detecting or labeling α-Klotho in biological milieu has long been a challenge, however, hampering the understanding of its role. Here, we developed branched peptides by single-shot parallel automated fast-flow synthesis that recognize α-Klotho with improved affinity relative to their monomeric versions. These peptides were further shown to selectively label Klotho for live imaging in kidney cells. Our results demonstrate that automated flow technology enables rapid synthesis of complex peptide architectures, showing promise for future detection of α-Klotho in physiological settings.     

Assembly of Alzheimer's Aβ peptide into nanostructured amyloid fibrils

The self-assembly of β-amyloid (Aβ) peptide into highly ordered amyloid fibril structures represents one of the pathological hallmarks of Alzheimer's disease. This process leads to the transient stabilization of ordered or disordered intermediates, which are thought to act as the main pathogenic culprits in neurodegenerative amyloid disease. This review describes recent results from different biophysical techniques, ranging from structure determination to single-particle methods by which the outgrowth of individual fibrils can be followed, and it explains their contributions towards understanding the mechanism of assembly. Finally, we will outline emerging methods and molecules to specifically interfere with the assembly and pathogenic impact of Aβ peptide.     

Biomolecular Click Reactions Using a Minimal pH-Activated Catcher/Tag Pair for Producing Native-Sized Spider-Silk Proteins**

A type of protein/peptide pair known as Catcher/Tag pair spontaneously forms an intermolecular isopeptide bond which can be applied for biomolecular click reactions. Covalent protein conjugation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines, but it is essential to increase the current toolbox of orthogonal Catcher/Tag pairs to expand the range of applications further, for example, for controlled multiple-fragment ligation. We report here the engineering of novel Catcher/Tag pairs for protein ligation, aided by a crystal structure of a minimal CnaB domain from Lactobacillus plantarum. We show that a newly engineered pair, called SilkCatcher/Tag enables efficient pH-inducible protein ligation in addition to being compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the SilkCatcher/Tag pair in the production of native-sized highly repetitive spider-silk-like proteins with >90 % purity, which is not possible by traditional recombinant production methods.     

Phosphopeptide enrichment by IEF

In our efforts to improve the identification of phosphopeptides by MS we have used peptide IEF on IPG strips. Phosphopeptides derived from trypsin digests of single proteins as well as complex cellular protein mixtures can be enriched by IEF and recovered in excellent yields at the acidic end of an IPG strip. IPG peptide fractionation in combination with MS/MS analysis has allowed us to identify phosphopeptides from tryptic digests of a cellular protein extract.     

Investigating the interactions of the first 17 amino acid residues of Huntingtin with lipid vesicles using mass spectrometry and molecular dynamics

The first 17 amino acid residues of Huntingtin protein (Nt17 of htt) are thought to play an important role in the protein's function; Nt17 is one of two membrane binding domains in htt. In this study the binding ability of Nt17 peptide with vesicles comprised of two subclasses of phospholipids is studied using electrospray ionization ‐ mass spectrometry (ESI‐MS) and molecular dynamics (MD) simulations. Overall, the peptide is shown to have a greater propensity to interact with vesicles of phosphatidylcholine (PC) rather than phosphatidylethanolamine (PE) lipids. Mass spectra show an increase in lipid‐bound peptide adducts where the ordering of the number of such specie is 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) > 1‐palmitoyl‐2‐oleoyl‐glycero‐3‐phosphocholine (POPC) > 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3 phosphoethanolamine (POPE). MD simulations suggest that the compactness of the bilayer plays a role in governing peptide interactions. The peptide shows greater disruption of the DOPC bilayer order at the surface and interacts with the hydrophobic tails of lipid molecules via hydrophobic residues. Conversely, the POPE vesicle remains ordered and lipids display transient interactions with the peptide through the formation of hydrogen bonds with hydrophilic residues. The POPC system displays intermediate behavior with regard to the degree of peptide‐membrane interaction. Finally, the simulations suggest a helix stabilizing effect resulting from the interactions between hydrophobic residues and the lipid tails of the DOPC bilayer.     

Lysine‐directed staining of proteins for MS‐based analyses

Visualization of proteins and MS‐based analyses are elemental tasks in modern biochemistry. Nevertheless, reports about covalent protein dyes and their suitability for subsequent MS experiments remain scarce. In a recent work, we demonstrated that covalent prestaining of proteins with Uniblue A drastically speeds up proteomic workflows. The present study introduces dabsyl chloride as another truly MS‐compatible protein stain. Remarkably, although Uniblue A and dabsyl chloride employ different nucleophilic reaction mechanisms, both are highly specific for lysine residues. The predictable peptide modifications allow easy integration into state‐of‐the‐art bioinformatic workflows. Further, lysine‐directed derivatizations with hydrophobic reagents such as dabsyl chloride complement the cysteine‐directed ALiPHAT strategy for increasing the sensitivity of peptide identifications.     

The 3′-End of tRNA and Its Role in Protein Biosynthesis

In the course of protein biosynthesis, the 3′-ends of aminoacyl-tRNA (aa-tRNA) and peptidyl-tRNA specifically interact with macromolecules of the protein biosynthesis machinery. The 3′-end of tRNA consists of an invariant C-C-A single strand. Interaction of the aminoacyl-tRNA 3′-end with elongation factor Tu (EF-Tu) containing bound GTP is necessary for the formation of the aa-tRNA·EF-Tu·GTP complex and, after the complex binds to the ribosome, for the GTP hydrolysis. This process is followed by the specific binding of the aminoacyl-tRNA 3′-end to the aminoacyl (A) site of the ribosome. In this review, a model is proposed that involves Watson-Crick base pairing of the C? C sequence of the aminoacyl-tRNA 3′-end with a specific G? G sequence of the ribosomal 23S RNA. Similarly, peptidyl-tRNA binds with its 3′-end to the peptidyl (P) site of the ribosome. This binding may also involve Watson-Crick base pairing of the C-C-A sequence with a complementary sequence of 23S RNA. It is proposed that peptide bond formation is catalyzed by a functional site of the 23S RNA located near the 3′-ends of aminoacyl-tRNA and peptidyl-tRNA. A model is suggested in which two loops of the 23S RNA, brought into close proximity via folding, are involved both in binding the 3′-ends of the tRNAs and in catalyzing peptide bond formation. This model presumes a dynamic structure for ribosomal RNA, which is modulated by interaction with elongation factors and ribosomal proteins.