Protecting Triazabutadienes To Afford Acid Resistance

Recent work on triazabutadienes has shown that they have the ability to release aryl diazonium ions under exceptionally mild acidic conditions. There are instances that require that this release be prevented or minimized. Accordingly, a base‐labile protection strategy for the triazabutadiene is presented. It affords enhanced synthetic and practical utility of the triazabutadiene. The effects of steric and electronic factors in the rate of removal are discussed, and the triazabutadiene protection is shown to be compatible with the traditional acid‐labile protection strategy used in solid phase peptide synthesis.     

Site-Selective Aryl Diazonium Installation onto Protein Surfaces at Neutral pH using a Maleimide-Functionalized Triazabutadiene

Aryl diazonium cations are versatile bioconjugation reagents due to their reactivity towards electron-rich aryl residues and secondary amines, but historically their usage has been hampered by both their short lifespan in aqueous solution and the harsh conditions required to generate them in situ. Triazabutadienes address many of these issues as they are stable enough to endure multiple-step chemical syntheses and can persist for several hours in aqueous solution, yet upon UV-exposure rapidly release aryl diazonium cations under biologically-relevant conditions. This paper describes the synthesis of a novel maleimide-functionalized triazabutadiene suitable for site-selectively installing aryl diazonium cations into proteins at neutral pH; we show reaction with this molecule and a surface-cysteine of a thiol disulfide oxidoreductase. Through photoactivation of the site-selectively installed triazabutadiene motifs, we generate aryl diazonium functionality, which we further derivatize via azo-bond formation to electron-rich aryl species, showcasing the potential utility of this strategy for the generation of photoswitches or protein-drug conjugates.     

1,2,3-Triazabutadiene. VIII [1]. Untersuchungen zum Mechanismus der sensibilisierten Fotoisomerisierung von 1-Aryl-3-[3-methyl-benzthiazolinyliden-(2)]-triazenen

1,2,3-Triazabutadienes. VIII. Investigations of the Mechanism of the Sensitized Photoisomerization of 1-Aryl-3-[3-methyl-benzthiazolinyliden-(2)]-triazenes. It is possible to sensitize the photochemical cis-trans-isomerization of triazabutadienes by sensitizers with tripletenergies ranging from 40 to 80 kcal/mol. It was proved by flash photolysis that triazabutadienes quench the triplet states of sensitizers. The dependence of the photostationary state on the triplet energy of the sensitizer as well as the dependence of the quantum yields of the sensitized photoisomerization on the concentration of triazabutadiene were investigated, and the desactivation ratios of the sensitized photoisomerization were determinated. The found correspondence of desactivation ratios of the direct and sensitized photoisomerization make a triplet mechanism in both cases probable.     

1,2,3-Triazabutadiene. XIII. UV-vis-spektroskopische Untersuchungen zur Protonierung Z-E-isomerer 1-Aryl-3-[3-methylbenzthiazolinyliden-(2)]- und 1-Aryl-3-[1,3-dimethylbenzimidazolinyliden-(2)]-triazene

1,2,3-Triazabutadienes. XIII. U.V.-vis-Spectroscopic Investigations of the Protonation of Z-E-Isomeric 1-Aryl-3-[3-methylbenzthiazolinyliden-(2)]- and 1-Aryl-3-[1,3-dimethylbenzimidazolinyliden-(2)]-triazenes The u.v.-vis absorption spectra of Z-E-isomeric 1-aryl-3-[3-methylbenzthiazolinyliden-(2)]- and 1-aryl-3-[1,3-dimethyl-benzimidazolinyliden-(2)]-triazenes 1–3 resp. 4 show a pronounced halochromic effect in the presence of acids. The reason is the protonation of the N¹-atom of the triazabutadiene chain whereby the energetically favourable structure of a diazatrimethine is formed. The magnitude of the halochromic effect depends on the substituents in the aryl and benzo residue and on the Z-E-structure of the compounds.     

Computational Insight on the Interaction of Common Blood Proteins with Gold Nanoparticles

Protein interactions with engineered gold nanoparticles (AuNPs) and the consequent formation of the protein corona are very relevant and poorly understood biological phenomena. The nanoparticle coverage affects protein binding modalities, and the adsorbed protein sites influence interactions with other macromolecules and cells. Here, we studied four common blood proteins, i.e., hemoglobin, serum albumin, α1-antiproteinase, and complement C3, interacting with AuNPs covered by hydrophobic 11-mercapto-1-undecanesulfonate (MUS). We use Molecular Dynamics and the Martini coarse−grained model to gain quantitative insight into the kinetics of the interaction, the physico-chemical characteristics of the binding site, and the nanoparticle adsorption capacity. Results show that proteins bind to MUS−capped AuNPs through strong hydrophobic interactions and that they adapt to the AuNP surfaces to maximize the contact surface, but no dramatic change in the secondary structure of the proteins is observed. We suggest a new method to calculate the maximum adsorption capacity of capped AuNPs based on the effective surface covered by each protein, which better represents the realistic behavior of these systems.     

1,2,3-Triazabutadiene. V. Untersuchungen zum Mechanismus der thermischen cis-trans-Isomerisierung von substituierten 1-Aryl-3-[methylbenzthiazolinyliden-(2)]-triazenen

1,2,3-Triazabutadienes. V. Investigations of the Mechanism of the Thermal cis-trans Isomerisation of Substituted 1-aryl-3-[methylbenzthiazolinyliden-(2)]-triazenes The velocity of the thermal cis-trans-isomerisation of substituted 1-aryl-3-[methyl-benzthiazolinyliden-(2)]-triazenes was determined, varying the substituents in the aryl resp. benzo residue, the temperature and the solvent. Electron- withdrawing substituents in ortho- resp. para-position of the aryl residue and electron-releasing substituents in position 6 of the benzo residue increase the velocity of isomerisation as it do polar solvents and acid catalyst. In the aryl residue electron-releasing substituted and in meta-position electron-withdrawing substituted derivatives do not thermically isomerise in solution under the given conditions. From these results a polar rotation mechanism of the isomerisation around the NN-double bound of the triazabutadiene chain is concluded.     

The Role of F-Box Proteins during Viral Infection

The F-box domain is a protein structural motif of about 50 amino acids that mediates protein–protein interactions. The F-box protein is one of the four components of the SCF (SKp1, Cullin, F-box protein) complex, which mediates ubiquitination of proteins targeted for degradation by the proteasome, playing an essential role in many cellular processes. Several discoveries have been made on the use of the ubiquitin–proteasome system by viruses of several families to complete their infection cycle. On the other hand, F-box proteins can be used in the defense response by the host. This review describes the role of F-box proteins and the use of the ubiquitin–proteasome system in virus–host interactions.     

Main-chain complementarity in protein-protein recognition

We present a statistical analysis of protein structures basedon interatomic C_a distances. The overall distance distributionsreflect in detail the contents of sequence-specific substructuresmaintained by local interactions (such as -helixes) and longerrange interactions (such as disulfide bridges and ß-sheets).We also show that a volume scaling of the distances makes distancedistributions for protein chains of different length superimposable.Distance distributions were also calculated specifically foramino acids separated by a given number of residues. Specificfeatures in these distributions are visible for sequence separationsof up to 20 amino acid residues. A simple representation, whichpreserves most of the information in the distance distributions,was obtained using six parameters only. The parameters giverise to canonical distance intervals and when predicting coarse-graineddistance constraints by methods such as data-driven artificialneural networks, these should preferably be selected from theseintervals. We discuss the use of the six parameters for determiningor reconstructing 3-D protein structures.     

Distance distributions in proteins: a six-parameter representation

We present a statistical analysis of protein structures basedon interatomic C_a distances. The overall distance distributionsreflect in detail the contents of sequence-specific substructuresmaintained by local interactions (such as -helixes) and longerrange interactions (such as disulfide bridges and ß-sheets).We also show that a volume scaling of the distances makes distancedistributions for protein chains of different length superimposable.Distance distributions were also calculated specifically foramino acids separated by a given number of residues. Specificfeatures in these distributions are visible for sequence separationsof up to 20 amino acid residues. A simple representation, whichpreserves most of the information in the distance distributions,was obtained using six parameters only. The parameters giverise to canonical distance intervals and when predicting coarse-graineddistance constraints by methods such as data-driven artificialneural networks, these should preferably be selected from theseintervals. We discuss the use of the six parameters for determiningor reconstructing 3-D protein structures.     

Covalent Protein Labeling by SpyTag–SpyCatcher in Fixed Cells for Super‐Resolution Microscopy

Labeling proteins with high specificity and efficiency is a fundamental prerequisite for microscopic visualization of subcellular protein structures and interactions. Although the comparatively small size of epitope tags makes them less perturbative to fusion proteins, they require the use of large antibodies that often limit probe accessibility and effective resolution. Here we use the covalent SpyTag–SpyCatcher system as an epitope‐like tag for fluorescent labeling of intracellular proteins in fixed cells for both conventional and super‐resolution microscopy. We also applied this method to endogenous proteins by gene editing, demonstrating its high labeling efficiency and capability for isoform‐specific labeling.     

Search for the most stable folds of protein chains: I. Application of a self-consistent molecular field theory to a problem of protein three-dimensional structure prediction

We present a general approach to the prediction of 3-D foldsof protein chains from their amino acid sequences. The approachis based on the use of the self-consistent molecular field theoryfor long-range interactions, the use of 1-D statistical mechanicsfor short-range interactions and on the discovery that thereis and should only be a relatively small discrete set of foldingpatterns. This makes it possible to examine the full varietyof ‘potentially stable’ folds and to determine thethermodynamically stable structure. In this paper, we give thegeneral theoretical background of the approach. The encouragingresults of the application of this approach to ß-domainsare described in another paper.     

Rapid and easy development of versatile tools to study protein/ligand interactions

The system described here allows the expression of protein fragments into a solvent-exposed loop of a carrier protein, the beta-lactamase BlaP. When using Escherichia coli constitutive expression vectors, a positive selection of antibioresistant bacteria expressing functional hybrid beta-lactamases is achieved in the presence of beta-lactams making further screening of correctly folded and secreted hybrid beta-lactamases easier. Protease-specific recognition sites have been engineered on both sides of the beta-lactamase permissive loop in order to cleave off the exogenous protein fragment from the carrier protein by an original two-step procedure. According to our data, this approach constitutes a suitable alternative for production of difficult to express protein domains. This work demonstrates that the use of BlaP as a carrier protein does not alter the biochemical activity and the native disulphide bridge formation of the inserted chitin binding domain of the human macrophage chitotriosidase. We also report that the beta-lactamase activity of the hybrid protein can be used to monitor interactions between the inserted protein fragments and its ligands and to screen neutralizing molecules.     

Determining the roles of different chain fragments in recognition of immunoglobulin fold

We examine sequence-to-structure specificity of ß-structuralfragments of immunoglobulin domains. The structure specificityof separate chain fragments is estimated by computing the Z-scorevalues in recognition of the native structure in gapless threadingtests. To improve the accuracy of our calculations we use energyaveraging over diverse homologs of immunoglobulin domains. Weshow that the interactions between residues of ß-structureare more determinant in recognition of the native structurethan the interactions within the whole chain molecule. Thisresult distinguishes immunoglobulins from more typical proteinswhere the interactions between residues of the whole chain normallyrecognize the native fold more accurately than interactionsbetween the residues of the secondary structure residues alone[Reva,B. and Topiol,S. (2000) Biocomputing: Proceedings of thePacific Symposium. World Scientific Publishing Co., pp. 168–178].We also find that the predominant contributions of the secondarystructure are produced by the four central ß-strandsthat form the core of the molecule. The results of this studyallow us through quantitative means to understand the architectureof immunoglobulin molecules. Comparing the fold recognitiondata for different chain fragments one can say that ß-strandsform a rigid frame for immunoglobulin molecules, whereas loops,with no structural role, can develop a broad variety of bindingspecificities. It is well known that protein function is determinedby specific portions of a protein chain. This study suggeststhat the whole protein structure can be predominantly determinedby a few fragments of chain which form the structural frameworkof the molecule. This idea may help in better understandingthe mechanisms of protein evolution: strengthening a proteinstructure in the key framework-forming regions allows mutationsand flexibility in other chain regions.     

Fibrosis Protein-Protein Interactions from Google Matrix Analysis of MetaCore Network

Protein–protein interactions is a longstanding challenge in cardiac remodeling processes and heart failure. Here, we use the MetaCore network and the Google matrix algorithms for prediction of protein–protein interactions dictating cardiac fibrosis, a primary cause of end-stage heart failure. The developed algorithms allow identification of interactions between key proteins and predict new actors orchestrating fibroblast activation linked to fibrosis in mouse and human tissues. These data hold great promise for uncovering new therapeutic targets to limit myocardial fibrosis.     

Exploring protein-protein interactions with phage display

Protein–protein interactions mediate essentially all biological processes. A detailed understanding of these interactions is thus a major goal of modern biological chemistry. In recent years, genome sequencing efforts have revealed tens of thousands of novel genes, but the benefits of genome sequences will only be realized if these data can be translated to the level of protein function. While genome databases offer tremendous opportunities to expand our knowledge of protein–protein interactions, they also present formidable challenges to traditional protein chemistry methods. Indeed, it has become apparent that efficient analysis of proteins on a proteome‐wide scale will require the use of rapid combinatorial approaches. In this regard, phage display is an established combinatorial technology that is likely to play an even greater role in the future of biology. This article reviews recent applications of phage display to the analysis of protein–protein interactions. With combinatorial mutagenesis strategies, it is now possible to rapidly map the binding energetics at protein–protein interfaces through statistical analysis of phage‐displayed protein libraries. In addition, naïve phage‐displayed peptide libraries can be used to obtain small peptide ligands to essentially any protein of interest, and in many cases, these binding peptides act as antagonists or even agonists of natural protein functions. These methods are accelerating the pace of research by enabling the study of complex protein–protein interactions with simple molecular biology methods. With further optimization and automation, it may soon be possible to study hundreds of different proteins in parallel with efforts comparable to those currently expended on the analysis of individual proteins.     

Stability and Cell Permeability of Sulfonyl Fluorides in the Design of Lys-Covalent Antagonists of Protein-Protein Interactions

Recently we reported on aryl-fluorosulfates as possible stable and effective electrophiles for the design of lysine covalent, cell permeable antagonists of protein–protein interactions (PPIs). Here we revisit the use of aryl-sulfonyl fluorides as Lys-targeting moieties, incorporating these electrophiles in XIAP (X-linked inhibitor of apoptosis protein) targeting agents. We evaluated stability in buffer and reactivity with Lys311 of XIAP of various aryl-sulfonyl fluorides using biochemical and biophysical approaches, including displacement assays, mass spectrometry, SDS gel electrophoresis, and denaturation thermal shift measurements. To assess whether these modified electrophilic “warheads” can also react with Tyr, we repeated these evaluations with a Lys311Tyr XIAP mutant. Using a direct cellular assay, we could demonstrate that selected agents are cell permeable and interact covalently with their intended target in cell. These results suggest that certain substituted aryl-sulfonyl fluorides can be useful Lys- or Tyr-targeting electrophiles for the design of covalent pharmacological tools or even future therapeutics targeting protein–protein interactions.     

A Hamiltonian Replica Exchange Molecular Dynamics (MD) Method for the Study of Folding,Based on the Analysis of the Stabilization Determinants of Proteins

Herein, we present a novel Hamiltonian replica exchange protocol for classical molecular dynamics simulations of protein folding/unfolding. The scheme starts from the analysis of the energy-networks responsible for the stabilization of the folded conformation, by means of the energy-decomposition approach. In this framework, the compact energetic map of the native state is generated by a preliminary short molecular dynamics (MD) simulation of the protein in explicit solvent. This map is simplified by means of an eigenvalue decomposition. The highest components of the eigenvector associated with the lowest eigenvalue indicate which sites, named “hot spots”, are likely to be responsible for the stability and correct folding of the protein. In the Hamiltonian replica exchange protocol, we use modified force-field parameters to treat the interparticle non-bonded potentials of the hot spots within the protein and between protein and solvent atoms, leaving unperturbed those relative to all other residues, as well as solvent-solvent interactions. We show that it is possible to reversibly simulate the folding/unfolding behavior of two test proteins, namely Villin HeadPiece HP35 (35 residues) and Protein A (62 residues), using a limited number of replicas. We next discuss possible implications for the study of folding mechanisms via all atom simulations.     

Visualizing the Adenylation Activities and Protein–Protein Interactions of Aryl Acid Adenylating Enzymes

Structural and activity studies have revealed the dynamic and transient actions of carrier protein (CP) activity in primary and secondary metabolic pathways. CP-mediated interactions play a central role in nonribosomal peptide biosynthesis, as they serve as covalent tethers for amino acid and aryl acid substrates and enable the growth of peptide intermediates. Strategies are therefore required to study protein–protein interactions efficiently. Herein, we describe activity-based probes used to demonstrate the protein–protein interactions between aryl CP (ArCP) and aryl acid adenylation (A) domains as well as the substrate specificities of the aryl acid A domains. If coupled with in-gel fluorescence imaging, this strategy allows visualization of the protein–protein interactions required to recognize and transfer the substrate to the partner ArCP. This technique has potential for the analysis of protein–protein interactions within these biosynthetic enzymes at the molecular level and for use in the combinatorial biosynthesis of new nonribosomal peptides.