Functional Residues In Proteins Research Articles

Enzymatic Protein Immobilization for Nanobody Array.

Antibody arrays play a pivotal role in the detection and quantification of biomolecules, with their effectiveness largely dependent on efficient protein immobilization. Traditional methods often use heterobifunctional cross-linking reagents for attaching functional residues in proteins to corresponding chemical groups on the substrate surface. However, this method does not control the antibody's anchoring point and orientation, potentially leading to reduced binding efficiency and overall performance. Another method using anti-antibodies as intermediate molecules to control the orientation can be used but it demonstrates lower efficiency. Here, we demonstrate a site-specific protein immobilization strategy utilizing OaAEP1 (asparaginyl endopeptidase) for building a nanobody array. Moreover, we used a nanobody-targeting enhanced green fluorescent protein (eGFP) as the model system to validate the protein immobilization method for building a nanobody array. Finally, by rapidly enriching eGFP, this method further highlights its potential for rapid diagnostic applications. This approach, characterized by its simplicity, high efficiency, and specificity, offers an advancement in the development of surface-modified protein arrays. It promises to enhance the sensitivity and accuracy of biomolecule detection, paving the way for broader applications in various research and diagnostic fields.

Functional Sites Induce Long-Range Evolutionary Constraints in Enzymes.

Functional residues in proteins tend to be highly conserved over evolutionary time. However, to what extent functional sites impose evolutionary constraints on nearby or even more distant residues is not known. Here, we report pervasive conservation gradients toward catalytic residues in a dataset of 524 distinct enzymes: evolutionary conservation decreases approximately linearly with increasing distance to the nearest catalytic residue in the protein structure. This trend encompasses, on average, 80% of the residues in any enzyme, and it is independent of known structural constraints on protein evolution such as residue packing or solvent accessibility. Further, the trend exists in both monomeric and multimeric enzymes and irrespective of enzyme size and/or location of the active site in the enzyme structure. By contrast, sites in protein–protein interfaces, unlike catalytic residues, are only weakly conserved and induce only minor rate gradients. In aggregate, these observations show that functional sites, and in particular catalytic residues, induce long-range evolutionary constraints in enzymes.

Weighted protein residue networks based on joint recurrences between residues.

BackgroundWeighted and un-weighted protein residue networks can predict key functional residues in proteins based on the closeness centrality C and betweenness centrality B values for each residue. A static snapshot of the protein structure, and a cutoff distance, are used to define edges between the network nodes. In this work we apply the weighted network approach to study the β-Lactamase Inhibitory Protein (BLIP). Joint recurrences extracted from molecular dynamics MD trajectory positions of the protein residue carbon alpha atoms are used to define edge weights between nodes, and no cutoff distance is used. The results for B and C from our approach are compared with those extracted from an un-weighted network, and a weighted network that uses interatomic contacts to define edge weights between nodes, respectively.ResultsThe joint recurrence weighted network approach performs well in pointing out key protein residues. Furthermore, it seems to emphasize residues with medium to high relative solvent accessibility that lie in loop regions between secondary structure elements of the protein.ConclusionsProtein residue networks that use joint recurrences extracted from molecular dynamics simulations of a solvated protein perform well in pointing to hotspot residues and hotspot clusters. This approach uses no distance cutoff threshold, and does not exclude any interactions between the residues, including water-mediated interactions.

Chemical Proteomics with Sulfonyl Fluoride Probes Reveals Selective Labeling of Functional Tyrosines in Glutathione Transferases

Chemical probes have great potential for identifying functional residues in proteins in crude proteomes. Here we studied labeling sites of chemical probes based on sulfonyl fluorides (SFs) on plant and animal proteomes. Besides serine proteases and many other proteins, SF-based probes label Tyr residues in glutathione transferases (GSTs). The labeled GSTs represent four different GST classes that share less than 30% sequence identity. The targeted Tyr residues are located at similar positions in the promiscuous substrate binding site and are essential for GST function. The high selectivity of SF-based probes for functional Tyr residues in GSTs illustrates how these probes can be used for functional studies of GSTs and other proteins in crude proteomes.

Functional Residues in Proteins

We are delighted to present a special issue on the subject of functional residues of proteins. Finding focused articles within the time frame of a special issue is always challenging, which is further compounded by authors' reluctance in making their contributions to new journals. We have been fortunate enough that some leading researchers in the field agreed to support this special issue and contributed their work at our request. We present here nine articles on various aspects of the subject. We received some more submissions, which could not be accommodated because of a strict quality control, we tried to maintain. Finally compiled special issue starts with a very interesting commentary on posttranslated modifications leading to normal and disease-associated biological functions, contributed by T. M. Karve and A. K. Cheema. This is followed by the analysis of a specific biological system of ATP synthetase, contributed by Z. Ahmad et al., exploring its catalytic sites and focusing on charged residues. R. Prasad et al. then describe a related system of ABC transporters and review the state of the knowledge on its functional residues. Functional residues in a protein do not always follow a universal pattern, and this is demonstrated by R. Kumar et al. in describing the dynamic structure of estrogen receptors. This paper is then followed by a special group of functional residues, that is, cysteins in sulfurtransferases by N. Nagahara. Viola et al. present an excellent study of catalytic machinery in the biosynthesis of amino acids. Subsequntly, K. M. Fukasawa et al. then present their analysis of functionally relevant zinc-binding sites in metalloproteases. Subsequently, M. S. Khan et al. provide an elaborate discussion on the mechanism of serpin inhibition, specially focusing on the role of polymerization. Finally, M. A. Kabir et al. present an investigation of a eukaryotic chaperon, involved in protein-folding. Thus, the special issue covers a wide range of systems and describes an overview of some of the most significant studies on the subject of finding functional residues in proteins. We would like to express our most sincere gratitude to the authors who agreed to contribute and referees who provided useful feedback, allowing the papers to meet the high standards of publication. We hope that the readers of Journal of Amino Acids will find these articles of great interest and look forward to any comments to improve our future efforts. Shandar Ahmad Jung-Ying Wang Zulfiqar Ahmad Faizan Ahmad

High‐performance prediction of functional residues in proteins with machine learning and computed input features

One of the major challenges in genomics is to understand the function of gene products from their 3D structures. Computational methods are needed for the high-throughput prediction of the function of proteins from their 3D structure. Methods that identify active sites are important for understanding and annotating the function of proteins. Traditional methods exploiting either sequence similarity or structural similarity can be unreliable and cannot be applied to proteins with novel folds or low homology with other proteins. Here, we present a machine-learning application that combines computed electrostatic, evolutionary, and pocket geometric information for high-performance prediction of catalytic residues. Input features consist of our structure-based theoretical microscopic anomalous titration curve shapes (THEMATICS) electrostatics data, enhanced with sequence-based phylogenetic information from INTREPID and topological pocket information from ConCavity. Our THEMATICS-based input features are augmented with an additional metric, the theoretical buffer range. With the integration of the three different types of input, each of which performs admirably on its own, significantly better performance is achieved than that of any of these methods by itself. This combined method achieves 86.7%, 92.5%, and 93.8% recall of annotated functional residues at 5, 8, and 10% false-positive rates, respectively.

The use of electrospray mass spectrometry to identify an essential arginine residue in type II dehydroquinases

The arginine-specific reagent phenylglyoxal has been used to identify a hyper-reactive arginine residue which is essential for activity in the type II dehydroquinases of Streptomyces coelicolor and Aspergillus nidulans. Electrospray mass spectrometry was used both to characterise the phenylglyoxal modified protein, and to identify the phenylglyoxal modified peptides following enzymatic digestion. The advantages of using electrospray mass spectrometry for monitoring arginine modication aimed at identifying functional residues in proteins are discussed.

Identification of functional residues in proteins by charged-to-alanine scanning mutagenesis

“Charged-to-alanine” scanning mutagenesis, in which the charged amino acids in a protein (Asp, Glu, Arg, Lys, and His) are systematically substituted with alanine, is a rational mutagenesis strategy for identifying the functionally important regions of proteins. The targeting of the charged amino acids and their replacement with alanine are designed to minimize disruptions to the tertiary structure of the protein. Thus, the mutant proteins are more likely to be successfully expressed for analysis, and the probability of incorrectly assigning a functional role to a specific residue is reduced. The functionally important polar and neutral residues can be identified by the subsequent substitution of the residues flanking the functional charged residues identified. In this paper we describe the rationale behind the approach, the design of mutagenic oligonucleotides, and a detailed method for high-efficiency site-directed mutagenesis. The conclusions drawn from the analysis of two proteins by means of this technique are discussed in terms of the crystal structures of these proteins, which were solved after the completion of the mutagenesis analysis.