Structural Characterization of the Hemophore HasAp fromPseudomonas aeruginosa: NMR Spectroscopy Reveals Protein−Protein Interactions between Holo-HasAp and Hemoglobin†,‡

Background: NMR spectroscopy is one of the most powerful tools to study the structure and interaction properties of peptides and proteins from a dynamic perspective. Knowing the bioactive conformations of peptides is crucial in the drug discovery field to design more efficient analogue ligands and inhibitors of protein-protein interactions targeting therapeutically relevant systems. Objective: This review provides a toolkit to investigate peptide conformational properties by NMR. Methods: Articles cited herein, related to NMR studies of peptides and proteins were mainly searched through Pubmed and the web. More recent and old books on NMR spectroscopy written by eminent scientists in the field were consulted as well. Results: The review is mainly focused on NMR tools to gain the 3D structure of small unlabeled peptides. It is more application-oriented as it is beyond its goal to deliver a profound theoretical background. However, the basic principles of 2D homonuclear and heteronuclear experiments are briefly described. Protocols to obtain isotopically labeled peptides and principal triple resonance experiments needed to study them, are discussed as well. Conclusion: NMR is a leading technique in the study of conformational preferences of small flexible peptides whose structure can be often only described by an ensemble of conformations. Although NMR studies of peptides can be easily and fast performed by canonical protocols established a few decades ago, more recently we have assisted to tremendous improvements of NMR spectroscopy to investigate instead large systems and overcome its molecular weight limit.

Download Full-text

Genetic and Molecular Characterization of the Caenorhabditis elegans Gene, mel-26, a Postmeiotic Negative Regulator of MEI-1, a Meiotic-Specific Spindle Component

Genetics ◽

10.1093/genetics/150.1.119 ◽

1998 ◽

Vol 150 (1) ◽

pp. 119-128

Author(s):

M Rhys Dow ◽

Paul E Mains

Keyword(s):

Caenorhabditis Elegans ◽

Protein Interactions ◽

Negative Regulator ◽

Meiotic Spindle ◽

Loss Of Function ◽

Protein Protein Interactions ◽

Gain Of Function ◽

Recessive Mutations ◽

Female Germline

Abstract We have previously described the gene mei-1, which encodes an essential component of the Caenorhabditis elegans meiotic spindle. When ectopically expressed after the completion of meiosis, mei-1 protein disrupts the function of the mitotic cleavage spindles. In this article, we describe the cloning and the further genetic characterization of mel-26, a postmeiotic negative regulator of mei-1. mel-26 was originally identified by a gain-of-function mutation. We have reverted this mutation to a loss-of-function allele, which has recessive phenotypes identical to the dominant defects of its gain-of-function parent. Both the dominant and recessive mutations of mel-26 result in mei-1 protein ectopically localized in mitotic spindles and centrosomes, leading to small and misoriented cleavage spindles. The loss-of-function mutation was used to clone mel-26 by transformation rescue. As suggested by genetic results indicating that mel-26 is required only maternally, mel-26 mRNA was expressed predominantly in the female germline. The gene encodes a protein that includes the BTB motif, which is thought to play a role in protein-protein interactions.

Download Full-text

Proteomic Characterization of Protein-Protein Interactions

Austin Biology ◽

10.26420/austinbiol.2021.1027 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Veenstra TD ◽

Keyword(s):

Protein Interactions ◽

Disease Diagnosis ◽

Cell System ◽

Protein Protein Interactions ◽

Novel Technologies ◽

Cell Functions ◽

Single Protein ◽

A Cell ◽

Molecular Components

Identifying all the molecular components within a living cell is the first step into understanding how it functions. To further understand how a cell functions requires identifying the interactions that occur between these components. This fact is especially relevant for proteins. No protein within a human cell functions on its own without interacting with another biomolecule - usually another protein. While Protein-Protein Interactions (PPI) have historically been determined by examining a single protein per study, novel technologies developed over the past couple of decades are enabling high-throughput methods that aim to describe entire protein networks within cells. In this review, some of the technologies that have led to these developments are described along with applications of these techniques. Ultimately the goal of these technologies is to map out the entire circuitry of PPI within human cells to be able to predict the global consequences of perturbations to the cell system. This predictive capability will have major impacts on the future of both disease diagnosis and treatment.

Download Full-text

NMR Spectroscopy Provides a Novel Bioanalytical and Biophysical Approach towards the Characterization of Protein Interactions Involved in the Integration of RAS Signaling

Journal of Analytical & Bioanalytical Techniques ◽

10.4172/2155-9872.1000e122 ◽

2015 ◽

Vol 6 (5) ◽

Author(s):

Reena Chandrashekar

Keyword(s):

Nmr Spectroscopy ◽

Protein Interactions ◽

Ras Signaling ◽

Biophysical Approach

Download Full-text

Identification and Characterization of Protein‐Protein Interactions between Yeast Rap1p and the TFIID Complex

The FASEB Journal ◽

10.1096/fasebj.22.1_supplement.782.20 ◽

2008 ◽

Vol 22 (S1) ◽

Author(s):

Justin Harrison Layer ◽

Peter Anthony Weil

Keyword(s):

Protein Interactions ◽

Protein Protein Interactions ◽

Tfiid Complex ◽

Identification And Characterization

Download Full-text

Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

10.32920/ryerson.14668284.v1 ◽

2021 ◽

Author(s):

Syed N Shah

Keyword(s):

Protein Interactions ◽

Protein Complexes ◽

Affinity Purification ◽

Regulation Of Gene Expression ◽

Chromatin Assembly ◽

Histone Chaperones ◽

Protein Protein Interactions ◽

Selective Forces ◽

Assembly Proteins

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

Download Full-text

Characterization of COMMD protein–protein interactions in NF-κB signalling

Biochemical Journal ◽

10.1042/bj20051664 ◽

2006 ◽

Vol 398 (1) ◽

pp. 63-71 ◽

Cited By ~ 61

Author(s):

Prim de Bie ◽

Bart van de Sluis ◽

Ezra Burstein ◽

Karen J. Duran ◽

Ruud Berger ◽

...

Keyword(s):

Protein Interactions ◽

Copper Metabolism ◽

Nuclear Factor Κb ◽

Inhibitory Effect ◽

Protein Family ◽

Amino Acid Residues ◽

Protein Protein Interactions ◽

Necrosis Factor

COMMD [copper metabolism gene MURR1 (mouse U2af1-rs1 region 1) domain] proteins constitute a recently identified family of NF-κB (nuclear factor κB)-inhibiting proteins, characterized by the presence of the COMM domain. In the present paper, we report detailed investigation of the role of this protein family, and specifically the role of the COMM domain, in NF-κB signalling through characterization of protein–protein interactions involving COMMD proteins. The small ubiquitously expressed COMMD6 consists primarily of the COMM domain. Therefore COMMD1 and COMMD6 were analysed further as prototype members of the COMMD protein family. Using specific antisera, interaction between endogenous COMMD1 and COMMD6 is described. This interaction was verified by independent techniques, appeared to be direct and could be detected throughout the whole cell, including the nucleus. Both proteins inhibit TNF (tumour necrosis factor)-induced NF-κB activation in a non-synergistic manner. Mutation of the amino acid residues Trp24 and Pro41 in the COMM domain of COMMD6 completely abolished the inhibitory effect of COMMD6 on TNF-induced NF-κB activation, but this was not accompanied by loss of interaction with COMMD1, COMMD6 or the NF-κB subunit RelA. In contrast with COMMD1, COMMD6 does not bind to IκBα (inhibitory κBα), indicating that both proteins inhibit NF-κB in an overlapping, but not completely similar, manner. Taken together, these data support the significance of COMMD protein–protein interactions and provide new mechanistic insight into the function of this protein family in NF-κB signalling.

Download Full-text