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A Dynamic Model of pH-Induced Protein G'e Higher Order Structure Changes derived from Mass Spectrometric Analyses.

ABSTRACT: To obtain insight into pH change-driven molecular dynamics, we studied the higher order structure changes of protein G'e at the molecular and amino acid residue levels in solution by using nanoESI- and IM-mass spectrometry, CD spectroscopy, and protein chemical modification reactions (protein footprinting). We found a dramatic change of the overall tertiary structure of protein G'e when the pH was changed from neutral to acidic, whereas its secondary structure features remained nearly invariable. Limited proteolysis and surface-topology mapping of protein G'e by fast photochemical oxidation of proteins (FPOP) under neutral and acidic conditions reveal areas where higher order conformational changes occur on the amino-acid residue level. Under neutral solution conditions, lower oxidation occurs for residues of the first linker region, whereas greater oxidative modifications occur for amino-acid residues of the IgG-binding domains I and II. We propose a dynamic model of pH-induced structural changes in which protein G'e at neutral pH adopts an overall tight conformation with all four domains packed in a firm assembly, whereas at acidic pH, the three IgG-binding domains form an elongated alignment, and the N-terminal, His-tag-carrying domain unfolds. At the same time the individual IgG-binding domains themselves seem to adopt a more compacted fold. As the secondary structure features are nearly unchanged at either pH, interchange between both conformations is highly reversible, explaining the high reconditioning power of protein G'e-based affinity chromatography columns.

SUBMITTER: Yefremova Y

PROVIDER: S-EPMC5201196 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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A Dynamic Model of pH-Induced Protein G'e Higher Order Structure Changes derived from Mass Spectrometric Analyses.

Yefremova Yelena Y Al-Majdoub Mahmoud M Opuni Kwabena F M KF Koy Cornelia C Yan Yuetian Y Gross Michael L ML Glocker Michael O MO

Analytical chemistry 20151209 1

To obtain insight into pH change-driven molecular dynamics, we studied the higher order structure changes of protein G'e at the molecular and amino acid residue levels in solution by using nanoESI- and IM-mass spectrometry, CD spectroscopy, and protein chemical modification reactions (protein footprinting). We found a dramatic change of the overall tertiary structure of protein G'e when the pH was changed from neutral to acidic, whereas its secondary structure features remained nearly invariable ...[more]

PMID: 26606592

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Project description:Proteins adopt different higher-order structures (HOS) to enable their unique biological functions. Understanding the complexities of protein higher-order structures and dynamics requires integrated approaches, where mass spectrometry (MS) is now positioned to play a key role. One of those approaches is protein footprinting. Although the initial demonstration of footprinting was for the HOS determination of protein/nucleic acid binding, the concept was later adapted to MS-based protein HOS analysis, through which different covalent labeling approaches "mark" the solvent accessible surface area (SASA) of proteins to reflect protein HOS. Hydrogen-deuterium exchange (HDX), where deuterium in D2O replaces hydrogen of the backbone amides, is the most common example of footprinting. Its advantage is that the footprint reflects SASA and hydrogen bonding, whereas one drawback is the labeling is reversible. Another example of footprinting is slow irreversible labeling of functional groups on amino acid side chains by targeted reagents with high specificity, probing structural changes at selected sites. A third footprinting approach is by reactions with fast, irreversible labeling species that are highly reactive and footprint broadly several amino acid residue side chains on the time scale of submilliseconds. All of these covalent labeling approaches combine to constitute a problem-solving toolbox that enables mass spectrometry as a valuable tool for HOS elucidation. As there has been a growing need for MS-based protein footprinting in both academia and industry owing to its high throughput capability, prompt availability, and high spatial resolution, we present a summary of the history, descriptions, principles, mechanisms, and applications of these covalent labeling approaches. Moreover, their applications are highlighted according to the biological questions they can answer. This review is intended as a tutorial for MS-based protein HOS elucidation and as a reference for investigators seeking a MS-based tool to address structural questions in protein science.

Project description:Our previous work have shown that certain subpopulations of Klebsiella pneumoniae exhibit significant phenotypic changes under simulated microgravity (SMG), including enhanced biofilm formation and cellulose synthesis, which may be evoked by changes in gene expression patterns. It is well known that prokaryotic cells genomic DNA can be hierarchically organized into different higher-order three-dimensional structures, which can highly influence gene expression. It is remain elusive whether phenotypic changes induced by SMG in the subpopulations of K. pneumoniae are driven by genome higher-order structural changes. Here, we investigated the above-mentioned issue using the wild-type (WT) K. pneumoniae (WT was used as a control strain and continuously cultivated for 2 weeks under standard culture conditions of normal gravity) and two previous identified subpopulations (M1 and M2) obtained after 2 weeks of continuous incubation in a SMG device. By the combination of genome-wide chromosome conformation capture (Hi-C), RNA-seq and whole-genome methylation (WGS) analyses, we found that the along with the global chromosome interactions change, the compacting extent of M1, M2 subpopulations were much looser under SMG and even with an increase in active, open chromosome regions. In addition, transcriptome data showed that most differentially expressed genes (DEGs) were upregulated, whereas a few DEGs were downregulated in M1 and M2. The functions of both types DEGs were mainly associated with membrane fractions. Additionally, WGS analysis revealed that methylation levels were lower in M1 and M2. Using combined analysis of multi-omics data, we discovered that most upregulated DEGs were significantly enriched in the boundary regions of the variable chromosomal interaction domains (CIDs), in which genes regulating biofilm formation were mainly located. These results suggest that K. pneumoniae may regulate gene expression patterns through DNA methylation and changes in genome structure, thus resulting in new phenotypes in response to altered gravity.

Dataset Information

A Dynamic Model of pH-Induced Protein G'e Higher Order Structure Changes derived from Mass Spectrometric Analyses.

Publications

A Dynamic Model of pH-Induced Protein G'e Higher Order Structure Changes derived from Mass Spectrometric Analyses.

Similar Datasets

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