scholarly journals Functional Analysis of FRIGIDA Using Naturally Occurring Variation inArabidopsis thaliana

2019 ◽  
Author(s):  
Lei Zhang ◽  
José M Jiménez-Gómez
Keyword(s):  
Cellular Localization ◽  
Functional Characterization ◽  
Dna Polymorphisms ◽  
Single Amino Acid ◽  
Central Domain ◽  
Naturally Occurring ◽  
C Terminus ◽  
Natural Variants ◽  
Natural Sequence Variation ◽  
The Stability

AbstractTheFRIGIDAlocus (FRI, AT4G00650) has been extensively studied inArabidopsis thalianabecause of its role creating flowering time diversity. The FRI protein regulates flowering induction by binding partner proteins on its N- and C-terminus domains and creating a supercomplex that promotes transcription of the floral repressor FLC. Despite the knowledge accumulated on FRI, the function of the highly conserved central domain of the protein is still unknown. Functional characterization of naturally occurring DNA polymorphisms can provide useful information about the role of a protein and the localization of its operative domains. In the case of FRI, deleterious mutations are positively selected and widespread in nature, making them a powerful tool to study the function of the different domains of the protein. Here we explore natural sequence variation in the FRI locus in more than 1000 Arabidopsis accessions. We identify new mutations predicted to compromise the function of the protein and confirm our predictions by cloning 22 different alleles of FRI and expressing them in a common null genetic background. Our analysis allows us to pinpoint two single amino acid changes in the central domain that render the protein non-functional. We show that these two mutations determine the stability and cellular localization of the FRI protein. In summary, our work makes use of natural variants at the FRI locus to help understanding the function of the central domain of the FRI protein.

scholarly journals High-affinity binding of laurate to naturally occurring mutants of human serum albumin and proalbumin

1996 ◽  
Vol 320 (3) ◽  
pp. 911-916 ◽  
Author(s):  
Ulrich KRAGH-HANSEN ◽  
Anders Overgaard PEDERSEN ◽  
Monica GALLIANO ◽  
Lorenzo MINCHIOTTI ◽  
Stephen O. BRENNAN ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Single Amino Acid ◽  
High Affinity ◽  
Naturally Occurring ◽  
C Terminus ◽  
Heterozygous Carriers ◽  
Net Charges ◽  
Fatty Acid Anion ◽  
Normal Albumin ◽  
Dialysis Technique

Binding of laurate (n-dodecanoate) to genetic variants of albumin or its proprotein and to normal albumin isolated from the same heterozygous carriers was studied by a kinetic dialysis technique at physiological pH. The first stoichiometric association constant for binding to proalbumin Lille (Arg-2 → His) and albumin (Alb) Roma (Glu321 → Lys) was increased to 126% and 136% respectively compared with that for binding to normal albumin, whereas the constant for Alb Maku (Lys541 → Glu) was decreased to 80%. In contrast, normal laurate-binding properties were found for as many as nine other albumin variants with single amino acid substitutions. Because the net charges of all these mutants were different from that of normal albumin, the results suggest that the examples of modified laurate binding are not caused by long-range electrostatic effects. Rather, the three positions mentioned are located close to different binding sites for the fatty acid anion. The most pronounced effect was observed for the glycosylated Alb Casebrook, the binding constant of which was decreased to 20%. Binding to the glycosylated Alb Redhill was also decreased, but to a smaller extent (68%). These decreases in binding are caused by partial or total blocking of the high-affinity site by the oligosaccharides, by the negative charges of the oligosaccharides, and/or by conformational changes induced by these bulky moieties. Laurate binding to two chain-termination mutants (Alb Catania and Alb Venezia) was normal, indicating that the C-terminus of albumin is not important for binding. By using different preparations of normal albumin as controls in the binding experiments, it was also possible to compare the effect of various methods for isolation and defatting on laurate binding.

Functional characterization and structural modelling of peptidoglycan degrading β-N-acetyl-glucosaminidase from a dental isolate of Serratia marcescens

2020 ◽  
Vol 23 ◽  
Author(s):  
Aditi Rathee ◽  
Anil Panwar ◽  
Seema Kumari ◽  
Sanjay Chhibber ◽  
Ashok Kumar
Keyword(s):  
Functional Characterization ◽  
Major Change ◽  
Crystal Form ◽  
Bound State ◽  
Dynamics Simulation ◽  
Gram Positive ◽  
Structural Cell ◽  
Stability Structure ◽  
Arginine Residues ◽  
The Stability

Introduction:: Enzymatic degradation of peptidoglycan, a structural cell wall component of Gram-positive bacteria, has attracted considerable attention being a specific target for many known antibiotics. Methods:: Peptidoglycan hydrolases are involved in bacterial lysis through peptidoglycan degradation. β-N-acetylglucosaminidase, a peptidoglycan hydrolase, acts on O-glycosidic bonds formed by N-acetylglucosamine and N-acetyl muramic acid residues of peptidoglycan. Aim of present study was to study the action of β-N-acetylglucosaminidase, on methicillin- resistant Staphylococcus aureus (MRSA) and other Gram-negative bacteria. Results:: We investigated its dynamic behaviour using molecular dynamics simulation and observed that serine and alanine residues are involved in catalytic reaction in addition to aspartic acid, histidine, lysine and arginine residues. When simulated in its bound state, the RMSD values were found lesser than crystal form in the time stamp of 1000 picoseconds revealing its stability. Structure remained stably folded over 1000 picoseconds without undergoing any major change further confirming the stability of complex. Conclusion:: It can be concluded that enzymes belonging to this category can serve as a tool in eradicating Gram-positive pathogens and associated infections.

scholarly journals Anthocyanins Recovered from Agri-Food By-Products Using Innovative Processes: Trends, Challenges, and Perspectives for Their Application in Food Systems

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2632
Author(s):  
Henrique Silvano Arruda ◽  
Eric Keven Silva ◽  
Nayara Macêdo Peixoto Araujo ◽  
Gustavo Araujo Pereira ◽  
Glaucia Maria Pastore ◽  
...  
Keyword(s):  
Food Systems ◽  
Food Industry ◽  
Biological Properties ◽  
Extraction Techniques ◽  
Naturally Occurring ◽  
Current Review ◽  
Natural Colorants ◽  
Conventional Extraction ◽  
The Stability ◽  
By Products

Anthocyanins are naturally occurring phytochemicals that have attracted growing interest from consumers and the food industry due to their multiple biological properties and technological applications. Nevertheless, conventional extraction techniques based on thermal technologies can compromise both the recovery and stability of anthocyanins, reducing their global yield and/or limiting their application in food systems. The current review provides an overview of the main innovative processes (e.g., pulsed electric field, microwave, and ultrasound) used to recover anthocyanins from agri-food waste/by-products and the mechanisms involved in anthocyanin extraction and their impacts on the stability of these compounds. Moreover, trends and perspectives of anthocyanins’ applications in food systems, such as antioxidants, natural colorants, preservatives, and active and smart packaging components, are addressed. Challenges behind anthocyanin implementation in food systems are displayed and potential solutions to overcome these drawbacks are proposed.

scholarly journals Identification, heterologous production and bioactivity of lentinulin A and dendrothelin A, two natural variants of backbone N-methylated peptide macrocycle omphalotin A

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Emmanuel Matabaro ◽  
Hannelore Kaspar ◽  
Paul Dahlin ◽  
Daniel L. V. Bader ◽  
Claudia E. Murar ◽  
...  
Keyword(s):  
Natural Products ◽  
Lentinula Edodes ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Precursor Protein ◽  
Fungal Genome ◽  
Homologous Gene ◽  
C Terminus ◽  
Natural Variants ◽  
Recombinant Peptides

AbstractBackbone N-methylation and macrocyclization improve the pharmacological properties of peptides by enhancing their proteolytic stability, membrane permeability and target selectivity. Borosins are backbone N-methylated peptide macrocycles derived from a precursor protein which contains a peptide α-N-methyltransferase domain autocatalytically modifying the core peptide located at its C-terminus. Founding members of borosins are the omphalotins from the mushroom Omphalotus olearius (omphalotins A-I) with nine out of 12 L-amino acids being backbone N-methylated. The omphalotin biosynthetic gene cluster codes for the precursor protein OphMA, the protease prolyloligopeptidase OphP and other proteins that are likely to be involved in other post-translational modifications of the peptide. Mining of available fungal genome sequences revealed the existence of highly homologous gene clusters in the basidiomycetes Lentinula edodes and Dendrothele bispora. The respective borosins, referred to as lentinulins and dendrothelins are naturally produced by L. edodes and D. bispora as shown by analysis of respective mycelial extracts. We produced all three homologous peptide natural products by coexpression of OphMA hybrid proteins and OphP in the yeast Pichia pastoris. The recombinant peptides differ in their nematotoxic activity against the plant pathogen Meloidogyne incognita. Our findings pave the way for the production of borosin peptide natural products and their potential application as novel biopharmaceuticals and biopesticides.

scholarly journals Prediction of Functional Consequences of Missense Mutations in ANO4 Gene

2021 ◽  
Vol 22 (5) ◽  
pp. 2732
Author(s):  
Nadine Reichhart ◽  
Vladimir M. Milenkovic ◽  
Christian H. Wetzel ◽  
Olaf Strauß
Keyword(s):  
Transmembrane Protein ◽  
Functional Characterization ◽  
Missense Mutations ◽  
Mutant Proteins ◽  
Functional Consequences ◽  
New Perspective ◽  
The Stability ◽  
Dynamics Simulations ◽  
And Function

The anoctamin (TMEM16) family of transmembrane protein consists of ten members in vertebrates, which act as Ca2+-dependent ion channels and/or Ca2+-dependent scramblases. ANO4 which is primarily expressed in the CNS and certain endocrine glands, has been associated with various neuronal disorders. Therefore, we focused our study on prioritizing missense mutations that are assumed to alter the structure and stability of ANO4 protein. We employed a wide array of evolution and structure based in silico prediction methods to identify potentially deleterious missense mutations in the ANO4 gene. Identified pathogenic mutations were then mapped to the modeled human ANO4 structure and the effects of missense mutations were studied on the atomic level using molecular dynamics simulations. Our data show that the G80A and A500T mutations significantly alter the stability of the mutant proteins, thus providing new perspective on the role of missense mutations in ANO4 gene. Results obtained in this study may help to identify disease associated mutations which affect ANO4 protein structure and function and might facilitate future functional characterization of ANO4.

scholarly journals Natural variants in SARS-CoV-2 Spike protein pinpoint structural and functional hotspots with implications for prophylaxis and therapeutic strategies

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Suman Pokhrel ◽  
Benjamin R. Kraemer ◽  
Scott Burkholz ◽  
Daria Mochly-Rosen
Keyword(s):  
Amino Acid ◽  
Severe Disease ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Severe Respiratory Distress ◽  
S Protein ◽  
Individual Sequences ◽  
Natural Variants ◽  
Novel Coronavirus ◽  
The Individual

AbstractIn December 2019, a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of pneumonia with severe respiratory distress and outbreaks in Wuhan, China. The rapid and global spread of SARS-CoV-2 resulted in the coronavirus 2019 (COVID-19) pandemic. Earlier during the pandemic, there were limited genetic viral variations. As millions of people became infected, multiple single amino acid substitutions emerged. Many of these substitutions have no consequences. However, some of the new variants show a greater infection rate, more severe disease, and reduced sensitivity to current prophylaxes and treatments. Of particular importance in SARS-CoV-2 transmission are mutations that occur in the Spike (S) protein, the protein on the viral outer envelope that binds to the human angiotensin-converting enzyme receptor (hACE2). Here, we conducted a comprehensive analysis of 441,168 individual virus sequences isolated from humans throughout the world. From the individual sequences, we identified 3540 unique amino acid substitutions in the S protein. Analysis of these different variants in the S protein pinpointed important functional and structural sites in the protein. This information may guide the development of effective vaccines and therapeutics to help arrest the spread of the COVID-19 pandemic.

Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative

1992 ◽  
Vol 12 (2) ◽  
pp. 444-454
Author(s):  
S M Ruben ◽  
R Narayanan ◽  
J F Klement ◽  
C H Chen ◽  
C A Rosen
Keyword(s):  
Dna Binding ◽  
Complex Formation ◽  
Functional Characterization ◽  
Transcriptional Activator ◽  
Single Amino Acid ◽  
Nf Kappa B ◽  
Mobility Shift ◽  
Activation Domain ◽  
Transcriptional Activator Protein

The NF-kappa B transcription factor complex is composed of two proteins, designated p50 and p65, both having considerable homology to the product of the rel oncogene. We present evidence that the p65 subunit is a potent transcriptional activator in the apparent absence of the p50 subunit, consistent with in vitro results demonstrating that p65 can interact with DNA on its own. To identify the minimal activation domain, chimeric fusion proteins between the DNA binding domain of the yeast transcriptional activator protein GAL4 and regions of the carboxy terminus of p65 were constructed, and their transcriptional activity was assessed by using a GAL4 upstream activation sequence-driven promoter-chloramphenicol acetyltransferase fusion. This analysis suggests that the boundaries of the activation domain lie between amino acids 415 and 550. Moreover, single amino acid changes within residues 435 to 459 greatly diminished activation. Similar to other activation domains, this region contains a leucine zipper-like motif as well as an overall net negative charge. To identify those residues essential for DNA binding, we made use of a naturally occurring derivative of p65, lacking residues 222 to 231 (hereafter referred to as p65 delta), and produced via an alternative splice site. Gel mobility shift analysis using bacterially expressed p65, p65 delta, and various mutants indicates that residues 222 to 231 are important for binding to kappa B DNA. Coimmunoprecipitation analysis suggests that these residues likely contribute to the multimerization function required for homomeric complex formation or heteromeric complex formation with p50 in that no association of p65 delta with itself or with p50 was evident. However, p65 delta was able to form weak heteromeric complexes with p65 that were greatly reduced in their ability to bind DNA. On the basis of these findings, we suggest that subtle changes within the proposed multimerization domain can elicit different effects with the individual Rel-related proteins and that a potential role of p65 delta may be to negatively regulate NF-kappa B function through formation of nonfunctional heteromeric complexes.

scholarly journals Functional characterization of natural variants found on the major stress inducible 70-kDa heat shock gene, HSPA1A, in humans

2018 ◽  
Vol 506 (4) ◽  
pp. 799-804 ◽  
Author(s):  
Ryan Oliverio ◽  
Peter Nguyen ◽  
Brianna Kdeiss ◽  
Sara Ord ◽  
Amanda J. Daniels ◽  
...  
Keyword(s):  
Heat Shock ◽  
Functional Characterization ◽  
Heat Shock Gene ◽  
Natural Variants ◽  
Shock Gene
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