Molecular Biology Commons^™

Feronia Receptor-Like Kinase Regulates Rho Gtpase Signaling Of Root Hair Development, Q. Duan, D. Kita, C. Li, Alice Cheung, H. M. Wu

Alice Cheung

Plant RHO GTPases (RAC/ROPs) mediate multiple extracellular signals ranging from hormone to stress and regulate diverse cellular processes important for polarized cell growth, differentiation, development, reproduction, and responses to the environment. They shuttle between the GDP-bound inactive state and the GTP-bound activated state and their activation is predominantly mediated by a family of guanine nucleotide exchange factors (GEFs) referred to as ROPGEFs. Using the Arabidopsis ROPGEF1 as bait, we identified members of a receptor-like kinase (RLK) family as potential upstream regulators for RAC/ROP signaling. NADPH oxidase-derived reactive oxygen species (ROS) are emerging as important regulators for growth and development and …

An Interdomain Sector Mediating Allostery In Hsp70 Molecular Chaperones, Robert G. Smock, Olivier Rivoire, William P. Russ, Joanna F. Swain, Stanislas Leibler, Rama Ranganathan, Lila Gierasch

Lila Gierasch

Allosteric coupling between protein domains is fundamental to many cellular processes. For example, Hsp70 molecular chaperones use ATP binding by their actin-like N-terminal ATPase domain to control substrate interactions in their C-terminal substrate-binding domain, a reaction that is critical for protein folding in cells. Here, we generalize the statistical coupling analysis to simultaneously evaluate co-evolution between protein residues and functional divergence between sequences in protein sub-families. Applying this method in the Hsp70/110 protein family, we identify a sparse but structurally contiguous group of co-evolving residues called a ‘sector’, which is an attribute of the allosteric Hsp70 sub-family that links the …

Role Of Hsp70 Atpase Domain Intrinsic Dynamics And Sequence Evolution In Enabling Its Functional Interactions With Nefs, Ying Liu, Lila Gierasch, Ivet Bahar

Lila Gierasch

12 Hide Figures Abstract Author Summary Introduction Materials and Methods Results Discussion Supporting Information Acknowledgments Author Contributions References Reader Comments (0) Figures Abstract Catalysis of ADP-ATP exchange by nucleotide exchange factors (NEFs) is central to the activity of Hsp70 molecular chaperones. Yet, the mechanism of interaction of this family of chaperones with NEFs is not well understood in the context of the sequence evolution and structural dynamics of Hsp70 ATPase domains. We studied the interactions of Hsp70 ATPase domains with four different NEFs on the basis of the evolutionary trace and co-evolution of the ATPase domain sequence, combined with elastic …

Molecular Basis For Distinct Pathways For Protein Import Into Arabidopsis Chloroplasts, Danny Schnell, C. Rounds, H. Inoue

Danny Schnell

The translocons at the outer envelope membrane of chloroplasts (TOCs) initiate the import of thousands of nucleus-encoded proteins into the organelle. The identification of structurally and functionally distinct TOC complexes has led to the hypothesis that the translocons constitute different import pathways that are required to coordinate the import of sets of proteins whose expression varies in response to organelle biogenesis and physiological adaptation. To test this hypothesis, we examined the molecular basis for distinct TOC pathways by analyzing the functional diversification among the Toc159 family of TOC receptors. We demonstrate that the N-terminal A-domains of the Toc159 receptors regulate …

Determinants For Stop-Transfer And Post-Import Pathways For Protein Targeting To The Chloroplast Inner Envelope Membrane, Antonio A. B. Viana, Ming Li, Danny Schnell

Danny Schnell

he inner envelope membrane (IEM) of the chloroplast plays key roles in controlling metabolite transport between the organelle and cytoplasm and is a major site of lipid and membrane synthesis within the organelle. IEM biogenesis requires the import and integration of nucleus-encoded membrane proteins. Previous reports have led to the conclusion that membrane proteins are inserted into the IEM during protein import from the cytoplasm via a stop-transfer mechanism or are completely imported into the stroma and then inserted into the IEM in a post-import mechanism. In this study, we examined the determinants for each pathway by comparing the targeting …

Systematic Discovery Of Regulatory Motifs In Fusarium Graminearum By Comparing Four Fusarium Genomes, Lokesh Kumar, Andrew Breakspear, Corby Kistler, Li-Jun Ma, Xiaohui Xie

Li-Jun Ma

Background

Fusarium graminearum (Fg), a major fungal pathogen of cultivated cereals, is responsible for billions of dollars in agriculture losses. There is a growing interest in understanding the transcriptional regulation of this organism, especially the regulation of genes underlying its pathogenicity. The generation of whole genome sequence assemblies for Fg and three closely related Fusarium species provides a unique opportunity for such a study.

Results

Applying comparative genomics approaches, we developed a computational pipeline to systematically discover evolutionarily conserved regulatory motifs in the promoter, downstream and the intronic regions of Fg genes, based on the multiple alignments of sequenced Fusarium …

Examination Of Promotor Hypermethylation Patterns In Magnetically Enriched Exfoliated Breast Milk Epithelial Cells, Chung M. Wong

Masters Theses 1911 - February 2014

Suppression of genes involved in DNA repair, tumor suppression and detoxification through epigenetic modifications has been implicated in the etiology of cancer. As such analysis of promoter methylation patterns in genes frequently down regulated in breast cancer in non-cancerous subjects may serve as an indicator of breast cancer risk. CpG-island hypermethylation of single genes has been detected in cells isolated from nipple aspirate and ductal lavage, yet both isolation methods yield insufficient cells to complete an extensive analysis on any one donor sample.

As an alternative we have turned to magnetic separation of human mammary epithelial cells from breast milk. …

Anti-Diabetic Potentials Of Phenolic Enriched Chilean Potato And Select Herbs Of Apiaceae And Lamiaceae Families, Fahad Saleem

Masters Theses 1911 - February 2014

The incidence of diabetes mellitus and cardiovascular diseases is increasing at a worrisome rate globally. Diabetes mellitus is known to occur due to high blood glucose levels, caused by defects in insulin levels. Adult on-set type II diabetes, which is closely associated with obesity, is reported to be 90-95% of all diabetic cases and linked to diet and lifestyle factors. A large population of the developed and developing countries is now being effected by this epidemic. Natural sources of phenolic antioxidants and inhibitors of digestive enzymes from food sources have potential for low cost dietary management of type II diabetes. …

In Vivo Labeling Of A Model Β-Clam Protein With A Fluorescent Amino Acid, Mangayarkarasi Periasamy

Masters Theses 1911 - February 2014

Proteins can be labeled with different tags to enable their structural and functional investigations. In addition, labeling proteins at specific sites helps in studying the conformational dynamics of these molecules. A plethora of methods is available to facilitate labeling, choice of which largely depends on the requirements and the anticipated end results. In general, the various labeling methods can be classified into four different classes based on the stage at which labeling is performed, namely post translational labeling, non-ribosomal synthesis, in vitro translation and in vivo translation. Interestingly all these techniques use different unnatural amino acids for this purpose.

Protein …

Probing Local Structural Fluctuations In Myoglobin By Size-Dependent Thiol-Disulfide Exchange, Margaret M. Stratton, S N. Loh, T A. Cutler, J H. Ha

Biochemistry & Molecular Biology Department Faculty Publication Series

All proteins undergo local structural fluctuations (LSFs) or breathing motions. These motions are likely to be important for function but are poorly understood. LSFs were initially defined by amide hydrogen exchange (HX) experiments as opening events, which expose a small number of backbone amides to (1)H/(2)H exchange, but whose exchange rates are independent of denaturant concentration. Here, we use size-dependent thiol-disulfide exchange (SX) to characterize LSFs in single cysteine-containing variants of myoglobin (Mb). SX complements HX by providing information on motions that disrupt side chain packing interactions. Most importantly, probe reagents of different sizes and chemical properties can be used …

The 1.9 Å Structure Of Human Α-N-Acetylgalactosaminidase The Molecular Basis Of Schindler And Kanzaki Diseases, Nathaniel E. Clark, Scott Garman

Scott Garman

alpha-N-acetylgalactosaminidase (alpha-NAGAL; E.C. 3.2.1.49) is a lysosomal exoglycosidase that cleaves terminal alpha-N-acetylgalactosamine residues from glycopeptides and glycolipids. In humans, a deficiency of alpha-NAGAL activity results in the lysosomal storage disorders Schindler disease and Kanzaki disease. To better understand the molecular defects in the diseases, we determined the crystal structure of human alpha-NAGAL after expressing wild-type and glycosylation-deficient glycoproteins in recombinant insect cell expression systems. We measured the enzymatic parameters of our purified wild-type and mutant enzymes, establishing their enzymatic equivalence. To investigate the binding specificity and catalytic mechanism of the human alpha-NAGAL enzyme, we determined three crystallographic complexes with different …

Myosin Xi Is Essential For Tip Growth In Physcomitrella Patens, L Vidali, Gm Burkart, Rc Augustine, E Kerdavid, E Tuzel, M Bezanilla

Magdalena Bezanilla

Class XI myosins are plant specific and responsible for cytoplasmic streaming. Because of the large number of myosin XI genes in angiosperms, it has been difficult to determine their precise role, particularly with respect to tip growth. The moss Physcomitrella patens provides an ideal system to study myosin XI function. P. patens has only two myosin XI genes, and these genes encode proteins that are 94% identical to each other. To determine their role in tip growth, we used RNA interference to specifically silence each myosin XI gene using 5′ untranslated region sequences. We discovered that the two myosin XI …

On The Mechanism Of Protein Fold-Switching By A Molecular Sensor, Margaret M. Stratton, S N. Loh

Biochemistry & Molecular Biology Department Faculty Publication Series

Alternate frame folding (AFF) is a mechanism by which conformational change can be engineered into a protein. The protein structure switches from the wild-type fold (N) to a circularly-permuted fold (N'), or vice versa, in response to a signaling event such as ligand binding. Despite the fact that the two native states have similar structures, their interconversion involves folding and unfolding of large parts of the molecule. This rearrangement is reported by fluorescent groups whose relative proximities change as a result of the order-disorder transition. The nature of the conformational change is expected to be similar from protein to protein; …