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At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS2022

Evolution and folding of repeat proteins

Repeat proteins are made with tandem copies of similar amino acid stretches that fold into elongated architectures. These proteins constitute excellent model systems to investigate how evolution relates to structure, folding, and function. Here, we propose a scheme to map evolutionary information at the sequence level to a coarse-grained model for repeat-protein folding and use it to investigate the folding of thousands of repeat proteins. We model the energetics by a combination of an inverse Potts-model scheme with an explicit mechanistic model of duplications and deletions of repeats to calculate the evolutionary parameters of the system at the single-residue level. These parameters are used to inform an Ising-like model that allows for the generation of folding curves, apparent domain emergence, and occupation of intermediate states that are highly compatible with experimental data in specific case studies. We analyzed the folding of thousands of natural Ankyrin repeat proteins and found that a multiplicity of folding mechanisms are possible. Fully cooperative all-or-none transitions are obtained for arrays with enough sequence-similar elements and strong interactions between them, while noncooperative element-by-element intermittent folding arose if the elements are dissimilar and the interactions between them are energetically weak. Additionally, we characterized nucleation-propagation and multidomain folding mechanisms. We show that the global stability and cooperativity of the repeating arrays can be predicted from simple sequence scores.

Ezequiel A. Galpern↗
NASA NTRS2022

Resolving the fine structure in the energy landscapes of repeat proteins

Ankyrin (ANK) repeat proteins are coded by tandem occurrences of patterns with around 33 amino acids. They often mediate protein–protein interactions in a diversity of biological systems. These proteins have an elongated non-globular shape and often display complex folding mechanisms. This work investigates the energy landscape of representative proteins of this class made up of 3, 4 and 6 ANK repeats using the energy-landscape visualisation method (ELViM). By combining biased and unbiased coarse-grained molecular dynamics AWSEM simulations that sample conformations along the folding trajectories with the ELViM structure-based phase space, one finds a three-dimensional representation of the globally funnelled energy surface. In this representation, it is possible to delineate distinct folding pathways. We show that ELViMs can project, in a natural way, the intricacies of the highly dimensional energy landscapes encoded by the highly symmetric ankyrin repeat proteins into useful low-dimensional representations. These projections can discriminate between multiplicities of specific parallel folding mechanisms that otherwise can be hidden in oversimplified depictions.

Murilo N. Sanches↗
NASA NTRS2019

Size and Structure of the Sequence Space of Repeat Proteins

The coding space of protein sequences is shaped by evolutionary constraints set by requirements of function and stability. We show that the coding space of a given protein family— the total number of sequences in that family—can be estimated using models of maximum entropy trained on multiple sequence alignments of naturally occurring amino acid sequences. We analyzed and calculated the size of three abundant repeat proteins families, whose members are large proteins made of many repetitions of conserved portions of *30 amino acids. While amino acid conservation at each position of the alignment explains most of the reduction of diversity relative to completely random sequences, we found that correlations between amino acid usage at different positions significantly impact that diversity. We quantified the impact of different types of correlations, functional and evolutionary, on sequence diversity. Analysis of the detailed structure of the coding space of the families revealed a rugged landscape, with many local energy minima of varying sizes with a hierarchical structure, reminiscent of frustrated energy landscapes of spin glass in physics. This clustered structure indicates a multiplicity of subtypes within each family and suggests new strategies for protein design.

Jacopo Marchi↗
NASA NTRS2016

Microarray Data Analysis of Space Grown Arabidopsis Leaves for Genes Important in Vascular Patterning

Venation patterning in leaves is a major determinant of photosynthesis efficiency because of its dependency on vascular transport of photoassimilates, water, and minerals. Arabidopsis thaliana grown in microgravity show delayed growth and leaf maturation. Gene expression data from the roots, hypocotyl, and leaves of A. thaliana grown during spaceflight vs. ground control analyzed by Affymetrix microarray are available through NASAs GeneLab (GLDS-7). We analyzed the data for differential expression of genes in leaves resulting from the effects of spaceflight on vascular patterning. Two genes were found by preliminary analysis to be upregulated during spaceflight that may be related to vascular formation. The genes are responsible for coding an ARGOS like protein (potentially affecting cell elongation in the leaves), and an F-boxkelch-repeat protein (possibly contributing to protoxylem specification). Further analysis that will focus on raw data quality assessment and a moderated t-test may further confirm upregulation of the two genes and/or identify other gene candidates. Plants defective in these genes will then be assessed for phenotype by the mapping and quantification of leaf vascular patterning by NASAs VESsel GENeration (VESGEN) software to model specific vascular differences of plants grown in spaceflight.

Weitzeal, A. J.↗
NASA NTRS2000

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

NASA Discipline Plant Biology↗
NASA NTRS1994

Pr-specific phytochrome phosphorylation in vitro by a protein kinase present in anti-phytochrome maize immunoprecipitates

Protein kinase activity has repeatedly been found to co-purify with the plant photoreceptor phytochrome, suggesting that light signals received by phytochrome may be transduced or modulated through protein phosphorylation. In this study immunoprecipitation techniques were used to characterize protein kinase activity associated with phytochrome from maize (Zea mays L.). A protein kinase that specifically phosphorylated phytochrome was present in washed anti-phytochrome immunoprecipitates of etiolated coleoptile proteins. No other substrate tested was phosphorylated by this kinase. Adding salts or detergents to disrupt low-affinity protein interactions reduced background phosphorylation in immunoprecipitates without affecting phytochrome phosphorylation, indicating that the protein kinase catalytic activity is either intrinsic to the phytochrome molecule or associated with it by high-affinity interactions. Red irradiation (of coleoptiles or extracts) sufficient to approach photoconversion saturation reduced phosphorylation of immunoprecipitated phytochrome. Subsequent far-red irradiation reversed the red-light effect. Phytochrome phosphorylation was stimulated about 10-fold by a co-immunoprecipitated factor. The stimulatory factor was highest in immunoprecipitates when Mg2+ was present in immunoprecipitation reactions but remained in the supernatant in the absence of Mg2+. These observations provide strong support for the hypothesis that phytochrome-associated protein kinase modulates light responses in vivo. Since only phytochrome was found to be phosphorylated, the co-immunoprecipitated protein kinase may function to regulate receptor activity.

NASA Discipline Plant Biology↗
NASA NTRS1983

Information contained in protein shapes

The sequence of local conformations at C-alpha atoms of a protein has been considered as an informational message string. The total self-information contents and self-information per letter have been evaluated for 83 globular proteins whose structures are known from X-ray crystallography. The derived information contents provide a method of quantitating structural specificity of proteins. This method of analysis enables repeating, intricate structural features to be recognized. Among the globular proteins whose structures have been solved, high potential iron protein stands out with the largest three-letter dependence.

Sundaram, K.↗
NASA NTRS2000

A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin

Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant's responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N-(1-naph-thyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1, encodes a 419-amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein-protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level.

NASA Discipline Plant Biology↗
NASA NTRS1998

Decorin is a Zn(2+) Metalloprotein

Decorin is ubiquitously distributed in the extracellular matrix of mammals and a member of the proteoglycan family characterized by a core protein dominated by Leucine Rich Repeat motifs. We here demonstrate that decorin extracted from bovine tissues under denaturing conditions or produced in recombinant "native" form by cultured mammalian cells, has a high affinity for Zn(2+). Binding of Zn(2+) to decorin is demonstrated by Zn(2+) chelating chromatography and equilibrium dialyses. The Zn(2+) binding sites are localized to the N-terminal domain of the core protein that contains 4 Cys residues in the spacing reminiscent of a Zn finger. A recombinant 41 amino acid long peptide representing the N-terminal domain of decorin has full Zn(2+) binding activity and binds two Zn(2+) ions with an average K(D) of 3 x 10(exp -7) M. Biglycan, a proteoglycan that is structurally closely related to decorin contains a similar high affinity Zn(2+) binding segment, whereas the structurally more distantly related proteoglycans, epiphycan and osteoglycin, did not bind Zn(2+) with high affinity.

Yang, Vivian W.-C.↗
NASA NTRS2002

Regulation of period 1 expression in cultured rat pineal

The aim of the present study was to investigate the in vitro expression of Period 1 (Per1), Period 2 (Per2) and arylalkylamine N-acetyltransferase (AA-NAT) genes in the rat pineal gland to understand the mechanism(s) regulating the expression of these genes in this organ. Pineals, when maintained in vitro for 5 days, did not show circadian rhythmicity in the expression of any of the three genes monitored. Norepinephrine (NE) induced AA-NAT and Per1, whereas its effect on Per2 was negligible. Contrary to what was observed in other systems, NE stimulation did not induce circadian expression of Per1. The effect of NE on Per1 level was dose- and receptor subtype-dependent, and both cAMP and cGMP induced Per1. Per1 was not induced by repeated NE - or forskolin - stimulation. Protein synthesis was not necessary for NE-induced Per1, but it was for reduction of Per1 following NE stimulation. Per1 transcription in pinealocytes was activated by BMAL1/CLOCK. Our results indicate that important differences are present in the regulation of these genes in the mammalian pineal. Copyright 2002 S. Karger AG, Basel.

NASA Discipline Space Human Factors↗
NASA NTRS1993

Sequence and structural implications of a bovine corneal keratan sulfate proteoglycan core protein. Protein 37B represents bovine lumican and proteins 37A and 25 are unique

Amino acid sequence from tryptic peptides of three different bovine corneal keratan sulfate proteoglycan (KSPG) core proteins (designated 37A, 37B, and 25) showed similarities to the sequence of a chicken KSPG core protein lumican. Bovine lumican cDNA was isolated from a bovine corneal expression library by screening with chicken lumican cDNA. The bovine cDNA codes for a 342-amino acid protein, M(r) 38,712, containing amino acid sequences identified in the 37B KSPG core protein. The bovine lumican is 68% identical to chicken lumican, with an 83% identity excluding the N-terminal 40 amino acids. Location of 6 cysteine and 4 consensus N-glycosylation sites in the bovine sequence were identical to those in chicken lumican. Bovine lumican had about 50% identity to bovine fibromodulin and 20% identity to bovine decorin and biglycan. About two-thirds of the lumican protein consists of a series of 10 amino acid leucine-rich repeats that occur in regions of calculated high beta-hydrophobic moment, suggesting that the leucine-rich repeats contribute to beta-sheet formation in these proteins. Sequences obtained from 37A and 25 core proteins were absent in bovine lumican, thus predicting a unique primary structure and separate mRNA for each of the three bovine KSPG core proteins.

NASA Discipline Cell Biology↗
NASA NTRS2017

Novel Approach to Quantification of Telomere Length with Direct Nanopore Sequencing and PCR Amplification

The ends of human chromosomes contain telomeres, or tandem arrays of repeating DNA sequences capped by multiple associated proteins that protect chromosomal ends from degradation. Telomeres function to preserve genomic stability by preventing natural chromosomal ends from being recognized as broken DNA double-strand breaks and triggering inappropriate DNA damage responses. Mounting evidence shows telomere length is an inherited trait that decreases with cellular division and normal aging. In addition, telomere length also appears to be influenced by other factors such as cellular oxidative stress, radiation and mechanical unloading of tissues as in microgravity. To measure these potential effects of the space environment on telomere lengths and cellular aging and regenerative potential we developed a novel telomere measurement approach based on nanopore sequencing of PCR amplified bar-coded chromosome termini. Specifically, telomeres can be directly enriched using barcode sequences ligated to the end of a free end- repaired telomere using the WetLab-2 facility SmartCycler on ISS. Prior to the ligation and amplification protocol a proteinase K digestion of capping proteins followed by a single 95-degree C heat denaturation of the protease is included. After digestion and bar-code ligation, PCR amplification will initiate with the ligated barcoded sequence, suppressing amplification of intra-genomic fragments and resulting in long read barcoded telomere amplicons including the nanopore motor protein sequences. Purified PCR amplicons are then used for nanopore sequencing library generation by simple addition of motor proteins and sequencing library is loaded into the MinION nanopore DNA-sequencer. Amplicon sequence reads from the nanopore device can be base-called quickly on ISS due to barcoding ligation and subsequent PCR amplification enhancing the telomere sequence resolution. If successfully implemented on ISS this technique will provide a novel means of measuring regenerative ability of somatic stem cells in astronauts, and of determining whether spaceflight in microgravity alters their telomere lengths and causes premature cellular aging.

Ma, Kristin R.↗
NASA NTRS2022

Evaluating Mineral Lattices as Evolutionary Proxies for Metalloprotein Evolution

Protein coordinated iron-sulfur clusters drive electron flow within metabolic pathways for organisms throughout the tree of life. It is not known how iron-sulfur clusters were first incorporated into proteins. Structural analogies to iron-sulfde minerals present on early Earth, suggest a connection in the evolution of both proteins and minerals. The availability of large protein and mineral crystallographic structure data sets, provides an opportunity to explore co-evolution of proteins and minerals on a large-scale using informatics approaches. However, quantitative comparisons are confounded by the infnite, repeating nature of the mineral lattice, in contrast to metal clusters in proteins, which are fnite in size. We address this problem using the Niggli reduction to transform a mineral lattice to a fnite, unique structure that when translated reproduces the crystal lattice. Protein and reduced mineral structures were represented as quotient graphs with the edges and nodes corresponding to bonds and atoms, respectively. We developed a graph theory-based method to calculate the maximum common connected edge subgraph (MCCES) between mineral and protein quotient graphs. MCCES can accommodate differences in structural volumes and easily allows additional chemical criteria to be considered when calculating similarity. To account for graph size differences, we use the Tversky similarity index. Using consistent criteria, we found little similarity between putative ancient iron-sulfur protein clusters and iron-sulfur mineral lattices, suggesting these metal sites are not as evolutionarily connected as once thought. We discuss possible evolutionary implications of these findings in addition to suggesting an alternative proxy, mineral surfaces, for better understanding the coevolution of the geosphere and biosphere

Kenneth N. McGuinness↗
NASA NTRS1994

Transcription factor IID in the Archaea: sequences in the Thermococcus celer genome would encode a product closely related to the TATA-binding protein of eukaryotes

The first step in transcription initiation in eukaryotes is mediated by the TATA-binding protein, a subunit of the transcription factor IID complex. We have cloned and sequenced the gene for a presumptive homolog of this eukaryotic protein from Thermococcus celer, a member of the Archaea (formerly archaebacteria). The protein encoded by the archaeal gene is a tandem repeat of a conserved domain, corresponding to the repeated domain in its eukaryotic counterparts. Molecular phylogenetic analyses of the two halves of the repeat are consistent with the duplication occurring before the divergence of the archael and eukaryotic domains. In conjunction with previous observations of similarity in RNA polymerase subunit composition and sequences and the finding of a transcription factor IIB-like sequence in Pyrococcus woesei (a relative of T. celer) it appears that major features of the eukaryotic transcription apparatus were well-established before the origin of eukaryotic cellular organization. The divergence between the two halves of the archael protein is less than that between the halves of the individual eukaryotic sequences, indicating that the average rate of sequence change in the archael protein has been less than in its eukaryotic counterparts. To the extent that this lower rate applies to the genome as a whole, a clearer picture of the early genes (and gene families) that gave rise to present-day genomes is more apt to emerge from the study of sequences from the Archaea than from the corresponding sequences from eukaryotes.

NASA Discipline Exobiology↗
NASA NTRS2003

Sera from cancer patients contain two oscillating ECTO-NOX activities with different period lengths

ECTO-NOX protein's are cell surface-associated and growth-related hydroquinone oxidases with both protein disulfide-thiol interchange activity and the capacity to oxidize NAD(P)H. The activities of these ECTO-NOX proteins are not steady state but fluctuate to create a repeating pattern of oscillations. Two forms of ECTO-NOX activities have been distinguished. The constitutive ECTO-NOX (CNOX), is hormone responsive and refractory to quinone-site inhibitors. A tumor-associated NOX (tNOX) is unregulated, refractory to hormones and growth factors and responds to quinone-site inhibitors. CNOX proteins are widely distributed and exhibit oscillations in enzymatic activity with a period length of 24 min. tNOX proteins are cancer specific and exhibit oscillations with a period length of about 22 min. Our findings now demonstrate the presence of the novel oscillating tNOX activity in sera of patients with cancer whereas the constitutive NOX of non-cancer cells is present in sera of both cancer patients and healthy volunteers. We conclude that ECTO-NOX proteins in sera exhibit oscillatory characteristics similar to those of ECTO-NOX forms of the cell surface.

NASA Discipline Cell Biology↗
NASA NTRSDate not supplied

Conserved Function of RNA Binding Motif Protein48 (RBM48) and Armadillo Repeat Containing 7 (ARMC7) in Maize and Human U12 Splicing

Splicing of pre-mRNA is fundamental for genes containing introns. Emerging data points to a deeply conserved role of this process in eukaryotic cell differentiation and proliferation. The vast majority of introns termed U2-type introns are spliced by a major spliceosome; however, there also exist rare and more conserved U12-type introns that are spliced by a minor spliceosome. Mutations that disrupt U12 splicing inhibit cell differentiation in both maize endosperm and human blood cells. However, the mechanism underlying this process is not well understood. The maize RNA Binding Motif Protein 48 (RBM48) plays an essential role as a U12 splicing factor and is required for proper maize kernel development. Using human cell lines, CRISPR-Cas9 knockdown of RBM48 demonstrated a conserved function in human U12 intron splicing. RBM48 and Armadillo Repeat Containing 7 (ARMC7) protein interact in both maize and human as part of the activated minor spliceosome. Here we show RBM48 and ARMC7 co-localization in the nucleus of human cell lines. Vertebrate ARMC7 is normally localized in the cytosol, whereas RBM48 is found in the nucleus. Our data suggests that the interaction plays a role in regulating ARMC7 localization or the efficiency of U12 intron splicing. We also performed comprehensive transcriptome profiling and identified a common subset of conserved Minor Intron Containing Genes (MIGs) impacted in both human and maize RBM48 knockout mutants. Of importance, the vast majority of these MIGs are associated with developmental defects in both plants and animals. This suggests that aberrant splicing of these targets has a high likelihood of mediating abnormal cell phenotypes. These data support evolutionarily conserved U12 splicing mechanisms between maize and humans with both RBM48 and ARMC7 having roles in the activated spliceosome.

maize↗
NASA NTRS2000

Nutritional Evaluation of NASA's Rodent Food Bar Diet

Tests are being conducted on NASA's rodent Food Bar in preparation for long-term use as the rat and mouse diet aboard the International Space Station. Nutritional analyses are performed after the bars are manufactured and then repeated periodically to determine nutritional stability. The primary factors analyzed are protein, ash, fat, fiber, moisture, amino acids, fatty acids, and minerals. Nutrient levels are compared to values published in the National Research Council's dietary requirements for rodents, and also to those contained in several commonly used commercial rodent lab diets. The Food Bar is manufactured from a powdered diet to which moisture is added as it is processed through an extruder. The bars are dipped into potassium sorbate, vacuum-sealed, and irradiated. In order to determine nutrient changes during extrusion and irradiation, the powdered diet, the non-irradiated bars, and the irradiated bars are all analyzed. We have observed lower values for some nutrients (iodine, vitamin K, and iron) in the Food Bars compared with NRC requirements. Many nutrients in the Food Bars are contained at a higher level than levels in the NRC requirements. An additional factor we are investigating is the 26% moisture level in the Food Bars, which drops to about 15% within a week, compared to a stable 10% moisture in many standard lab chow diets. In addition to the nutritional analyses, the food bar is being fed to several strains of rats and mice, and feeding study and necropsy results are being observed (Barrett et al, unpublished data). Information from the nutritional analyses and from the rodent studies will enable us to recommend the formulation that will most adequately meet the rodent Food Bar requirements for long-term use aboard the Space Station.

Barrett, Joyce E.↗