A Ca2+-dependent bacterial antifreeze protein domain has a novel β-helical ice-binding fold

2008 ◽  
Vol 411 (1) ◽  
pp. 171-180 ◽  
Author(s):  
Christopher P. Garnham ◽  
Jack A. Gilbert ◽  
Christopher P. Hartman ◽  
Robert L. Campbell ◽  
Johanna Laybourn-Parry ◽  
...  
Keyword(s):  
Freezing Point ◽  
Protein Domain ◽  
Site Directed Mutagenesis ◽  
Hydrophobic Core ◽  
Amino Acid Region ◽  
Ice Binding ◽  
Binding Surface ◽  
The Antarctic ◽  
Antifreeze Activity ◽  
Acid Region

AFPs (antifreeze proteins) are produced by many organisms that inhabit ice-laden environments. They facilitate survival at sub-zero temperatures by binding to, and inhibiting, the growth of ice crystals in solution. The Antarctic bacterium Marinomonas primoryensis produces an exceptionally large (>1 MDa) hyperactive Ca2+-dependent AFP. We have cloned, expressed and characterized a 322-amino-acid region of the protein where the antifreeze activity is localized that shows similarity to the RTX (repeats-in-toxin) family of proteins. The recombinant protein requires Ca2+ for structure and activity, and it is capable of depressing the freezing point of a solution in excess of 2 °C at a concentration of 0.5 mg/ml, therefore classifying it as a hyperactive AFP. We have developed a homology-guided model of the antifreeze region based partly on the Ca2+-bound β-roll from alkaline protease. The model has identified both a novel β-helical fold and an ice-binding site. The interior of the β-helix contains a single row of bound Ca2+ ions down one side of the structure and a hydrophobic core down the opposite side. The ice-binding surface consists of parallel repetitive arrays of threonine and aspartic acid/asparagine residues located down the Ca2+-bound side of the structure. The model was tested and validated by site-directed mutagenesis. It explains the Ca2+-dependency of the region, as well its hyperactive antifreeze activity. This is the first bacterial AFP to be structurally characterized and is one of only five hyperactive AFPs identified to date.

scholarly journals Mutational analysis defines a C-terminal tail domain of RAP1 essential for Telomeric silencing in Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 138 (4) ◽  
pp. 1025-1040 ◽  
Author(s):  
C Liu ◽  
X Mao ◽  
A J Lustig
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Size Control ◽  
Telomere Maintenance ◽  
Site Directed Mutagenesis ◽  
In Vitro Mutagenesis ◽  
Tail Domain ◽  
Amino Acid Region ◽  
Telomeric Silencing ◽  
Acid Region

Abstract Alleles specifically defective in telomeric silencing were generated by in vitro mutagenesis of the yeast RAP1 gene. The most severe phenotypes occur with three mutations in the C-terminal 28 amino acids. Two of the alleles are nonsense mutations resulting in truncated repressor/activator protein 1 (RAP1) species lacking the C-terminal 25-28 amino acids; the third allele is a missense mutation within this region. These alleles define a novel 28-amino acid region, termed the C-terminal tail domain, that is essential for telomeric and HML silencing. Using site-directed mutagenesis, an 8-amino acid region (amino acids 818-825) that is essential for telomeric silencing has been localized within this domain. Further characterization of these alleles has indicated that the C-terminal tail domain also plays a role in telomere size control. The function of the C-terminal tail in telomere maintenance is not mediated through the RAP1 interacting factor RIF1: rap1 alleles defective in both the C-terminal tail and RIF1 interaction domains have additive effects on telomere length. Overproduction of SIR3, a dose-dependent enhancer of telomeric silencing, suppresses the telomeric silencing, but not length, phenotypes of a subset of C-terminal tail alleles. In contrast, an allele that truncates the terminal 28 amino acids of RAP1 is refractory to SIR3 overproduction. These results indicate that the C-terminal tail domain is required for SIR3-dependent enhancement of telomeric silencing. These data also suggest a distinct set of C-terminal requirements for telomere size control and telomeric silencing.

scholarly journals Combined molecular dynamics and neural network method for predicting protein antifreeze activity

2018 ◽  
Vol 115 (52) ◽  
pp. 13252-13257 ◽  
Author(s):  
Daniel J. Kozuch ◽  
Frank H. Stillinger ◽  
Pablo G. Debenedetti
Keyword(s):  
Neural Network ◽  
Molecular Dynamics ◽  
Thermal Hysteresis ◽  
Freezing Point ◽  
Dynamics Simulation ◽  
Ice Binding ◽  
Network Method ◽  
Physical Variables ◽  
Simple Neural Network ◽  
Antifreeze Activity

Antifreeze proteins (AFPs) are a diverse class of proteins that depress the kinetically observable freezing point of water. AFPs have been of scientific interest for decades, but the lack of an accurate model for predicting AFP activity has hindered the logical design of novel antifreeze systems. To address this, we perform molecular dynamics simulation for a collection of well-studied AFPs. By analyzing both the dynamic behavior of water near the protein surface and the geometric structure of the protein, we introduce a method that automatically detects the ice binding face of AFPs. From these data, we construct a simple neural network that is capable of quantitatively predicting experimentally observed thermal hysteresis from a trio of relevant physical variables. The model’s accuracy is tested against data for 17 known AFPs and 5 non-AFP controls.

scholarly journals Carrot ‘antifreeze’ protein has an irregular ice-binding site that confers weak freezing point depression but strong inhibition of ice recrystallization

2020 ◽  
Vol 477 (12) ◽  
pp. 2179-2192 ◽  
Author(s):  
Yannan Wang ◽  
Laurie A. Graham ◽  
Zhifu Han ◽  
Robert Eves ◽  
Audrey K. Gruneberg ◽  
...  
Keyword(s):  
Convex Surface ◽  
Freezing Point ◽  
Protein Structures ◽  
Freeze Tolerance ◽  
Antifreeze Protein ◽  
Site Directed Mutagenesis ◽  
Freezing Point Depression ◽  
Freezing Damage ◽  
Ice Recrystallization ◽  
Ice Binding

Ice-binding proteins (IBPs) are found in many biological kingdoms where they protect organisms from freezing damage as antifreeze agents or inhibitors of ice recrystallization. Here, the crystal structure of recombinant IBP from carrot (Daucus carota) has been solved to a resolution of 2.3 Å. As predicted, the protein is a structural homologue of a plant polygalacturonase-inhibiting protein forming a curved solenoid structure with a leucine-rich repeat motif. Unexpectedly, close examination of its surface did not reveal any large regions of flat, regularly spaced hydrophobic residues that characterize the ice-binding sites (IBSs) of potent antifreeze proteins from freeze-resistant fish and insects. An IBS was defined by site-directed mutagenesis of residues on the convex surface of the carrot solenoid. This imperfect site is reminiscent of the irregular IBS of grass ‘antifreeze’ protein. Like the grass protein, the carrot IBP has weak freezing point depression activity but is extremely active at nanomolar concentrations in inhibiting ice recrystallization. Ice crystals formed in the presence of both plant proteins grow slowly and evenly in all directions. We suggest that this slow, controlled ice growth is desirable for freeze tolerance. The fact that two plant IBPs have evolved very different protein structures to affect ice in a similar manner suggests this pattern of weak freezing point depression and strong ice recrystallization inhibition helps their host to tolerate freezing rather than to resist it.

scholarly journals Identification of Critical Residues in the Propeptide of LasA Protease of Pseudomonas aeruginosa Involved in the Formation of a Stable Mature Protease

2007 ◽  
Vol 189 (11) ◽  
pp. 3960-3968 ◽  
Author(s):  
Kerian K. Grande ◽  
Jean K. Gustin ◽  
Efrat Kessler ◽  
Dennis E. Ohman
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Enzymatic Activity ◽  
Insertional Mutagenesis ◽  
Site Directed Mutagenesis ◽  
Amino Terminal ◽  
Amino Acid Region ◽  
Lasa Protease ◽  
Mature Domain ◽  
Acid Region

ABSTRACT LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by Pseudomonas aeruginosa. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing lasA as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain.

scholarly journals Posttransition State Desolvation of the Hydrophobic Core of the src-SH3 Protein Domain

2003 ◽  
Vol 85 (1) ◽  
pp. 61-69 ◽  
Author(s):  
Weihua Guo ◽  
Sotiria Lampoudi ◽  
Joan-Emma Shea
Keyword(s):  
Protein Domain ◽  
Hydrophobic Core

On the thickness of the Antarctic ice, and its relations to that of the glacial epoch

2021 ◽  
pp. 1-8
Author(s):  
James CROLL ◽  
David SUGDEN
Keyword(s):  
Thermal Regime ◽  
Freezing Point ◽  
Ice Sheet ◽  
Heat Sources ◽  
Mean Velocity ◽  
Point Estimates ◽  
Glacial Epoch ◽  
Antarctic Continent ◽  
Global Sea Level ◽  
The Antarctic

ABSTRACT At a time when nobody has yet landed on the Antarctic continent (1879), this presentation and accompanying paper predicts the morphology, dynamics and thermal regime of the Antarctic ice sheet. Mathematical modelling of the ice sheet is based on the assumptions that the thickness of tabular icebergs reflects the average thickness of the ice at the margin and that the surface gradients are comparable to those of reconstructed former ice sheets in the Northern Hemisphere. The modelling shows that (a) ice is thickest near the centre at the South Pole and thins towards the margin; (b) the thickness at the pole is independent of the amount of snowfall at that place; and (c) the mean velocity at the margin, assuming a mean annual snowfall of two inches per year, is 400–500 feet per year. The thermal regime of the ice sheet is influenced by three heat sources – namely, the bed, the internal friction of ice flow and the atmosphere. The latter is the most significant and, since ice has a downwards as well as horizontal motion, this carries cold ice down into the ice sheet. Since the temperature at which ice melts is lowered by pressure at a rate of 0.0137 °F for every atmosphere of pressure (something known since 1784), much of the ice sheet and its base must be below the freezing point. Estimates of the thickness of ice at the centre depend closely on the surface gradients assumed and range between 3 and 24 miles. Such uncertainty is of concern since both the volume and gravitational attraction of the ice mass have an effect on global sea level. In order to improve our estimate of the volume of ice, we will have to wait 76 years for John Glen to develop a realistic flow law for ice.

Identification and analysis of two sequences encoding ice-binding proteins obtained from a putative bacterial symbiont of the psychrophilic Antarctic ciliate Euplotes focardii

2014 ◽  
Vol 26 (5) ◽  
pp. 491-501 ◽  
Author(s):  
Sandra Pucciarelli ◽  
Federica Chiappori ◽  
Raghul Rajan Devaraj ◽  
Guang Yang ◽  
Ting Yu ◽  
...  
Keyword(s):  
Amino Acid Level ◽  
Bacterial Symbiont ◽  
The Arctic ◽  
Glacial Ice ◽  
Mould Fungus ◽  
Lake Vostok ◽  
Ice Binding ◽  
Stigmatella Aurantiaca ◽  
Ice Binding Protein ◽  
The Antarctic

AbstractWe identified two ice-binding protein (IBP) sequences, named EFsymbAFP and EFsymbIBP, from a putative bacterial symbiont of the Antarctic psychrophilic ciliate Euplotes focardii. EFsymbAFP is 57.43% identical to the antifreeze protein (AFP) from the Stigmatella aurantiaca strain DW4/3-1, which was isolated from the Victoria Valley lower glacier. EFsymbIBP is 53.38% identical to the IBP from the Flavobacteriaceae bacterium strain 3519-10, isolated from the glacial ice of Lake Vostok. EFsymbAFP and EFsymbIBP are 31.73% identical at the amino acid level and are organized in tandem on the bacterial chromosome. The relatively low sequence identity and the tandem organization, which appears unique to this symbiont, suggest an occurrence of horizontal gene transfer (HGT). Structurally, EFsymbAFP and EFsymbIBP are similar to the AFPs from the snow mould fungus Typhula ishikariensis and from the Arctic yeast Leucosporidium sp. AY30. A phylogenetic analysis showed that EFsymbAFP and EFsymbIBP cluster principally with the IBP sequences from other Antarctic bacteria, supporting the view that these sequences belong to an Antarctic symbiontic bacterium of E. focardii. These results confirm that IBPs have a complex evolutionary history, which includes HGT events, most probably due to the demands of the environment and the need for rapid adaptation.

scholarly journals The yeast silent information regulator Sir4p anchors and partitions plasmids.

1997 ◽  
Vol 17 (12) ◽  
pp. 7061-7068 ◽  
Author(s):  
A Ansari ◽  
M R Gartenberg
Keyword(s):  
Coiled Coil ◽  
Axial Rotation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Interacting Protein ◽  
Segregation Behavior ◽  
Amino Acid Region ◽  
Dividing Cells ◽  
Nuclear Component ◽  
Acid Region

Circular plasmids containing telomeric TG1-3 arrays or the HMR E silencer segregate efficiently between dividing cells of the yeast Saccharomyces cerevisiae. Subtelomeric X repeats augment the TG1-3 partitioning activity by a process that requires the SIR2, SIR3, and SIR4 genes, which are also required for silencer-based partitioning. Here we show that targeting Sir4p to DNA directly via fusion to the bacterial repressor LexA confers efficient mitotic segregation to otherwise unstable plasmids. The Sir4p partitioning activity resides within a 300-amino-acid region (residues 950 to 1262) which precedes the coiled-coil dimerization motif at the extreme carboxy end of the protein. Using a topology-based assay, we demonstrate that the partitioning domain also retards the axial rotation of LexA operators in vivo. The anchoring and partitioning properties of LexA-Sir4p chimeras persist despite the loss of the endogenous SIR genes, indicating that these functions are intrinsic to Sir4p and not to a complex of Sir factors. In contrast, inactivation of the Sir4p-interacting protein Rap1p reduces partitioning by a LexA-Sir4p fusion. The data are consistent with a model in which the partitioning and anchoring domain of Sir4p (PAD4 domain) attaches to a nuclear component that divides symmetrically between cells at mitosis; DNA linked to Sir4p by LexA serves as a reporter of protein movement in these experiments. We infer that the segregation behavior of telomere- and silencer-based plasmids is, in part, a consequence of these Sir4p-mediated interactions. The assays presented herein illustrate two novel approaches to monitor the intracellular dynamics of nuclear proteins.

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