determining-ice-binding-planes-antifreeze-proteins-fluorescence-based

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Embryonic stem (ES) cell lines represent a population of undifferentiated pluripotent cells capable of multilineage differentiation in vitro. Although very useful for studying developmental processes, human ES cell lines have also been suggested as a potential and unlimited source for cellular transplantation and the treatment of human disease. The proteomic basis of embryonic stemness (pluripotentiality and multilineage differentiation) and the transitions that lead to specific cell lineages however, remain to be defined. As an important first step in defining these processes, we have performed a proteomic analysis of undifferentiated mouse R1 ES cell lines using pH 3-10, 4-7 and 6-11 two-dimensional electrophoresis gels, matrix-assisted laser desorption/ionization and tandem mass spectrometry. Of the 700 gel spots analyzed, 241 distinct protein species were identified corresponding to 218 unique proteins, with a significant proportion functionally related to protein expression.

Two-dimensional gel electrophoresis database of murine R1 embryonic stem cells.

Two-dimensional gel electrophoresis (2-DE) is widely used for initial protein separation in proteomics. Commercial products using neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis ((SDS-PAGE)/(Bis (2-hydroxyethyl) imino-tris (hydroxymethyl) methane-HCl, or Bis-Tris)) have greatly improved this technique, but cost and limited sizes restrict their applications. An "in-house" system is presented, resulting in better resolution, separation, and new spot visualization and improved resolution when compared to Tris-HCl gels. Their utility is demonstrated using albumin-depleted serum samples, rabbit heart left ventricle, and human immunodeficiency virus type 1 (HIV-1).

Improvements in two-dimensional gel electrophoresis by utilizing a low cost "in-house" neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis system.

Serum is a readily available source for diagnostic assays, but the identification of disease-specific serum biomarkers has been impeded by the dominance of human serum albumin and immunoglobulins (Igs) in the serum proteome. There is a need to reduce the technical variation in serum processing and analysis to allow for a reproducible analysis of large cohorts. To this end, we have developed a rapid and reproducible procedure for sample preparation and high-resolution two-dimensional gel electrophoresis to analyze human serum. Serum is centrifuged at high speed to remove lipids and aggregated proteins, incubated with protein G resin to remove IgG, precipitated with NaCl/ethanol to deplete albumin, and slowly resolubilized in a sodium dodecyl sulfate (SDS)/N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) buffer. The delipidated and IgG/albumin depleted serum proteins are focused on pH 4-7 linear large immobilized pH gradient strips, and then resolved by Bis-Tris SDS-polyacrylamide gel electrophoresis. The robustness and reproducibility of the optimized procedure was determined for three individual serum samples on three consecutive days. An image analysis of the nine silver-stained gels demonstrated that the intensity and localization of protein spots are highly reproducible. Our IgG and albumin depletion procedure will aid in screening the patient sera for normal biological variation and disease-specific biomarkers.

A robust, streamlined, and reproducible method for proteomic analysis of serum by delipidation, albumin and IgG depletion, and two-dimensional gel electrophoresis.

Antifreeze proteins (AFPs) bind to the surface of ice crystals and lower the non-equilibrium freezing temperature of the icy solution below its melting point. We have recently reported the discovery of three novel hyperactive AFPs from a bacterium, a primitive insect and a fish, which, like two hyperactive AFPs previously recognized in beetles and moths, are considerably better at depressing the freezing point than most fish AFPs. When cooled below the non-equilibrium freezing temperature, ice crystals formed in the presence of any of five distinct, moderately active fish AFPs grow suddenly along the c-axis. Ice crystals formed in the presence of any of the five evolutionarily and structurally distinct hyperactive AFPs remain stable to lower temperatures, and then grow explosively in a direction normal to the c-axis when cooled below the freezing temperature. We argue that this one consistent distinction in the behaviour of these two classes of AFPs is the key to hyperactivity. Whereas both AFP classes bind irreversibly to ice, the hyperactive AFPs are better at preventing ice growth out of the basal planes.

The basis for hyperactivity of antifreeze proteins.

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 degrees 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 beta-roll from alkaline protease. The model has identified both a novel beta-helical fold and an ice-binding site. The interior of the beta-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.AFPS

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

Loss-of-function mutations in calpain 3 have been shown to cause limb-girdle muscular dystrophy type 2A (LGMD2A), an autosomal recessive disorder that results in gradual wasting of the muscles of the hip and shoulder areas. Due to the inherent instability of calpain 3, recombinant expression of the full-length enzyme has not been possible, making in vitro analysis of specific LGMD2A-causing mutations difficult. However, because calpain 3 is highly similar in amino acid sequence to calpain 2, the recently solved crystal structure of full-length, Ca2+-bound, calpastatin-inhibited rat calpain 2 has allowed us to model calpain 3 as a Ca2+-bound homodimer. The model revealed three distinct areas of the enzyme that undergo a large conformational change upon Ca2+ binding. Located in these areas are several residues that undergo mutation to cause LGMD2A. We investigated the in vitro effects of six of these mutations by making the corresponding mutations in rat calpain 2. All six mutations examined in this study resulted in a decrease in enzyme activity. All but one of the mutations caused an increased rate of autoproteolytic degradation of the enzyme as witnessed by SDS-PAGE, indicating the decrease in enzyme activity is caused, at least in part, by an increase in the rate of autoproteolytic degradation. The putative in vivo effects of these mutations on calpain 3 activity are discussed with respect to their ability to cause LGMD2A.

Limb-girdle muscular dystrophy type 2A can result from accelerated autoproteolytic inactivation of calpain 3.

By binding to the surface of ice crystals, type III antifreeze protein (AFP) can depress the freezing point of fish blood to below that of freezing seawater. This 7-kDa globular protein is encoded by a multigene family that produces two major isoforms, SP and QAE, which are 55% identical. Disruptive mutations on the ice-binding site of type III AFP lower antifreeze activity but can also change ice crystal morphology. By attaching green fluorescent protein to different mutants and isoforms and by examining the binding of these fusion proteins to single-crystal ice hemispheres, we show that type III AFP has a compound ice-binding site. There are two adjacent, flat, ice-binding surfaces at 150Â° to each other. One binds the primary prism plane of ice; the other, a pyramidal plane. Steric mutations on the latter surface cause elongation of the ice crystal as primary prism plane binding becomes dominant. SP isoforms naturally have a greatly reduced ability to bind the prism planes of ice. Mutations that make the SP isoforms more QAE-like slow down the rate of ice growth. On the basis of these observations we postulate that other types of AFP also have compound ice-binding sites that enable them to bind to multiple planes of ice.

Compound ice-binding site of an antifreeze protein revealed by mutagenesis and fluorescent tagging.

Durch Bindung an die Oberfl&auml;che von Eiskristallen, Typ III Frostschutzmittel Protein (AFP) kann den Gefrierpunkt Fische dr&uuml;cken Blut bis unter dem Gefrierpunkt Meerwasser. Diese kugelige 7-kDa-Protein wird von einer multigene Familie kodiert, das produziert zwei gro&szlig;e Isoformen, SP und QAE, die 55 % identisch sind. Disruptive Mutationen auf die Eis-Bindungsstelle des Typs III AFP Frostschutzmittel Aktivit&auml;t senken k&ouml;nnen aber auch &Auml;nderung Eis Kristall Morphologie. Durch Anf&uuml;gen des gr&uuml;n fluoreszierenden Proteins zu verschiedenen Mutanten und Isoformen und untersuchen die Bindung des diese Schmelzverfahrensproteine zu Einzel-Kristall Eis Hemisph&auml;ren zeigen wir, dass Typ III AFP eine zusammengesetzte Eis-Bindungsstelle. Es gibt zwei angrenzende, flache, Eis-Bindung Oberfl&auml;chen bei 150 &deg; zueinander. Man bindet das prim&auml;re Prism-Flugzeug von Eis; andererseits ein pyramidaler Flugzeug. Sterische Mutationen auf der letzteren Oberfl&auml;che bewirken Dehnung der Eiskristall als prim&auml;re Prism Flugzeug Bindung dominant wird. SP haben Isoformen nat&uuml;rlich eine stark reduzierte F&auml;higkeit, die Prisma-Flugzeuge des Eises zu binden. Mutationen, die den SP-Isoformen weitere QAE machen-wie die Wachstumsrate Eis verlangsamen. Auf der Grundlage dieser Beobachtungen postulieren wir, dass andere Arten von AFP auch zusammengesetzte Eis-Bindungsstellen, die erm&ouml;glichen, auf mehrere Ebenen des Eises zu binden.

Zusammengesetzte Eis-Bindungsstelle eines Frostschutzmittel-Proteins durch Mutagenese und fluoreszierende tagging aufgedeckt.

The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca(2+)-bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFPice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the "anchored clathrate" mechanism of AFP action.

Anchored clathrate waters bind antifreeze proteins to ice.

Ice nucleation proteins (INPs) allow water to freeze at high subzero temperatures. Due to their large size (>120 kDa), membrane association, and tendency to aggregate, an experimentally-determined tertiary structure of an INP has yet to be reported. How they function at the molecular level therefore remains unknown.

Novel dimeric β-helical model of an ice nucleation protein with bridged active sites.

Cellular microtubules are marked by abundant and evolutionarily conserved post-translational modifications that have the potential to tune their functions. This review focuses on the astonishing chemical complexity introduced in the tubulin heterodimer at the post-translational level and summarizes the recent advances in identifying the enzymes responsible for these modifications and deciphering the consequences of tubulin's chemical diversity on the function of molecular motors and microtubule associated proteins.

The chemical complexity of cellular microtubules: tubulin post-translational modification enzymes and their roles in tuning microtubule functions.

Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis. Its main structural element is an irregular β-helix with six loops of 18 or more residues that lies alongside an α-helix. β-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the β-helical fold is the nonsequential arrangement of loops that places the N- and C termini inside the solenoid of β-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi.

Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation.

Type III antifreeze proteins (AFPs) can be sub-divided into three classes of isoforms. SP and QAE2 isoforms can slow, but not stop, the growth of ice crystals by binding to pyramidal ice planes. The other class (QAE1) binds both pyramidal and primary prism planes and is able to halt the growth of ice. Here we describe the conversion of a QAE2 isoform into a fully-active QAE1-like isoform by changing four surface-exposed residues to develop a primary prism plane binding site. Molecular dynamics analyses suggest that the basis for gain in antifreeze activity is the formation of ice-like waters on the mutated protein surface.

Engineering a naturally inactive isoform of type III antifreeze protein into one that can stop the growth of ice.

A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca(2+) levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca(2+)-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice.

Re-evaluation of a bacterial antifreeze protein as an adhesin with ice-binding activity.

Tubulin is subject to a reversible post-translational modification involving polyglutamylation and deglutamylation of glutamate residues in its C-terminal tail. This process plays key roles in regulating the function of microtubule associated proteins, neuronal development, and metastatic progression. This study describes the synthesis and testing of three phosphinic acid-based inhibitors that have been designed to inhibit both the glutamylating and deglutamylating enzymes. The compounds were tested against the polyglutamylase TTLL7 using tail peptides as substrates (100 μM) and the most potent inhibitor displayed an IC₅₀ value of 150 μM. The incorporation of these compounds into tubulin C-terminal tail peptides may lead to more potent TTLL inhibitors.

Phosphinic acid-based inhibitors of tubulin polyglutamylases.

A Ca(2+) -dependent 1.5-MDa antifreeze protein present in an Antarctic Gram-negative bacterium, Marinomonas primoryensis (MpAFP), has recently been reassessed as an ice-binding adhesin. The non-ice-binding region II (RII), one of five distinct domains in MpAFP, constitutes ~ 90% of the protein. RII consists of ~ 120 tandem copies of an identical 104-residue sequence. We used the Protein Homology/analogy Recognition Engine server to define the boundaries of a single 104-residue RII construct (RII monomer). CD demonstrated that Ca(2+) is required for RII monomer folding, and that the monomer is fully structured at a Ca(2+) /protein molar ratio of 10 : 1. The crystal structure of the RII monomer was solved to a resolution of 1.35 Å by single-wavelength anomalous dispersion and molecular replacement methods with Ca(2+) as the heavy atom to obtain phase information. The RII monomer folds as a Ca(2+) -bound immunoglobulin-like β-sandwich. Ca(2+) ions are coordinated at the interfaces between each RII monomer and its symmetry-related molecules, suggesting that these ions may be involved in the stabilization of the tandemly repeated RII. We hypothesize that > 600 Ca(2+) ions help to rigidify the chain of 104-residue repeats in order to project the ice-binding domain of MpAFP away from the bacterial cell surface. The proposed role of RII is to help the strictly aerobic bacterium bind surface ice in an Antarctic lake for better access to oxygen and nutrients. This work may give insights into other bacterial proteins that resemble MpAFP, especially those of the large repeats-in-toxin family that have been characterized as adhesins exported via the type I secretion pathway.

Article	Year
Two-dimensional gel electrophoresis database of murine R1 embryonic stem cells. Proteomics\| PubMed ID: 15378706	2004
Improvements in two-dimensional gel electrophoresis by utilizing a low cost "in-house" neutral pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Proteomics\| PubMed ID: 15887185	2005
A robust, streamlined, and reproducible method for proteomic analysis of serum by delipidation, albumin and IgG depletion, and two-dimensional gel electrophoresis. Proteomics\| PubMed ID: 15924293	2005
The basis for hyperactivity of antifreeze proteins. Cryobiology\| PubMed ID: 16887111	2006
A Ca2+-dependent bacterial antifreeze protein domain has a novel beta-helical ice-binding fold. The Biochemical journal\| PubMed ID: 18095937	2008
Limb-girdle muscular dystrophy type 2A can result from accelerated autoproteolytic inactivation of calpain 3. Biochemistry\| PubMed ID: 19226146	2009
Compound ice-binding site of an antifreeze protein revealed by mutagenesis and fluorescent tagging. Biochemistry\| PubMed ID: 20853841	2010
Anchored clathrate waters bind antifreeze proteins to ice. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 21482800	2011
Novel dimeric β-helical model of an ice nucleation protein with bridged active sites. BMC structural biology\| PubMed ID: 21951648	2011
The chemical complexity of cellular microtubules: tubulin post-translational modification enzymes and their roles in tuning microtubule functions. Cytoskeleton (Hoboken, N.J.)\| PubMed ID: 22422711	2012
Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22645341	2012
Engineering a naturally inactive isoform of type III antifreeze protein into one that can stop the growth of ice. FEBS letters\| PubMed ID: 23017208	2012
Re-evaluation of a bacterial antifreeze protein as an adhesin with ice-binding activity. PloS one\| PubMed ID: 23144980	2012
Phosphinic acid-based inhibitors of tubulin polyglutamylases. Bioorganic & medicinal chemistry letters\| PubMed ID: 23777780	2013
Role of Ca²⁺ in folding the tandem β-sandwich extender domains of a bacterial ice-binding adhesin. The FEBS journal\| PubMed ID: 24024640	2013

Christopher P. Garnham

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