Structural Studies of Antarctic Fish Antifreeze Glycoproteins by One- and Two-Dimensional NMR Spectroscopy

Dill, Kilian; Huang, Le Hui; Bearden, Daniel W.; Feeney, Robert E.

doi:10.1080/07328309208017809

Cited by 12 publications

(11 citation statements)

References 24 publications

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“…Even though the study on synthetic AFGPs by Nishimura and coworkers presents a consistent picture the situation for naturally occurring AFGPs seems to be more complicated [167]. Structural studies on these molecules, isolated from fish, have a long history yielding conflicting interpretations [170][171][172][173][174][175].…”

Section: Antifreeze Glycoproteinsmentioning

confidence: 99%

Conformation of Glycopeptides and Glycoproteins

Meyer¹,

Moeller²

2007

ChemInform

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Glycosylation is the most frequent post-translational modification found in proteins. Glycoproteins are involved in a highly diverse spectrum of biological functions, ranging from protein folding and molecular and cellular recognition to attack of pathogens and regulation of biological half-life. Consequently, understanding the structural role of glycosylation provides key insight into biological processes as well as ideas for medicinal applications. Here, we review the current methodology to arrive at highresolution structures of glycopeptides and glycoproteins. To achieve this, significant obstacles have to be overcome like preparation of sufficient amounts of sufficiently pure and homogeneous material, analyzing highly ambiguous spectroscopic data, and dealing with molecules that show local or global disorder. We will provide an overview of our knowledge of glycoprotein and glycopeptide structure, and illustrate with selected examples the performance and limitations of current methodology.

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Section: Antifreeze Glycoproteinsmentioning

confidence: 99%

Conformation of Glycopeptides and Glycoproteins

Meyer¹,

Moeller²

2007

ChemInform

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“…3) are not due to a structural difference. 2D NMR studies (300 MHz) [89] allowed further refinement of this data and measurement of amide exchange rates which ruled out significant strong hydrogen bonding involving the amide protons in aqueous solutions. Comparison of AFGP amide vibrational frequencies with those observed and calculated for beta and gamma‐turns in other peptides suggests that AFGPs contain substantial turn structure [90] while NMR studies on model glycopeptides showed an intramolecular hydrogen bond between the amide proton of N ‐acetylgalactosamine and the carbonyl oxygen of the Thr to which the sugar is attached [91].…”

Section: Solution Conformationmentioning

confidence: 99%

‘Antifreeze’ glycoproteins from polar fish

Harding

Anderberg

Haymet

2003

European Journal of Biochemistry

255

200

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Antifreeze glycoproteins (AFGPs) constitute the major fraction of protein in the blood serum of Antarctic notothenioids and Arctic cod. Each AFGP consists of a varying number of repeating units of (Ala-Ala-Thr) n , with minor sequence variations, and the disaccharide b-D-galactosyl-(1fi3)-a-N-acetyl-D-galactosamine joined as a glycoside to the hydroxyl oxygen of the Thr residues. These compounds allow the fish to survive in subzero ice-laden polar oceans by kinetically depressing the temperature at which ice grows in a noncolligative manner. In contrast to the more widely studied antifreeze proteins, little is known about the mechanism of ice growth inhibition by AFGPs, and there is no definitive model that explains their properties. This review summarizes the structural and physical properties of AFGPs and advances in the last decade that now provide opportunities for further research in this field.High field NMR spectroscopy and molecular dynamics studies have shown that AFGPs are largely unstructured in aqueous solution. While standard carbohydrate degradation studies confirm the requirement of some of the sugar hydroxyls for antifreeze activity, the importance of following structural elements has not been established: (a) the number of hydroxyls required, (b) the stereochemistry of the sugar hydroxyls (i.e. the requirement of galactose as the sugar), (c) the acetamido group on the first galactose sugar, (d) the stereochemistry of the b-glycosidic linkage between the two sugars and the a-glycosidic linkage to Thr, (e) the requirement of a disaccharide for activity, and (f) the Ala and Thr residues in the polypeptide backbone. The recent successful synthesis of small AFGPs using solution methods and solidphase chemistry provides the opportunity to perform key structure-activity studies that would clarify the important residues and functional groups required for activity.Genetic studies have shown that the AFGPs present in the two geographically and phylogenetically distinct Antarctic notothenioids and Arctic cod have evolved independently, in a rare example of convergent molecular evolution. The AFGPs exhibit concentration dependent thermal hysteresis with maximum hysteresis (1.2°C at 40 mgAEmL )1 ) observed with the higher molecular mass glycoproteins. The ability to modify the rate and shape of crystal growth and protect cellular membranes during lipid-phase transitions have resulted in identification of a number of potential applications of AFGPs as food additives, and in the cryopreservation and hypothermal storage of cells and tissues.

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“…Furthermore, AFGP-8 is the only AFGP found in the tissue samples outside the blood stream (Burcham et al, 1986;Mulvihill et al, 1980). The x-ray structure for AFGP-8 is not yet known, but experimental work on the structural elucidation of this AFGP using a variety of experimental techniques, such as nuclear magnetic resonance (NMR) and ultra-ultraviolet circular dichroism (CD) (Bush et al, 1981(Bush et al, , 1984Rao and Bush, 1987;Filira et al, 1990;Dill et al, 1992;Mimura et al, 1992;Lane et al, 1998) have yielded rather confusing and conflicting messages, from the standpoint of structural biology. NMR work by Bush et al and Lane et al suggested that, because NMR spectra show an absence of long-range coupling, AFGP-8 does not have well-defined structure at all.…”

Section: Introductionmentioning

confidence: 99%

The Dynamics, Structure, and Conformational Free Energy of Proline-Containing Antifreeze Glycoprotein

Nguyen

Colvin

Yeh

et al. 2002

Biophysical Journal

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Recent NMR studies of the solution structure of the 14-amino acid antifreeze glycoprotein AFGP-8 have concluded that the molecule lacks long-range order. The implication that an apparently unstructured molecule can still have a very precise function as a freezing inhibitor seems startling at first consideration. To gain insight into the nature of conformations and motions in AFGP-8, we have undertaken molecular dynamics simulations augmented with free energy calculations using a continuum solvation model. Starting from 10 different NMR structures, 20 ns of dynamics of AFGP were explored. The dynamics show that AFGP structure is composed of four segments, joined by very flexible pivots positioned at alanine 5, 8, and 11. The dynamics also show that the presence of prolines in this small AFGP structure facilitates the adoption of the poly-proline II structure as its overall conformation, although AFGP does adopt other conformations during the course of dynamics as well. The free energies calculated using a continuum solvation model show that the lowest free energy conformations, while being energetically equal, are drastically different in conformations. In other words, this AFGP molecule has many structurally distinct and energetically equal minima in its energy landscape. In addition, conformational, energetic, and hydrogen bond analyses suggest that the intramolecular hydrogen bonds between the N-acetyl group and the protein backbone are an important integral part of the overall stability of the AFGP molecule. The relevance of these findings to the mechanism of freezing inhibition is discussed.

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Structural Studies of Antarctic Fish Antifreeze Glycoproteins by One- and Two-Dimensional NMR Spectroscopy

Cited by 12 publications

References 24 publications

Conformation of Glycopeptides and Glycoproteins

Conformation of Glycopeptides and Glycoproteins

‘Antifreeze’ glycoproteins from polar fish

The Dynamics, Structure, and Conformational Free Energy of Proline-Containing Antifreeze Glycoprotein

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