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1.
Int J Mol Sci ; 22(9)2021 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-34066830

RESUMEN

Protein solubility is based on the compatibility of the specific protein surface with the polar aquatic environment. The exposure of polar residues to the protein surface promotes the protein's solubility in the polar environment. The aquatic environment also influences the folding process by favoring the centralization of hydrophobic residues with the simultaneous exposure to polar residues. The degree of compatibility of the residue distribution, with the model of the concentration of hydrophobic residues in the center of the molecule, with the simultaneous exposure of polar residues is determined by the sequence of amino acids in the chain. The fuzzy oil drop model enables the quantification of the degree of compatibility of the hydrophobicity distribution observed in the protein to a form fully consistent with the Gaussian 3D function, which expresses an idealized distribution that meets the preferences of the polar water environment. The varied degrees of compatibility of the distribution observed with the idealized one allow the prediction of preferences to interactions with molecules of different polarity, including water molecules in particular. This paper analyzes a set of proteins with different levels of hydrophobicity distribution in the context of the solubility of a given protein and the possibility of complex formation.


Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Agregado de Proteínas , Proteínas Anticongelantes Tipo III/química , Proteínas Fimbrias/química , Hemoglobinas/química , Humanos , Proteínas de la Membrana/química , Modelos Moleculares , Dominios Proteicos , Solubilidad
2.
Phys Chem Chem Phys ; 21(39): 21836-21846, 2019 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-31552400

RESUMEN

Antifreeze proteins (AFPs) inhibit ice growth in various organisms at subzero temperature. Recently, AFPs as a hydrate inhibitor have been a topic of intense discussion, while the detailed mechanism remains obscure. The present work aims to explore molecular insight into the adsorption and inhibition of an AFP III on methane hydrate. Three polar, hydrophilic, and neutral amino acids (Asn14, Thr18, and Gln44) are mutated to elucidate the molecular mechanism of AFP III antifreeze activity. Another triple mutation is also designed to investigate the effect of the side chain. Atomistic molecular dynamics simulations provide detailed structural and dynamical aspects of protein residues and water molecules at the hydrate/water interface. Initially, it was proposed that the AFP III operates by the adsorption-inhibition mechanism on hydrates, almost similar to that of ice. The exchange of amide and hydroxyl groups by mutagenesis alters the shape of the side chain and the capability of hydrogen bonding and demonstrates that hydrogen bonds are not directly responsible for the AFP III antifreeze activity. Moreover, we deciphered that the length of the pendant group is an important factor in the entrapment of the AFP III on the hydrate cages, which is compatible with van der Waals interactions between the side chains and hydrate surface. The results suggest that this interaction is sensitive to the geometry and shape of the hydrate-binding surface (HBS) of the AFP, which implies that the interface between hydrates and the AFP is relatively rigid.


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Metano/química , Simulación de Dinámica Molecular , Adsorción , Secuencia de Aminoácidos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Mutación , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Propiedades de Superficie , Temperatura
3.
Phys Chem Chem Phys ; 20(42): 26926-26933, 2018 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-30260363

RESUMEN

Type III antifreeze proteins (AFP III) have been widely recognized as one class of ice-binding proteins produced by several biological organisms to withstand freezing conditions. Besides their ability to restrict ice growth through their ice-binding site (IBS), AFP III have also been shown to possess a great propensity for hydrophobic surfaces such as the air-water interface. Yet, it is not known whether AFP III adsorb with a specific orientation and how hydrophobic interactions affect the IBS. Molecular insights on the accessibility of the IBS and its interactions with water are important for understanding AFP III action in vivo but also for their application as ice-inhibiting agents for deicing, frozen food storage, as well as for long-term blood and organ cryo-preservation. Here, the orientation of fish AFP III adsorbed at the air-water interface has been studied using a combination of molecular dynamics (MD) simulations and vibrational sum-frequency generation (SFG) spectroscopy together with spectral calculations. The SFG/MD analysis indicated that when AFP III adsorbs at the air-water interface, it mostly retains its native state and orients with a tilt angle of 120° with respect to the surface normal. We found that the IBS is only partially solvated, leaving the pyramidal ice plane binding domain exposed to the vapor phase. These findings suggest that interactions with hydrophobic interfaces (e.g., cell membranes, polymers) could lead to the partial decoupling of the IBS from water and, to some extent, to a loss of AFP III antifreezing activity.


Asunto(s)
Proteínas Anticongelantes Tipo III/metabolismo , Hielo , Agua/metabolismo , Adsorción , Animales , Proteínas Anticongelantes Tipo III/química , Sitios de Unión , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Perciformes , Unión Proteica , Estructura Secundaria de Proteína , Análisis Espectral , Tensión Superficial , Vibración , Agua/química
4.
Phys Chem Chem Phys ; 20(10): 6996-7006, 2018 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-29468240

RESUMEN

We report on a molecular dynamics study on the relation between the structure and the orientational (and hydrogen bond) dynamics of hydration water around the ocean pout AFP III anti-freeze protein. We find evidence for an increasing tetrahedral structure from the area opposite to the ice binding site (IBS) towards the protein IBS, with the strongest signal of tetrahedral structure around the THR-18 residue of the IBS. The tetrahedral structural parameter mostly positively correlates with increased reorientation decay times. Interestingly, for several key (polar) residues that are not part of the IBS but are in its vicinity, we observe a decrease of the reorientation time with increasing tetrahedral structure. A similar anti-correlation is observed for the hydrogen-bonded water molecules. These effects are enhanced at a lower temperature. We interpret these results in terms of the structure-making and structure-breaking residues. Moreover, we investigate the tetrahedral structure and dynamics of waters at a partially dehydrated IBS, and for the protein adsorbed at the air-water interface. We find that the mutation changes the preferred protein orientation upon adsorption at an air-water interface. These results are in agreement with the water-air Vibration Sum Frequency Generation spectroscopic experiments showing a strongly reduced tetrahedral signal upon mutation at the IBS.


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Simulación de Dinámica Molecular , Agua/química , Sitios de Unión , Congelación , Enlace de Hidrógeno , Cinética , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad
5.
Comput Biol Chem ; 73: 13-24, 2018 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-29413812

RESUMEN

Antifreeze proteins are a class of biological molecules of interest in many research and industrial applications due to their highly specialized function, but there is little information of their stability and properties under varied pH derived from computational studies. To gain novel insights in this area, we conducted molecular dynamics (MD) simulations with the antifreeze protein 1KDF at varied temperatures and pH. Water solvation and H-bond formation around specific residues - ASN14, THR18 and GLN44 - involved in its antifreeze activity were extensively studied. We found that at pH1 there was a disruption in water solvation around the basal and the ice binding surfaces of the molecule. This was induced by a small change in the secondary structure propensities of some titrable residues, particularly GLU35. This change explains the experimentally observed reduction in antifreeze activity previously reported for this protein at pH1. We also found that THR18 showed extremely low H-bond formation, and that the three antifreeze residues all had very low average H-bond lifetimes. Our results confirm long-standing assumptions that these small, compact molecules can maintain their antifreeze activity in a wide range of pH, while demonstrating the mechanism that may reduce antifreeze activity at low pH. This aspect is useful when considering industrial and commercial use of antifreeze proteins subject to extreme pH environments, in particular in food industrial applications.


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Simulación de Dinámica Molecular , Concentración de Iones de Hidrógeno , Conformación Proteica , Solubilidad , Temperatura , Agua/química
6.
Biochem Biophys Res Commun ; 495(1): 1055-1060, 2018 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-29137985

RESUMEN

The aim of this study was to examine the effect of chemical cationization on the structure and function of antifreeze protein III (AFP III) over an extreme temperature range (-40°C to +90°C) using far-UV synchrotron radiation circular dichroism (SRCD) and ice recrystallization inhibition (IRI) assays. Chemical cationization was able to produce a modified AFP III with a net cationic charge at physiological pH that had enhanced resistance to denaturation at elevated temperatures, with no immediate negative impact on protein structure at subzero temperatures. Furthermore, cationized AFP III retained an IRI activity similar to that of native AFP III. Consequently, chemical cationization may provide a pathway to the development of more robust antifreeze proteins as supplementary cryoprotectants in the cryopreservation of clinically relevant cells.


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Proteínas Anticongelantes Tipo III/ultraestructura , Criopreservación/métodos , Cristalización/métodos , Hielo , Electricidad Estática , Ensayo de Materiales , Conformación Proteica , Desnaturalización Proteica , Relación Estructura-Actividad , Propiedades de Superficie , Temperatura
7.
J Biomol Struct Dyn ; 35(16): 3591-3604, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27882844

RESUMEN

Antifreeze proteins (AFPs), found in certain vertebrates, plants, fungi and bacteria have the ability to permit their survival in subzero environments by thermal hysteresis mechanism. However, the exact mechanism of ice growth inhibition is still not clearly understood. Here, four long explicit molecular dynamics (MD) simulations have been carried out at two different temperatures (277 and 298 K) with and without glycan to study the conformational rigidity of the Ocean pout type III antifreeze protein in aqueous medium and the structural arrangements of water molecules hydrating its ice-binding surface. It is found that irrespective of the temperature the ice-binding surface (IBS) of the protein is relatively more rigid than its non ice-binding surface (NonIBS) in its native and glycosylated form. Hydrophilic residues N14, T18 and Q44 are essential to antifreeze activity. Radial distribution, density distribution function and nearest neighbor orientation plots with respect to individual two surfaces confirm that density of water molecule near these binding surface in native and glycosylated form are relatively more than the nonbinding surface. The glycosylated form shows a strong peak than the native one. From rotational auto correlation function of water molecules around ice-binding sites, it is prominent that with increase in temperature, strong interaction between the water oxygen and the hydrogen bond acceptor group on the protein-binding surface decreases. This provides a possible molecular reason behind the ice-binding activity of ocean pout at the prism plane of ice.


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Proteínas de Peces/química , Simulación de Dinámica Molecular , Agua/química , Animales , Sitios de Unión , Glicosilación , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Perciformes/fisiología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Electricidad Estática , Temperatura , Termodinámica
8.
FEBS Lett ; 590(23): 4202-4212, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27718246

RESUMEN

The quaternary-amino-ethyl 1 (QAE1) isoforms of type III antifreeze proteins (AFPs) prevent the growth of ice crystals within organisms living in polar regions. We determined the antifreeze activity of wild-type and mutant constructs of the Japanese notched-fin eelpout (Zoarces elongates Kner) AFP8 (nfeAFP8) and characterized the structural and dynamics properties of their ice-binding surface using NMR. We found that the three constructs containing the V20G mutation were incapable of stopping the growth of ice crystals and exhibited structural changes, as well as increased conformational flexibility, in the first 310 helix (residues 18-22) of the sequence. Our results suggest that the inactive nfeAFP8s are incapable of anchoring water molecules due to the unusual and flexible backbone conformation of their primary prism plane-binding surface.


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Proteínas Anticongelantes Tipo III/metabolismo , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Perciformes , Secuencia de Aminoácidos , Animales , Proteínas Anticongelantes Tipo III/genética , Proteínas de Peces/genética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutación , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Temperatura
9.
J Chem Phys ; 145(7): 075101, 2016 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-27544127

RESUMEN

Water molecules from the solvation shell of the ice-binding surface are considered important for the antifreeze proteins to perform their function properly. Herein, we discuss the problem whether the extent of changes of the mean properties of solvation water can be connected with the antifreeze activity of the protein. To this aim, the structure of solvation water of a type III antifreeze protein from Macrozoarces americanus (eel pout) is investigated. A wild type of the protein is used, along with its three mutants, with antifreeze activities equal to 54% or 10% of the activity of the native form. The solvation water of the ice-binding surface and the rest of the protein are analyzed separately. To characterize the structure of solvation shell, parameters describing radial and angular characteristics of the mutual arrangement of the molecules were employed. They take into account short-distance (first hydration shell) or long-distance (two solvation shells) effects. The obtained results and the comparison with the results obtained previously for a hyperactive antifreeze protein from Choristoneura fumiferana lead to the conclusion that the structure and amino acid composition of the active region of the protein evolved to achieve two goals. The first one is the modification of the properties of the solvation water. The second one is the geometrical adjustment of the protein surface to the specific crystallographic plane of ice. Both of these goals have to be achieved simultaneously in order for the protein to perform its function properly. However, they seem to be independent from one another in a sense that very small antifreeze activity does not imply that properties of water become different from the ones observed for the wild type. The proteins with significantly lower activity still modify the mean properties of solvation water in a right direction, in spite of the fact that the accuracy of the geometrical match with the ice lattice is lost because of the mutations. Therefore, we do not observe any correlation between the antifreeze activity and the extent of modification of the properties of solvation water.


Asunto(s)
Proteínas Anticongelantes Tipo III , Agua/química , Animales , Proteínas Anticongelantes Tipo III/química , Proteínas Anticongelantes Tipo III/genética , Proteínas Anticongelantes Tipo III/metabolismo , Antenas de Artrópodos/química , Dominio Catalítico , Mutación , Perciformes , Solventes/química
10.
J Reprod Dev ; 61(1): 1-6, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25311466

RESUMEN

Embryos obtained via superovulation are necessary for mammalian artificial reproduction, and viability is a key determinant of success. Nonfreezing storage at 4 C is possible, but currently used storage solutions can maintain embryo viability for only 24-48 h. Here we found that 10 mg/ml antifreeze protein (AFP) dissolved in culture medium 199 with 20% (v/v) fetal bovine serum and 25 mM HEPES could keep bovine embryos alive for 10 days at 4 C. We used a recombinant AFP isolated from the notched-fin eelpout (Zoarces elongatus Kner). Photomicroscopy indicated that the AFP-embryo interaction was enhanced at 37 C. Embryos pre-warmed with the AFP solution at 37 C for 60 min maintained high viability, whereas those that were not pre-warmed could live no longer than 7 days. Thus, short-term storage of bovine embryos was achieved by a combination of AFP-containing medium and controlled pre-warming.


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Criopreservación/métodos , Animales , Bovinos , Supervivencia Celular , Frío , Medios de Cultivo , Técnicas de Cultivo de Embriones , Proteínas de Peces/química , Microscopía Fluorescente , Perciformes , Proteínas Recombinantes/química , Factores de Tiempo
11.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 12): 3266-72, 2014 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-25478844

RESUMEN

The 1.8 Šresolution neutron structure of deuterated type III antifreeze protein in which the methyl groups of leucine and valine residues are selectively protonated is presented. Comparison between this and the 1.85 Šresolution neutron structure of perdeuterated type III antifreeze protein indicates that perdeuteration improves the visibility of solvent molecules located in close vicinity to hydrophobic residues, as cancellation effects between H atoms of the methyl groups and nearby heavy-water molecules (D2O) are avoided.


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Proteínas de Peces/química , Difracción de Neutrones/métodos , Perciformes , Animales , Deuterio/química , Modelos Moleculares , Perciformes/metabolismo , Protones , Solventes/química , Agua/química
12.
J Phys Chem B ; 118(30): 8962-71, 2014 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-25051212

RESUMEN

We performed time- and polarization-resolved pump-probe and two-dimensional infrared (2D-IR) experiments to study the dynamics of the amide I vibration of a 7 kDa type-III antifreeze protein. In the pump-probe experiments, we used femtosecond mid-infrared pulses to investigate the vibrational relaxation dynamics of the amide mode. The transient spectra show the presence of two spectral components that decay with different lifetimes, indicative of the presence of two distinct amide subbands. The 2D-IR experiments reveal the coupling between the two bands in the form of cross-peaks. On the basis of previous work by Demirdöven et al. ( J. Am. Chem. Soc. 2004 , 126 , 7981 - 7990 ), we assign the observed bands to the two infrared-active modes α(-) and α(+) found in protein ß-sheets. The amplitudes of the cross-peak were found to increase with delay time, indicating that the cross-peaks originate from population transfer between the coupled amide oscillators. The time constant of the energy transfer was found to be 6-7 ps.


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Animales , Anisotropía , Elasticidad , Cinética , Modelos Moleculares , Perciformes , Estructura Secundaria de Proteína , Espectrofotometría Infrarroja , Temperatura , Vibración , Difracción de Rayos X
14.
FEBS Lett ; 586(21): 3876-81, 2012 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-23017208

RESUMEN

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.


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Hielo/análisis , Mutación , Secuencia de Aminoácidos , Animales , Proteínas Anticongelantes Tipo III/genética , Proteínas Anticongelantes Tipo III/metabolismo , Sitios de Unión , Anguilas , Escherichia coli/genética , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Ingeniería de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Relación Estructura-Actividad
15.
J Mol Recognit ; 24(4): 724-32, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21472814

RESUMEN

Antifreeze proteins (AFPs) inhibit ice growth at sub-zero temperatures. The prototypical type-III AFPs have been extensively studied, notably by X-ray crystallography, solid-state and solution NMR, and mutagenesis, leading to the identification of a compound ice-binding surface (IBS) composed of two adjacent ice-binding sections, each which binds to particular lattice planes of ice crystals, poisoning their growth. This surface, including many hydrophobic and some hydrophilic residues, has been extensively used to model the interaction of AFP with ice. Experimentally observed water molecules facing the IBS have been used in an attempt to validate these models. However, these trials have been hindered by the limited capability of X-ray crystallography to reliably identify all water molecules of the hydration layer. Due to the strong diffraction signal from both the oxygen and deuterium atoms, neutron diffraction provides a more effective way to determine the water molecule positions (as D(2) O). Here we report the successful structure determination at 293 K of fully perdeuterated type-III AFP by joint X-ray and neutron diffraction providing a very detailed description of the protein and its solvent structure. X-ray data were collected to a resolution of 1.05 Å, and neutron Laue data to a resolution of 1.85 Å with a "radically small" crystal volume of 0.13 mm(3). The identification of a tetrahedral water cluster in nuclear scattering density maps has allowed the reconstruction of the IBS-bound ice crystal primary prismatic face. Analysis of the interactions between the IBS and the bound ice crystal primary prismatic face indicates the role of the hydrophobic residues, which are found to bind inside the holes of the ice surface, thus explaining the specificity of AFPs for ice versus water.


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Hielo , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Difracción de Neutrones , Neutrones
16.
Biochemistry ; 49(42): 9063-71, 2010 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-20853841

RESUMEN

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 150° 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.


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Proteínas de Peces/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Proteínas Anticongelantes Tipo III/genética , Sitios de Unión/genética , Clonación Molecular , Proteínas de Peces/genética , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Hielo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Perciformes/genética , Perciformes/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Homología de Secuencia de Aminoácido
17.
Proteins ; 78(14): 2973-83, 2010 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-20737588

RESUMEN

Kinetic measurement of protein folding is limited by the method used to trigger folding. Traditional methods, such as stopped flow, have a long mixing dead time and cannot be used to monitor fast folding processes. Here, we report a compound, 4-(bromomethyl)-6,7-dimethoxycoumarin, that can be used as a "photolabile cage" to study the early stages of protein folding. The folding process of a protein, RD1, including kinetics, enthalpy, and volume change, was studied by the combined use of a phototriggered caging strategy and time-resolved photoacoustic calorimetry. The cage caused unfolding of the photolabile protein, and then a pulse UV laser (∼10(-9) s) was used to break the cage, leaving the protein free to refold and allowing the resolving of two folding events on a nanosecond time scale. This strategy is especially good for monitoring fast folding proteins that cannot be studied by traditional methods.


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Cumarinas/química , Rayos Láser , Pliegue de Proteína , Rayos Ultravioleta , Secuencia de Aminoácidos , Calorimetría , Dicroismo Circular , Simulación por Computador , Cinética , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
18.
Biophys J ; 99(2): 609-18, 2010 Jul 21.
Artículo en Inglés | MEDLINE | ID: mdl-20643081

RESUMEN

It has been suggested that above a critical protein concentration, fish Type III antifreeze protein (AFP III) self-assembles to form micelle-like structures that may play a key role in antifreeze activity. To understand the complex activity of AFP III, a comprehensive description of its association state and structural organization in solution is necessary. We used analytical ultracentrifugation, analytical size-exclusion chromatography, and dynamic light scattering to characterize the interactions and homogeneity of AFP III in solution. Small-angle neutron scattering was used to determine the low-resolution structure in solution. Our results clearly show that at concentrations up to 20 mg mL(-1) and at temperatures of 20 degrees C, 6 degrees C, and 4 degrees C, AFP III is monomeric in solution and adopts a structure compatible with that determined by crystallography. Surface tension measurements show a propensity of AFP III to localize at the air/water interface, but this surface activity is not correlated with any aggregation in the bulk. These results support the hypothesis that each AFP III molecule acts independently of the others, and that specific intermolecular interactions between monomers are not required for binding to ice. The lack of attractive interactions between monomers may be functionally important, allowing for more efficient binding and covering of the ice surface.


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Proteínas Anticongelantes Tipo III/metabolismo , Aire , Animales , Cromatografía en Gel , Luz , Modelos Moleculares , Peso Molecular , Difracción de Neutrones , Dispersión de Radiación , Dispersión del Ángulo Pequeño , Soluciones , Tensión Superficial , Ultracentrifugación , Agua/química
19.
Artículo en Inglés | MEDLINE | ID: mdl-20516595

RESUMEN

Antifreeze proteins (AFPs) are found in different species from polar, alpine and subarctic regions, where they serve to inhibit ice-crystal growth by adsorption to ice surfaces. Recombinant North Atlantic ocean pout (Macrozoarces americanus) AFP has been used as a model protein to develop protocols for amino-acid-specific hydrogen reverse-labelling of methyl groups in leucine and valine residues using Escherichia coli high-density cell cultures supplemented with the amino-acid precursor alpha-ketoisovalerate. Here, the successful methyl protonation (methyl reverse-labelling) of leucine and valine residues in AFP is reported. Methyl-protonated AFP was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Crystals were grown in D(2)O buffer by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed in a few 24 h exposures a very low background and clear small spots up to a resolution of 1.80 A from a crystal of dimensions 1.60 x 0.38 x 0.38 mm corresponding to a volume of 0.23 mm(3).


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Peces , Protones , Secuencia de Aminoácidos , Animales , Proteínas Anticongelantes Tipo III/genética , Cristalización , Expresión Génica , Leucina/química , Metilación , Datos de Secuencia Molecular , Difracción de Neutrones , Valina/química
20.
Artículo en Inglés | MEDLINE | ID: mdl-19342793

RESUMEN

The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D(2)O by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed that with a few exposures of 24 h a very low background and clear small spots up to a resolution of 1.85 A were obtained using a ;radically small' perdeuterated AFP D crystal of dimensions 0.70 x 0.55 x 0.35 mm, corresponding to a volume of 0.13 mm(3).


Asunto(s)
Proteínas Anticongelantes Tipo III/química , Proteínas Anticongelantes Tipo III/aislamiento & purificación , Deuterio/química , Difracción de Neutrones , Perciformes/metabolismo , Animales , Cristalización , Electroforesis en Gel de Poliacrilamida , Océanos y Mares
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