Many flavoproteins are non-covalent complexes between FMN and an apoprotein. To understand better the stability of flavoproteins, we have studied the energetics of the complex between FMN and the apoflavodoxin from Anabaena PCC 7119 by a combination of site-directed mutagenesis, titration calorimetry, equilibrium binding constant determinations, and x-ray crystallography. Comparison of the strength of the wild type and mutant apoflavodoxin-FMN complexes and that of the complexes between wild type apoflavodoxin and shortened FMN analogues (riboflavin and lumiflavin) allows the dissection of the binding energy into contributions associated with the different parts of the FMN molecule. The estimated contribution of the phosphate is greatest, at 7 kcal mol ؊1 ; that of the isoalloxazine is of around 5-6 kcal mol ؊1 (mainly due to interaction with Trp-57 and Tyr-94 in the apoprotein) and the ribityl contributes least: around 1 kcal mol ؊1 . The stabilization of the complex is both enthalpic and entropic although the enthalpy contribution is dominant. Both the phosphate and the isoalloxazine significantly contribute to the enthalpy of binding. The ionic strength does not have a large effect on the stability of the FMN complex because, although it weakens the phosphate interactions, it strengthens the isoalloxazine-protein hydrophobic interactions. Phosphate up to 100 mM does not affect the strength of the riboflavin complex, which suggests the isoalloxazine and phosphate binding sites may be independent in terms of binding energy. Interestingly, we find crystallographic evidence of flexibility in one of the loops (57-62) involved in isoalloxazine binding.Many proteins use tightly bound cofactors to perform their biological functions. Flavoproteins, redox proteins carrying a flavin group, are one example (1). Although there are several flavoproteins where the flavin is covalently bound (2), the large majority consist on tight, but non-covalent, complexes of apoprotein and flavin. The flavins in flavoproteins derive from riboflavin and come in two lengths. The short one, flavin mononucleotide (FMN; Fig. 1) is just 5Ј phosphoriboflavin; the long one is flavin adenine dinucleotide (FAD). In either flavin, the redox properties are confined to the isoalloxazine ring, the rest of the molecule being important for binding. FMN and FAD are not usually interchangeable. On binding to the protein, the redox properties of the flavin are tailored to suit the particular requirements of the redox reaction where the protein is involved (3-5).One of the smallest and better known flavoproteins is flavodoxin. Flavodoxin is an ␣/␤ electron transfer protein, involved in both photosynthetic and non-photosynthetic reactions, that carries a molecule of non-covalently bound FMN as its only redox center (6, 7). The FMN can be removed from the protein, and the resulting apoflavodoxin can be easily reconstituted. Although there are several studies on the interaction of FMN with apoflavodoxins (4, 6, 8 -10), a detailed analysis of the energetics of...

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Certain bacteria promote the formation of ice in super-cooled water by means of ice nucleators which contain a unique protein associated with the cell membrane. Ice nucleators in general are believed to act by mimicking the structure of an ice crystal surface, thus imposing an ice-like arrangement on the water molecules in contact with the nucleating surface and lowering the energy necessary for the initiation of ice formation. Quantitative investigation of the bacterial ice-nucleating process has recently been made possible by the discovery of certain bacteria that shed stable membrane vesicles with ice nucleating activity. The opposite effect, inhibition of ice formation, has been described for a group of glycoproteins found in different fish and insect species. This group of substances, termed antifreeze glycoproteins (AFGPs), promotes the supercooling of water with no appreciable effect on the equilibrium freezing point or melting temperature. Substantial evidence now indicates that AFGPs act by binding to a growing ice crystal and slowing crystal growth. As the ice-nucleating protein surface is believed to have a structure similar to an embryonic ice crystal, AFGPs might be predicted to interact directly with a bacterial ice-nucleating site. We report here that AFGPs from the antarctic fish Dissostichus mawsoni inhibit the ice-nucleating activity of membrane vesicles from the bacterium Erwinia herbicola. The inhibition effect shows saturation at high concentration of AFGP and conforms to a simple binding reaction between the AFGP and the nucleation centre.

Differential scanning calorimetry has been performed with Palinurus vulgaris haemocyanin monomers and hexamers. The denaturation of the protein is irreversible. Both the temperature of the transition maximum and the enthalpy are lower for the monomer than for the hexamer. A scan rate dependence of the temperature of the maxima is found for both the monomer and the hexamer; for the hexamer at least, this can be explained in terms of a two-state kinetic model. Some comments are made as to the use of equilibrium thermodynamics in the analysis of irreversible scanning calorimetric traces.Haemocyanins are oligomeric copper-bearing proteins which transport and store oxygen in a great number of arthropods and molluscs. The basic unit for arthropod haemocyanin is the hexamer, with a subunit mass of around 75 kDa. Mollusc haemocyanins are cylindrical with a subunit mass of around 400 kDa. Haemocyanins have been extensively studied as they make excellent models for investigating the multiple linkages upon which the functional behaviour of proteins is based (for a review see [l]).From a structural point of view, extensive spectroscopic studies have been carried out to elucidate the main features of the binuclear, copper-oxygen binding site. The structure of the haemocyanin of the arthropod Panulirus interruptus has been resolved by X-ray to a resolution of 0.32 nm [2] and it appears that all arthropod haemocyanins share a common architecture, according to which the basic subunit is folded into three distinct alligned domains, with the active site in the middle one.With the structural knowledge available, it seemed worthwhile exploring the energetic aspects of the folding and assembly of the molecule. Thus, we report here on the results of differential scanning calorimetry of the thermal denaturation of P . vulgaris haemocyanin in both the hexameric and monomeric forms. Differential scanning calorimetry has been widely used to study folding-unfolding processes in proteins (for reviews see [3, 41). For complex proteins that undergo reversible denaturation, the deconvolution of the experimental heat capacity profile leads to the thermodynamic characterization of those intermediate states that are significantly populated in the unfolding process, and to the definition of the structural blocks of the protein that undergo unfolding in a more or less independent fashion (see for instance . In this case, however, we have found the differential scanning calorimetry traces corresponding to the thermal denaturation of P. vulgaris haemocyanin to be irreversible and strongly scan-rate-dependent. Therefore, the analysis of these calorimetric traces has been carried out using a recently proposed approach [11, 121 which takes into account the possible kinetic character of the irreversible thermal denaturation of proteins.Correspondence to A. Parody-Morreale, Departamento de Quimica-Fisica, Facultad de Ciencias, E-18071 Granada, Spain MATERIALS AND METHODS Extraction and purification of proteinHaemocyanin was purified from lobsters (Palin...

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A detailed account of the assembly of a thermopile heat-conduction isothermal titration calorimeter to be used in the study of biomolecular interactions is given. The instrument is built according to the twin-cell principle with a sensitivity of 5 pJ. Its reaction vessel volume is only 200 {il. Stirring heats of the reaction vessel are below 5 pJ.

dbstract. mis paper continues the description of an isothermal titration calorimeter already outlined in the previous paper. We have determined its thermal parameters and report here on the performance of the calorimeter and the method of measuring via electrical compensation of the thermal effect. We have also calculated the effective volume of the reaction vessel and the heat of stirring of the reaction mixture in the vessel, and of injecting water into water. Finally, correct working of the calorimeter has been tested by a dilution experiment of a saccharose solution.