Characterization of the Membranes of Thermoplasma acidophilum

Thermoplasma acidophilum grows optimally under aeration at 59 C and pH 2. Both intact cells and membranes disaggregate below pH 1 and above pH 5, producing no sedimentable particles. Increase in ionic strength at pH 5 or below results in cellular lysis and membrane disaggregation. Membranous components produced by lysis at alkaline pH reaggregate upon reduction of both pH and ionic strength. Osmotic environment plays little role in cellular stability. Membranes prepared by sonic lysis at pH 5 exhibit vesicular structures and are composed of multiple proteins. Although the amino acid composition of the membrane proteins is similar to other mycoplasmal membranes, the number of free amino and carboxyl groups is less than half of those in Acholeplasma. Reduction of the number of free carboxyl groups results in membrane stabilization over a wide range of pH. Increase in the number of free amino groups reverses the stability of membranes relative to pH. Acidophily in Thermoplasma can be related to a significant reduction in repulsing negative charges on the membrane proteins.     

The denaturation of circular enterocin AS-48 by urea and guanidinium hydrochloride

The unfolding thermodynamics of the circular enterocin protein AS-48, produced by Enterococcus faecalis, has been studied. The native structure of the 70-amino-acid-long protein turned out to be extremely stable against heat and denaturant-induced unfolding. At pH 2.5 and low ionic strength, it denatures at 102 degrees C, while at 25 degrees C, the structure only unfolds in 6.3 M guanidinium hydrochloride (GuHCl) and does not unfold even in 8 M urea. A comparison of its thermal unfolding in water and in the presence of urea shows a good correspondence between the two deltaGw(298) values, which are about 30 kJ mol(-1) at pH 2.5 and low ionic strength. The stability of the structure is highly dependent upon ionic strength and so GuHCl acts both as a denaturant and a stabilising agent. This seems to be why the deltaGw(298) value calculated from the unfolding data in GuHCl is twice as high as in the absence of this salt. At least part of the high stability of native AS-48 can almost certainly be put down to its circular organization since other structural features are quite normal for a protein of this size.     

Energetics of solvent and ligand-induced conformational changes in alpha-lactalbumin

The energetics of structural changes in the holo and apo forms of a-lactalbumin and the transition between their native and denatured states induced by binding Ca2+ and Na+ have been studied by differential scanning and isothermal titration microcalorimetry and circular dichroism spectroscopy under various solvent conditions. Removal of Ca2+ from the protein enhances its sensitivity to pH and ionic conditions due to noncompensated negative charge-charge interactions at the cation binding site, which significantly reduces its overall stability. At neutral pH and low ionic strength, the native structure of apo-alpha-lactalbumin is stable below 14 C and undergoes a conformational change to a native-like molten globule intermediate at temperatures above 25 degrees C. The denaturation of either holo- or apo-alpha-lactalbumin is a highly cooperative process that is characterized by an enthalpy of similar magnitude when calculated at the same temperature. Measured by direct calorimetric titration, the enthalpy of Ca2+-binding to apo-LA at pH 7.5 is -7.1 kJ mol(-1) at 5.0 degrees C. which is essentially invariant to protonation effects. This small enthalpy effect infers that stabilization of alpha-lactalbumin by Ca2+ is primarily an entropy driven process, presumably arising from electrostatic interactions and the hydration effect. In contrast to the binding of calcium, the interaction of sodium with apo-LA does not produce a noticeable heat effect and is characterized by its ionic nature rather than specific binding to the metal-binding site. Characterization of the conformational stability and ligand binding energetics of alpha-lactalbumin as a function of solvent conditions furnishes significant insight regarding the molecular flexibility and regulatory mechanism mediated by this protein.     

Structural basis for the cold adaptation of psychrophilic M37 lipase from Photobacterium lipolyticum

The M37 lipase from Photobacterium lipolyticum shows an extremely low activation energy and strong activity at low temperatures, with optimum activity seen at 298 K and more than 75% of the optimum activity retained down to 278 K. Though the M37 lipase is most closely related to the filamentous fungal lipase, Rhizomucor miehei lipase (RML) at the primary structure level, their activity characteristics are completely different. In an effort to identify structural components of cold adaptation in lipases, we determined the crystal structure of the M37 lipase at 2.2 A resolution and compared it to that of nonadapted RML. Structural analysis revealed that M37 lipase adopted a folding pattern similar to that observed for other lipase structures. However, comparison with RML revealed that the region beneath the lid of the M37 lipase included a significant and unique cavity that would be occupied by a lid helix upon substrate binding. In addition, the oxyanion hole was much wider in M37 lipase than RML. We propose that these distinct structural characteristics of M37 lipase may facilitate the lateral movement of the helical lid and subsequent substrate hydrolysis, which might explain its low activation energy and high activity at low temperatures.     

Ionic strength-dependent denaturation of Thermomyces lanuginosus lipase induced by SDS

Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denaturation by sodium dodecyl sulfate (SDS). We have found that at neutral pH, structural integrity is strongly dependent on ionic strength. In 10 mM phosphate buffer and SDS, the lipase exhibits a far-UV CD spectrum similar to other proteins denatured in this surfactant while the near-UV CD spectrum shows a complete loss of tertiary structure, observations supported by steady state fluorescence spectroscopy. However, when increasing the ionic strength by the addition of NaCl, the lipase was rendered resistant towards SDS denaturation, as observed by all techniques employed. The effect of salt on the critical micelle concentration (CMC) of SDS was observed to correlate with the effect on the degree of SDS-induced denaturation. This finding is compatible with the notion that the concentration of SDS monomers is a crucial factor for SDS–lipase interactions. The presented results are important for the understanding and improvement of protein stability in surfactant systems.     

Denaturation of α-lactalbumin and myoglobin by the anionic biosurfactant rhamnolipid

Glycolipid biosurfactants (GBS) are promising environmentally friendly alternatives to chemical surfactants. Surfactants interact with proteins in many applications, often leading to significant changes in protein properties. Given GBS' marked difference in structure compared to traditional chemical surfactants, it is of interest to investigate their impact on protein structure and stability. Here we combine spectroscopic and calorimetric studies to analyze the interactions between the anionic GBS rhamnolipid (RL) and two model proteins α-lactalbumin in the Ca^2 +-free apo-form (αLA) and myoglobin (Mb), whose interactions with traditional surfactants are well known. RL denatures αLA at sub-cmc concentrations (0.1–1 mM) while Mb is only denatured above the cmc, i.e. in the presence of RL micelles. Denaturation leads to increased α-helicity, similar to the effect of SDS. The proteins bind approximately the same amount of RL by weight as SDS. However, RL employs a denaturation mechanism which combines features from non-ionic surfactants (very slow unfolding kinetics and few unfolding steps) with those of SDS (unfolding below the cmc in the case of αLA and the ability to unfold stable proteins in the case of Mb). We ascribe these features to RL's weakly acidic carboxylic head group and complex hydrophobic tail, which lead to a low cmc and low protein affinity. These features restrict the concentration range where RL monomers can bind and denature proteins while still allowing micelles to bind and denature to a significant extent.     

Structural basis for pH dependent monomer-dimer transition of 3,4-dihydroxy 2-butanone-4-phosphate synthase domain from Mycobacterium tuberculosis

3,4-dihydroxy 2-butanone 4-phosphate synthase (DHBPS) and GTP cyclohydrolase-II (GTPCH-II) are the two initial enzymes involved in riboflavin biosynthesis pathway, which has been shown to be essential for the pathogens. In Mycobacterium tuberculosis (Mtb), the ribA2 gene (Rv1415) encodes for the bi-functional enzyme with DHBPS and GTPCH-II domains at N- and C-termini, respectively. We have determined three crystal structures of Mtb-DHBPS domain in complex with phosphate and glycerol at pH 6.0, with sulphate at pH 4.0 and with zinc and sulphate at pH 4.0 at 1.8, 2.06 and 2.06 ? resolution, respectively. The hydrodynamic volume and enzyme activity studies revealed that the Mtb-DHBPS domain forms a functional homo-dimer between the pH 6.0 and 9.0, however, at pH 5.0 and below, it forms a stable inactive monomer in solution. Furthermore, the functional activity of Mtb-DHBPS and its dimeric state could be restored by increasing the pH between 6.0 and 9.0. The comparison of crystal structures determined at different pH revealed that the overall three-dimensional structure of Mtb-DHBPS monomer remains the same. However, the length of the α6-helix at pH 6.0 has increased from 15 to 22 ? in pH 4.0 by increasing the number of amino acids contributing to the α6-helix from 11 to 15, achieving a higher structural stability at pH 4.0. Taken together our experiments strongly suggest that the Mtb-DHBPS domain can transit between inactive monomer to active dimer depending upon its pH values, both in solution as well in crystal structure.     

NMR Structure of a Monomeric Intermediate on the Evolutionarily Optimized Assembly Pathway of a Small Trimerization Domain

Efficient formation of specific intermolecular interactions is essential for self-assembly of biological structures. The foldon domain is an evolutionarily optimized trimerization module required for assembly of the large, trimeric structural protein fibritin from phage T4. Monomers consisting of the 27 amino acids comprising a single foldon domain subunit spontaneously form a natively folded trimer. During assembly of the foldon domain, a monomeric intermediate is formed on the submillisecond time scale, which provides the basis for two consecutive very fast association reactions. Mutation of an intermolecular salt bridge leads to a monomeric protein that resembles the kinetic intermediate in its spectroscopic properties. NMR spectroscopy revealed essentially native topology of the monomeric intermediate with defined hydrogen bonds and side-chain interactions but largely reduced stability compared to the native trimer. This structural preorganization leads to an asymmetric charge distribution on the surface that can direct rapid subunit recognition. The low stability of the intermediate allows a large free-energy gain upon trimerization, which serves as driving force for rapid assembly. These results indicate different free-energy landscapes for folding of small oligomeric proteins compared to monomeric proteins, which typically avoid the transient population of intermediates.     

The pH-dependent structural variation of complementarity-determining region H3 in the crystal structures of the Fv fragment from an anti-dansyl monoclonal antibody.

The Fv fragment from an anti-dansyl antibody was optimally crystallized into two crystal forms having slightly different lattice dimensions at pH 5.25 and 6.75. The two crystal structures were determined and refined at high resolution at 112 K (at 1.45 A for the crystal at pH 5.25 and at 1.55 A for that at pH 6.75). In the two crystal structures, marked differences were identified in the first half of CDRH3 s having an amino acid sequence of Ile95H-Tyr96H-Tyr97H-His98H-Tyr99H-Pro1 00H-Trp100aH-Phe100bH-Ala101H- Tyr102H. NMR pH titration experiments revealed the p Kavalues of four histidine residues (His27dL, His93L, His55H and His98H) exposed to solvent. Only His98H (p Ka=6.3) completely changed its protonation state between the two crystallization conditions. In addition, the environmental structures including hydration water molecules around the four histidine residues were carefully compared. While the hydration structures around His27dL, His93L and His55H were almost invariant between the two crystal structures, those around His98Hs showed great difference in spite of the small conformational difference of His98H between the two crystal structures. These spectroscopic and crystallographic findings suggested that the change in the protonation state in His98H was responsible for the structural differences between pH 5.25 and 6.75. In addition, the most plausible binding site of the dansyl group was mapped into the present structural models with our previous NMR experimental results. The complementarity-determining regions H1, H3 and the N-terminal region in the VH domain formed the site. The side-chain of Tyr96H occupied the site and interacted with Phe27H of H1, giving a clue for the binding mode of the dansyl group in the site.     

Role of the C-terminal chain in human interferongamma stability: an electrostatic study

Electrostatic interactions in two structures of human interferon gamma (hIFNgamma), corresponding to interferon molecule alone and bound to its receptor, were analyzed on the basis of a continuum dielectric model. It was found that a number of titratable groups, mainly basic, show large pK shifts and remain in their neutral forms at physiologically relevant pH. The fact that these groups are largely common to both structures and that most of them belong to the set of most conserved sites suggests that this is a property inherent to the hIFNgamma molecule rather than an artifact of the crystal packing. His111 was also found deprotonated at neutral pH. It was concluded that receptor recognition involving His111 is driven by aromatic coupling of His111 and Tyr52 from the receptor rather than by electrostatic interactions. The structure corresponding to hIFNgamma in complex with its receptor shows a reduction in number and in degree of desolvation of the buried titratable sites. This finding suggested that on receptor binding, hIFNgamma adopts energetically more favorable, relaxed, conformation. It was experimentally shown that in contrast to the full-size hIFNgamma, the construct having 21 amino acid residues deleted from the C-terminus is soluble. The hydrophobicity profile analysis suggested that factors other than the exposure of hydrophobic parts of the molecule are responsible for the low stability and propensity for aggregation. On the basis of these results, it was assumed that the electrostatic influence of the C-terminal part contributes particularly to the low solvent exposure of the titratable groups, and hence to the low structural stability and propensity for aggregation of the recombinant hIFNgamma. Proteins 2001;43:125-133.     

pH dependence of stability of the 10th human fibronectin type III domain: a computational study

We present detailed computational studies based on electrostatic calculations to evaluate the origins of pKa values and the pH dependence of stability for the 10th type III domain of human fibronectin (FNfn10). One of our goals is to validate the calculation protocols by comparison to experimental data (Koide, A.; Jordan, M. R.; Horner, S.; Batori, V.; Koide, S. Biochemistry 2001, 40, 10326-10333). Another goal is to evaluate the sensitivity of the calculated ionization free energies and apparent pKa values on local structural fluctuations, which do not alter the structural convergence to a particular architecture, by using a complete ensemble of solution NMR structures and the NMR average minimized structure of FNfn10 (Main, A. L.; Harvey, T. S.; Baron, M.; Boyd, J.; Campbell, I. D. Cell 1992, 71, 671-678). Our calculations demonstrate that, at high ionic strength, FNfn10 is more stable at low pH compared to neutral pH, in overall agreement with experimental data. This behavior is attributed to contributions from unfavorable Coulombic interactions in a surface patch for the pairs Asp7-Glu9 and Asp7-Asp23. The unfavorable interactions are decreased at low pH, where the acidic residues become neutral, and are further decreased at high ionic strength because of increased screening by salt ions. Elimination of the unfavorable interactions in the theoretical mutants Asp7Asn (D7N) and Asp7Lys (D7K) produce higher calculated stabilities at neutral pH and any ionic strength compared to the wild-type, in agreement with the experimental data. We also discuss subtleties in the calculated apparent pKa values and ionization free energies, which are not in agreement with the experimental data. This work demonstrates that comparative electrostatic calculations can provide rapid predictions of pH-dependent properties of proteins and can be significant aids in guiding the design of proteins with tailored properties.     

pH-dependent aggregation of cutinase is efficiently suppressed by 1,8-ANS

We have studied the thermal stability of the triglyceride-hydrolyzing enzyme cutinase from F. solani pisi at pH values straddling the pI (pH 8.0). At the pI, increasing the protein concentration from 5 to 80 microM decreases the apparent melting temperature by 19 degrees C. This effect vanishes at pH values more than one unit away from pI. In contrast to additives such as detergents and osmolytes, the hydrophobic fluorophore 1,8-ANS completely and saturably suppresses this effect, restoring 70% of enzymatic activity upon cooling. ANS binds strongly to native cutinase as a noncompetitive inhibitor with up to 5 ANS per cutinase molecule. Only the first ANS molecule stabilizes cutinase; however, the last 4 ANS molecules decrease Tm by up to 7 degrees C. Similar pI-dependent aggregation and suppression by ANS is observed for T. lanuginosus lipase, but not for lysozyme or porcine alpha-amylase, suggesting that this behavior is most prevalent for proteins with affinity for hydrophobic substrates and consequent exposure of hydrophobic patches. Aggregation may be promoted by a fluctuating ensemble of native-like states associating via intermolecular beta-sheet rich structures unless blocked by ANS. Our data highlight the chaperone activity of small molecules with affinity for hydrophobic surfaces and their potential application as stabilizers at appropriate stoichiometries.     

The effects of ionic strength on the self-association of human spectrin.

The self-association of human spectrin has been studied by means of sedimentation equilibrium in the analytical ultracentrifuge at pH 7.5 and over a range of ionic strength from 0.009 to 1.0 M. Increasing ionic strength above 0.1 M reduces the equilibrium constants for all of the measurable steps in the self-association reaction. These results support the concept of charge-charge interactions stabilizing the tetramer and higher oligomers with respect to the heterodimer. In addition, increasing ionic strength brought about a dissociation of the heterodimer to component polypeptide chains. Dissociation to the heterodimers is also enhanced with a decrease in ionic strength below 0.05 M. This low ionic strength-dependent dissociation is consistent with generalised electrostatic repulsion; however, this effect also correlates with some loss of alpha-helical content as revealed by circular dichroism. The secondary, tertiary and quaternary structures may all be partially disrupted by electrostatic free energy at low ionic strength.     

Integrin Engagement by the Helical RGD Motif of the Helicobacter pylori CagL Protein Is Regulated by pH-induced Displacement of a Neighboring Helix

Arginine-aspartate-glycine (RGD) motifs are recognized by integrins to bridge cells to one another and the extracellular matrix. RGD motifs typically reside in exposed loop conformations. X-ray crystal structures of the Helicobacter pylori protein CagL revealed that RGD motifs can also exist in helical regions of proteins. Interactions between CagL and host gastric epithelial cell via integrins are required for the translocation of the bacterial oncoprotein CagA. Here, we have investigated the molecular basis of the CagL-host cell interactions using structural, biophysical, and functional analyses. We solved an x-ray crystal structure of CagL that revealed conformational changes induced by low pH not present in previous structures. Using analytical ultracentrifugation, we found that pH-induced conformational changes in CagL occur in solution and not just in the crystalline environment. By designing numerous CagL mutants based on all available crystal structures, we probed the functional roles of CagL conformational changes on cell surface integrin engagement. Together, our data indicate that the helical RGD motif in CagL is buried by a neighboring helix at low pH to inhibit CagL binding to integrin, whereas at neutral pH the neighboring helix is displaced to allow integrin access to the CagL RGD motif. This novel molecular mechanism of regulating integrin-RGD motif interactions by changes in the chemical environment provides new insight to H. pylori-mediated oncogenesis.     

Crystal structure and properties of CYP231A2 from the thermoacidophilic archaeon Picrophilus torridus

The crystal structure of a cytochrome P450 from the thermoacidophile Picrophilus torridus, CYP231A2 (PTO1399), has been solved. This structure reveals a wide open substrate access channel. To better understand ligand-induced structural transitions in CYP231A2, protein-ligand interactions were investigated using 4-phenylimidazole. Comparison of the ligand-free and -bound CYP231A2 structures shows conformational changes where the F and G helices swing as a single rigid body about a pivot point at the N-terminal end of the F helix, allowing the F helix region to dip toward the heme, resulting in closer contacts with the ligand. Thermal melting data illustrate that the melting temperature for CYP231A2 increases nearly 10 degrees C upon ligand binding, thus illustrating that the closed conformation is substantially more stable. Furthermore, spectroscopic data indicate that the active site is stable at pH 4.5, although, unusually, the thiolate ligand to the iron can be reversibly protonated. CYP231A2 does not exhibit structural features normally associated with thermophilic proteins such as an increase in salt bridge networks or extensive aromatic clustering. The increase in thermal stability instead is best correlated with the smaller size and shorter loops in CYP231A2 compared to other P450s.     

Structural characterization of an early fusion intermediate of influenza virus hemagglutinin

The hemagglutinin (HA) envelope protein of influenza virus mediates viral entry through membrane fusion in the acidic environment of the endosome. Crystal structures of HA in pre- and postfusion states have laid the foundation for proposals for a general fusion mechanism for viral envelope proteins. The large-scale conformational rearrangement of HA at low pH is triggered by a loop-to-helix transition of an interhelical loop (B loop) within the fusion domain and is often referred to as the "spring-loaded" mechanism. Although the receptor-binding HA1 subunit is believed to act as a "clamp" to keep the B loop in its metastable prefusion state at neutral pH, the "pH sensors" that are responsible for the clamp release and the ensuing structural transitions have remained elusive. Here we identify a mutation in the HA2 fusion domain from the influenza virus H2 subtype that stabilizes the HA trimer in a prefusion-like state at and below fusogenic pH. Crystal structures of this putative early intermediate state reveal reorganization of ionic interactions at the HA1-HA2 interface at acidic pH and deformation of the HA1 membrane-distal domain. Along with neutralization of glutamate residues on the B loop, these changes cause a rotation of the B loop and solvent exposure of conserved phenylalanines, which are key residues at the trimer interface of the postfusion structure. Thus, our study reveals the possible initial structural event that leads to release of the B loop from its prefusion conformation, which is aided by unexpected structural changes within the membrane-distal HA1 domain at low pH.     

Salt bridges regulate both dimer formation and monomeric flexibility in HdeB and may have a role in periplasmic chaperone function

Escherichia coli and Gram-negative bacteria that live in the human gut must be able to tolerate rapid and large changes in environmental pH. Low pH irreversibly denatures and precipitates many bacterial proteins. While cytoplasmic proteins are well buffered against such swings, periplasmic proteins are not. Instead, it appears that some bacteria utilize chaperone proteins that stabilize periplasmic proteins, preventing their precipitation. Two highly expressed and related proteins, HdeA and HdeB, have been identified as acid-activated chaperones. The structure of HdeA is known and a mechanism for activation has been proposed. In this model, dimeric HdeA dissociates at low pH, and the exposed dimeric interface binds exposed hydrophobic surfaces of acid-denatured proteins, preventing their irreversible aggregation. We now report the structure and biophysical characterization of the HdeB protein. The monomer of HdeB shares a similar structure with HdeA, but its dimeric interface is different in composition and spatial location. We have used fluorescence to study the behavior of HdeB as pH is lowered, and like HdeA, it dissociates to monomers. We have identified one of the key intersubunit interactions that controls pH-induced monomerization. Our analysis identifies a structural interaction within the HdeB monomer that is disrupted as pH is lowered, leading to enhanced structural flexibility.     

Enigmatic thermotropic phase behavior of highly asymmetric mixed-chain phosphatidylcholines that form mixed-interdigitated gel phases.

Twelve saturated mixed-chain phosphatidylcholines have been identified for which the thermotropic phase behavior observed upon cooling from the L alpha phase is dependent upon the thermal history of the sample in the gel phase. If fully hydrated samples of these lipids are cooled and soon thereafter examined by differential scanning calorimetry, one observes a single highly cooperative endotherm (the chain-melting phase transition) upon heating, and on subsequent cooling, a single exotherm that may occur at temperatures as much as 4-6 degrees C below that of the single endotherm observed upon heating. In contrast, if the samples are incubated in the gel state at low temperatures for prolonged periods of time, one observes a single heating endotherm as before, but two sharp exotherms upon cooling. The latter transitions occur at temperatures close to that of the single endotherm observed upon heating and the single cooling exotherm observed prior to incubation in the gel state. The combined enthalpy of the two cooling exotherms is the same as that of the single heating endotherm or the single cooling exotherm initially observed. Infrared spectroscopic and X-ray diffraction studies indicate that the structural conversions characteristic of liquid-crystalline/gel phase transitions occur at both of those cooling exotherms. Of the 12 lipids that exhibit this unusual behavior, nine fulfill the previously defined structural requirements for the formation of the so-called mixed-interdigitated gel phase, and there is evidence in the literature that one of the three remaining lipids also forms such a structure. Infrared spectroscopic studies of the other two lipids indicate that their gel phases exhibit spectroscopic features that closely resemble those of lipids that meet the previously defined structural criteria for the formation of mixed-interdigitated gel phases and that differ markedly from those of both saturated symmetric-chain and saturated mixed-chain phosphatidylcholines that do not normally form mixed-interdigitated gel phases. Also, electron density reconstructions based on small-angle X-ray diffraction studies of the gel phases of those two lipids indicate that the thickness of their gel phase bilayers is consistent with their forming mixed-interdigitated gel phases. Thus the unusual thermotropic phase behavior described here may be a general characteristic of phosphatidylcholines that form mixed-interdigitated gel phases. This unusual behavior is not associated with any major change in any of several physical properties of these lipid bilayers but may arise from an alteration of the size and/or structure of microdomains present in the liquid-crystalline phase.     

Structural change of bacteriorhodopsin in the purple membrane above pH 10 decreases heterogeneity of the irreversible photobleaching components

Kinetic investigations of irreversible photobleaching of bacteriorhodopsin (bR) in purple membrane (PM) at high temperature have previously shown two kinds of bR species upon light illumination. The bR species consist of kinetically fast- and slow-denatured components, whose proportions were dependent upon structural changes in dark, as shown by CD. In order to elucidate electrostatic contribution on the heterogeneous stability and the bR structure in PM, photobleaching behaviour and structural changes over a wide pH range were investigated by kinetics as well as various spectroscopic techniques. Kinetics revealed that photobleaching below pH 9 obeyed double-exponential functions, whereas measurements above pH 10 were characterized by a single-decay component. FT-IR deconvoluted spectra showed a alpha(II)-to-alpha(I) transition in the transmembrane helices around pH 10. Near-IR Raman scattering spectra demonstrated the equilibrium shift of retinal isomers from all trans to 13-cis form. Near-UV CD spectra suggested configurational changes in the aromatic residues around the retinal pocket. An exciton-to-positive transition in visible CD spectrum was observed. This indicates disorganization in the 2D-crystalline lattice of PM, which occurred concomitantly with the changes above pH 10. A model for the changes in kinetic behaviour and molecular structure around pH 10 is discussed, focusing on changes in charge distribution upon alkalinization.     

Structural basis for antifreeze activity of ice-binding protein from arctic yeast

Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ～25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-? resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96-115 form a long α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic loop region ((243)PFVPAPEVV(251)). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn(185) provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins.