Covalent cross-linking of proteins without chemical reagents

A facile method for the formation of zero-length covalent cross-links between protein molecules in the lyophilized state without the use of chemical reagents has been developed. The cross-linking process is performed by simply sealing lyophilized protein under vacuum in a glass vessel and heating at 85 degrees C for 24 h. Under these conditions, approximately one-third of the total protein present becomes cross-linked, and dimer is the major product. Chemical and mass spectroscopic evidence obtained shows that zero-length cross-links are formed as a result of the condensation of interacting ammonium and carboxylate groups to form amide bonds between adjacent molecules. For the protein examined in the most detail, RNase A, the cross-linked dimer has only one amide cross-link and retains the enzymatic activity of the monomer. The in vacuo cross-linking procedure appears to be general in its applicability because five different proteins tested gave substantial cross-linking, and co-lyophilization of lysozyme and RNase A also gave a heterogeneous covalently cross-linked dimer.     

Molecular pathology of dityrosine cross-links in proteins: structural and functional analysis of four proteins

The dityrosine bond (DT) is an oxidative covalent cross-link between two tyrosines. DT cross-linking is increasingly identified as a marker of oxidative stress, aging and disease, and has been detected in diverse pathologies. While DT cross- linked proteins have been documented, the consequences of the DT link on the structure and function of the so modified proteins are yet to be understood. With this in view, we have studied the properties of intermolecular DT-dimers of four proteins of diverse functions, namely the enzyme ribonuclease A, the signal protein calmodulin, and the eye lens proteins alpha- and gamma B-crystallins. We find that DT is formed through radical reactions and type I photosensitization (including .OH, O2- and OONO-), but not by 1O2 and NO, (which modify his, trp and met more readily). Tyr residues on the surface of the protein make DT bonds (intra- and intermolecular) most readily and preferentially. The conformation of each of these DT-dimers, monitored by spectroscopy, is seen not to be significantly altered in comparison to that of the parent monomer, but the structural stability of the DT cross-linked molecule is lower than that of the parent native monomer. The DT-dimer is denatured at a lower temperature, and at lower concentrations of urea or guanidinium chloride. The effect of DT-cross-linking on the biological activities of these proteins was next studied. The enzymatic activity of the DT-dimer of ribonuclease A is not lost but lowered. DT-dimerization of lens alpha-crystallin did not significantly affect the chaperone-like ability; it inhibits the self-aggregation and precipitation of target proteins just as well as the parent, unmodified alpha-crystallin does. DT-dimerization of gamma B-crystallin is however seen to lead to more ready aggregation and precipitation, a point of interest in cataract. In the case of calmodulin, we could generate both intermolecular and intramolecular DT cross-linking, and study both the DT-dimer and DT-monomer. The DT-dimer binds smooth muscle light chain kinase and also Ca2+, but less efficiently and over a broad concentration range than the native monomer. The intramolecular DT-monomer is weaker in all these respects, presumably since it is structurally more constrained. These results suggest that DT cross-linking of globular proteins weakens their structural stability and compromises (though does not abolish) their biological activity, both of which are pathologically relevant. The intramolecular DT cross-link would appear to lead to more severe structural and functional consequences.     

Unfolding and refolding of bovine beta-lactoglobulin monitored by hydrogen exchange measurements.

Bovine beta-lactoglobulin (beta-LG) is a widely studied protein belonging to the lipocalin family, whose structural characterisation has been reported by X-ray crystallography and NMR studies at physiological and acidic pH, respectively. Bovine beta-LG consists of nine antiparallel beta-sheets and a terminal alpha-helix segment. The beta-sheets form a calyx structure with a hydrophobic buried cluster conferring stability to the protein while a hydrophobic surface patch provides stabilising interactions between the barrel and the flanking terminal helix. Here, the stability and the folding properties of bovine beta-LG in the presence of a chemical denaturant is probed. The analysis of the NMR spectra recorded in aqueous solution with increasing amounts of urea revealed that the intensities of the backbone cross-peaks decrease upon increasing urea concentration, while their secondary shifts do not change significantly on going from 0 to 5 M urea, thus suggesting the presence of slow exchange between native and unfolded protein. Hydrogen exchange measurements at different urea concentrations were performed in order to evaluate the exchange rates of individual backbone amide protons. The opening reactions that determine protein exchange can be computed for the most slowly exchanging hydrogen atoms, and the measured exchange rates and the corresponding free energies can be expressed in terms of the equilibrium energetic for the global transition and the local fluctuations. Most of the residues converge to define a common isotherm identifying a unique cooperative folding unit, encompassing all the strands, except strand betaI, and the terminal region of the helix. The amides that do not join the same global unfolding isotherm are characterised by low DeltaGH20op and especially by low m values, indicating that they are already substantially exposed in the native state. A two-state unfolding model N <==> U is therefore proposed for this rather big protein, in agreement with CD data. Renaturation studies show that the unfolding mechanism is reversible up to 6 M urea and suggest a similar unfolding and refolding pathway. Present results are discussed in light of the hypothesis of an alpha-->beta transition proposed for bovine beta-LG refolding.     

Effects of gamma radiation on beta-lactoglobulin: oligomerization and aggregation

The conformational changes and aggregation process of beta-lactoglobulin (beta-LG) subjected to gamma irradiation are presented. Beta-LG in solutions of different protein concentrations (3 and 10 mg/ml) and in solid state with different water activities (a(w)) (0.22; 0.53; 0.74) was irradiated using a Cobalt-60 radiation source at dose level of 1-50 kGy. Small-angle X-ray scattering (SAXS) was used to study the conformational changes of beta-LG due to the irradiation treatment. The irradiated protein was also examined by high performance size exclusion chromatography (HPSEC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing and reducing conditions and fluorescence. SAXS analysis showed that the structural conformation of irradiated beta-LG in solid state at different a(w) and dose level was essentially the same as the nonirradiated beta-LG. The scattering data also showed that the irradiation of beta-LG in solution promoted the formation of oligomers. Interestingly, from the data analysis and model building, it could be shown that the formed oligomers are linear molecules, built by linear combinations of beta-LG dimers (tetramers, hexamers, etc). The formation of oligomers was also evidenced by SDS-PAGE analysis and HPSEC chromatograms, in which products with higher molecular mass than that of the dimeric beta-LG were detected. Formation of intermolecular cross-linking between tyrosyl radicals are proposed to be at least partially responsible for this occurrence. From the results it could be shown that the samples irradiated in solution presented some conformational changes under gamma irradiation, resulting in well ordered oligomers and aggregates formed by cross-linking of beta-LG dimers subunits, while the samples irradiated in the solid state were not modified.     

Formation of a polymethylene bis(disulfide) intersubunit cross-link between cysteine-281 residues in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase using octamethylene bis(methane[35S]thiosulfonate)

The synthesis of a radioactive cross-linking agent, S,S'-octamethylene bis(methane[35S]thiosulfonate) (OBMTS), is described. The route of synthesis can be generally used in the synthesis of 35S-labeled thiosulfonates for the selective modification of thiols in proteins. Glyceraldehyde-3-phosphate dehydrogenase (G3PD) reacts asymmetrically with the bifunctional inhibitor. Initially two molecules of OBMTS react rapidly with the active-site thiol, Cys-149, on two of the four subunits to inhibit the enzyme completely without cross-linking. This is followed by the modification of four Cys-281 residues to incorporate two cross-links into the tetramer. Reduction of modified G3PD with 5 mM dithioerythritol under nondenaturing conditions released the inhibitor blocking the active-site thiol and completely restored enzyme activity while leaving the cross-link intact. Sodium dodecyl sulfate (Na-DodSO4) gel electrophoresis of the cross-linked enzyme under nonreducing conditions showed a dimer (Mr 72000) as the major species which was only cleaved by reduction in Na-DodSO4 containing beta-mercaptoethanol. The monomer formed was still radioactive, showing that the first disulfide in the cross-link was reduced at a much faster rate than the second disulfide. The latter was only reduced by using vigorous conditions. The location of the intersubunit cross-linked residues was established by isolation of the cyanogen bromide and tryptic subdigest peptides containing modified Cys-281. There were identified by molecular weight, amino terminal sequence, and amino acid composition.     

Cross-linking experiments with the adenosine triphosphatase of sarcoplasmic reticulum.

The proteins of sarcoplasmic reticulum were cross-linked by rapid oxidation of thiol groups with I2. About two-thirds of the thiols were oxidized without any significant cross-linking, implying an extensive formation of intramolecular disulphide bonds. When the thiols were completely oxidized at room temperature a series of oligomers containing up to five molecules were observed, as well as large aggregates which were excluded from the gels. Complete oxidation at -10 degrees C left most of the ATPase (adenosine triphosphatase) as monomer. Similar results were obtained when copper-phenanthroline complexes or dimethyl suberimidate were used as cross-linking reagents. We conclude that most of the cross-linked species arise by linking of randomly colliding ATPase molecules which are present in the membrane at very high concentration.     

Proteins can adopt totally different folded conformations.

The three-dimensional structure of a protein is determined by interactions between its amino acids and by interactions of the amino acids with molecules of the environment. The great influence of the latter interactions is demonstrated for the enzyme phosphoglycerate kinase from yeast (PGK). In the native state, PGK is a compact, bilobal molecule; 35% and 13% of its amino acids are organised in the form of alpha-helices and beta-sheets, respectively. The molecules unfold at acidic pH and low ionic strength forming random-walk structures with a persistence length of 3 nm. More than 90% of the amino acid residues of the ensemble have phi,psi-angles corresponding to those of a straight beta-chain. Upon addition of 50% (v/v) trifluoroethanol to the acid-unfolded protein, the entire molecule is transformed into a rod-like, flexible alpha-helix. Addition of anions, such as chloride or trichloroacetate, to the acid-unfolded protein leads to the formation of amyloid-like fibres over a period of many hours when the anion concentration exceeds a critical limit. Half of the amino acid residues are then organised in beta-sheets. Both of the non-natively folded states of PGK contain more regular secondary structure than the native one. The misfolding starts in both cases from the acid-unfolded state, in which the molecules are essentially more expanded than in other denatured states, e.g. those effected by temperature or guanidine hydrochloride.     

Glycation cross-linking induced mechanical–enzymatic cleavage of microscale tendon fibers

Recent molecular modeling data using collagen peptides predicted that mechanical force transmitted through intermolecular cross-links resulted in collagen triple helix unwinding. These simulations further predicted that this unwinding, referred to as triple helical microunfolding, occurred at forces well below canonical collagen damage mechanisms. Based in large part on these data, we hypothesized that mechanical loading of glycation cross-linked tendon microfibers would result in accelerated collagenolytic enzyme damage. This hypothesis is in stark contrast to reports in literature that indicated that individually mechanical loading or cross-linking each retards enzymatic degradation of collagen substrates. Using our Collagen Enzyme Mechano-Kinetic Automated Testing (CEMKAT) System we mechanically loaded collagen-rich tendon microfibers that had been chemically cross-linked with sugar and tested for degrading enzyme susceptibility. Our results indicated that cross-linked fibers were > 5 times more resistant to enzymatic degradation while unloaded but became highly susceptible to enzyme cleavage when they were stretched by an applied mechanical deformation.     

Vibrational spectroscopic characteristics of secondary structure polypeptides in liquid water: constrained MD simulation studies

Using the constrained MD simulation method in combination with quantum chemistry calculation, Hessian matrix reconstruction, and fragmentation approximation methods, we established a computational scheme for numerical simulations of amide I IR absorption, vibrational circular dichroism (VCD), and 2D IR photon echo spectra of peptides in solution. Six different secondary structure peptides, i.e., alpha-helix, 3(10)-helix, pi-helix, antiparallel and parallel beta-sheets, and polyproline II (P(II)), are considered, and the vibrational characteristic features in their linear and nonlinear spectra in the amide I band region are discussed. Isotope-labeling effects on IR and VCD spectra are notable only for alpha- and pi-helical peptides due to the strong vibrational couplings between two nearest neighboring amide I local oscillators. The amplitudes of difference 2D IR spectra are shown to be strongly dependent on both the extent of mode delocalization and the relative orientation of local mode transition dipoles determined by secondary structure.     

Oxidative modification of nucleoside diphosphate kinase and its identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Nucleoside diphosphate kinase (NDPK, Nm23) has been implicated as a multifunctional protein. However, the regulatory mechanism of NDPK is poorly understood. We have examined the modification of NDPK in oxidative stresses. We found that oxidative stresses including diamide and H(2)O(2) treatment cause disulfide cross-linking of NDPK inside cells. This cross-linking was reversible in response to mild oxidative stress, and irreversible to strong stress. This suggests that disulfide cross-linked NDPK may be a possible mechanism in the modification of cellular regulation. To confirm this idea, oxidative modification of NDPK has been performed in vitro using purified human NDPK H(2)O(2) inactivated the nucleoside diphosphate (NDP) kinase activity of NDPK by producing intermolecular disulfide bonds. Disulfide cross-linking of NDPK also dissociated the native hexameric structure into a dimeric form. The oxidation sites were identified by the analysis of tryptic peptides of oxidized NDPK, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Intermolecular cross-linking between Cys109-Cys109, which is highly possible based on the X-ray crystal structure of NDPK-A, and oxidations of four methionine residues were identified in H(2)O(2)-treated NDPK. This cross-linkng was confirmed using mutant C109A (NDPK-A(C109A)) which had similar enzymatic activity as a wild NDPK-A. Mutant NDPK-A(C109A) was not cross-linked and was not easily denatured by the oxidant. Therefore, enzymatic activity and the quaternary structure of NDPK appear to be regulated by cross-linking with oxidant. These findings suggest one of the regulatory mechanisms of NDPK in various cellular processes.     

Preferential nitration with tetranitromethane of a specific tyrosine residue in penicillinase from Staphylococcus aureus PCl. Evidence that the preferentially nitrated residue is not part of the active site but that loss of activity is due to intermolecular cross-linking.

1. Nitration of tyrosine residues of staphylococal penicillinase was accompanied by a partial loss of enzymic activity, which was not readily explained by nitration of a single residue. 2. Loss of activity correlated with low recovery of tyrosine plus nitrotyrosine, which was consistent with cross-linking. 3. The fraction of treated enzyme that was eluted from Sephadex G-75 earlier than native penicillinase was similar to the fraction of enzyme activity lost. Protein eluted in positions corresponding to monomer, dimer and higher oligomers respectively showed major bands in corresponding positions in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicating that the increase in molecular weight was due to intermolecular cross-linking. Monomeric enzyme containing up to 4 mol of nitrotyrosine/mol retained full catalytic activity. Dimeric enzyme retained 50% of normal activity, whereas higher oligomers retained an average of 8-15% of normal activity. 4. Monomeric enzyme isolated after treatment with equimolar tetranitromethane was nitrated predominantly at tyrosine-72.5. Reaction of reduced nitrated monomer with 1,5-difluoro-2,4-dinitrobenzene gave a monomeric, apparently cross-linked product with full catalytic activity. 6. It is concluded that tyrosine-72 plays no part in the active site. Its preferential nitration may be due to its being insufficiently exposed to be available for intermolecular cross-linking. This poperty may make it useful for attachment of a reporter group.     

Depth-dependent analysis of membranes using benzophenone-based phospholipids

Any attempt to probe the membrane hydrophobic core with chemical reagents necessitates the use of reactive intermediates like carbenes and nitrenes, which can insert into C-H bonds. Several photoactivable reagents based on carbenes and nitrenes have been reported. However, the high reactivity of these reagents, often leads to very low insertion yields. We report here a high degree of cross-linking (35-40%) achieved with three benzophenone-based phospholipids and analyze the carbon functionalization data using a multiple Gaussian function. These phospholipids are so designed so as to permit depth-dependent labeling in membranes. Single bilayer vesicles were prepared from these phospholipids and dimyristoylphosphatidylcholine. The cross-linked product was isolated and characterized by mass spectroscopy. The results obtained indicated that the cross-linked product was dominated by dimeric product formed by intermolecular cross-linking. The Gaussian analysis used here provides insight into the relative depths of the probes inside the membrane.     

Heat-induced secondary structure and conformation change of bovine serum albumin investigated by Fourier transform infrared spectroscopy

Fourier transform infrared (FT-IR) spectra were measured for an aqueous solution (pD = 5.40) of defatted monomer bovine serum albumin (BSA) over a temperature range of 25-90 degrees C to investigate temperature-induced secondary structure and conformation changes. The curve fitting method combined with the Fourier self-deconvolution technique allowed us to explore details of the secondary structure and conformation changes in defatted BSA. Particularly striking in the FT-IR spectra was an observation of the formation of an irreversible intermolecular beta-sheet of BSA on heating above 70 degrees C. A band at 1630 cm(-1) in the spectra was assigned to short-segment chains connecting alpha-helical segments. The transition temperature for the short-segment chains connecting alpha-helical segments is lower by 17-18 degrees C, when compared to those of the alpha-helix, turn, and intermolecular beta-sheet structures of BSA, suggesting that the alpha-helix and turn structures of BSA are cooperatively denatured on heating. Moreover, the results give an important feature in heat-induced denaturation of BSA that the conformation changes occur twice around both 57 and 75 degrees C. The appearance of two peaks is interpreted by the collapse of the N-terminal BSA domain due to the crevice in the vicinity between domains I and II at low-temperature transition and by the change in cooperative unit composed of the other two BSA domains at high-temperature transition.     

Molecular pathology of dityrosine cross-links in proteins: Structural and functional analysis of four proteins

The dityrosine bond (DT) is an oxidative covalent cross-link between two tyrosines. DT cross-linking is increasingly identified as a marker of oxidative stress, aging and disease, and has been detected in diverse pathologies. While DT cross- linked proteins have been documented, the consequences of the DT link on the structure and function of the so modified proteins are yet to be understood. With this in view, we have studied the properties of intermolecular DT-dimers of four proteins of diverse functions, namely the enzyme ribonuclease A, the signal protein calmodulin, and the eye lens proteins alpha- and gamma B-crystallins. We find that DT is formed through radical reactions and type I photosensitization (including OH, O₂ ^– and OONO^–), but not by ¹O₂ and NO₂ ^– (which modify his, trp and met more readily). Tyr residues on the surface of the protein make DT bonds (intra- and intermolecular) most readily and preferentially. The conformation of each of these DT-dimers, monitored by spectroscopy, is seen not to be significantly altered in comparison to that of the parent monomer, but the structural stability of the DT cross-linked molecule is lower than that of the parent native monomer. The DT-dimer is denatured at a lower temperature, and at lower concentrations of urea or guanidinium chloride. The effect of DT-cross-linking on the biological activities of these proteins was next studied. The enzymatic activity of the DT-dimer of ribonuclease A is not lost but lowered. DT-dimerization of lens alpha-crystallin did not significantly affect the chaperone-like ability; it inhibits the self-aggregation and precipitation of target proteins just as well as the parent, unmodified alpha-crystallin does. DT-dimerization of gamma B-crystallin is however seen to lead to more ready aggregation and precipitation, a point of interest in cataract. In the case of calmodulin, we could generate both intermolecular and intramolecular DT cross-linking, and study both the DT-dimer and DT-monomer. The DT-dimer binds smooth muscle light chain kinase and also Ca²⁺, but less efficiently and over a broad concentration range than the native monomer. The intramolecular DT-monomer is weaker in all these respects, presumably since it is structurally more constrained. These results suggest that DT cross-linking of globular proteins weakens their structural stability and compromises (though does not abolish) their biological activity, both of which are pathologically relevant. The intramolecular DT cross-link would appear to lead to more severe structural and functional consequences.     

Estimation of protein secondary structure from the laser Raman amide I spectrum

A new method for estimating protein secondary structure from the laser Raman spectrum has been developed whereby the amide I Raman band of a protein is analyzed directly as a linear combination of amide I bands of proteins whose secondary structures are known. For 14 proteins, analyzed by removing each one from the reference set and analyzing its structure in terms of the remaining proteins, the average correlation coefficients between the Raman and X-ray diffraction estimates of helix, beta-strand, turn, and undefined were 0.98, 0.98, 0.82 and 0.35, respectively. Significant correlations were also observed for distinctions between alpha-helix (0.98) and disordered helix (0.82), and between parallel (0.82) and antiparallel (0.97) beta-sheets. The average standard deviation of these Raman estimates from the X-ray values is less than 4%. In addition, a singular value analysis of 20 Raman amide I spectra indicates that there may be as many as nine significant independent pieces of information present in the amide I region.     

Observation of Solvent Penetration during Cold Denaturation of E.?coli Phosphofructokinase-2

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N₂ 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner.     

Changes in structure and in interactions of heat-treated bovine beta-lactoglobulin

Heat stress on structure and ligand binding of beta-LG has been studied by fluorescence, circular dichroism and gel electrophoresis at pH 6.5. Native PAGE gel electrophoresis shows that denaturation of beta-LG is reversible up to 75 degrees C then it becomes irreversible due to aggregation of beta-LG. Formation of aggregated beta-LG is completed at 95 degrees C. Circular dichroism results indicate that formation of aggregated beta-LG is accompanied by the scrambling of disulfide bonds (creation of new intramolecular and intermolecular disulfide bridges and rearrangement of old intramolecular disulfide bridges). Addition of ethanolic retinol causes a change in polarity of the solution and favors transformation of the beta<-->alpha structure. In the presence of retinol, the alpha-helix content of the secondary structure of heat-treated beta-LG is increased and the major portion of its secondary structure is helical. Fluorescence results show that heat-treated beta-LG at 95 degrees C can still bind retinol. The refolding of the tertiary structure of beta-LG heat-denatured at 95 degrees C may recreate a retinol binding site. Surprisingly, the affinity of the new site for retinol is higher than that of native beta-LG; however, the apparent molar ratio is lower than one. The binding properties of beta-LG for terpenoids have been measured after its heat treatment at 20, 75 and 95 degrees C. The intensity of tryptophan emission at 330 nm was changed only in the case of the interaction with beta-ionone. Other ligands probably cannot bind to beta-LG or they bind in a binding site far from the tryptophan residues, hence not affecting its fluorescence.     

Mono- and disaccharides enhance the activity and enantioselectivity of Burkholderia cepacia lipase in organic solvent but do not significantly affect its conformation

Sucrose, trehalose, and mannitol were colyophilized with lipase from Burkholderia cepacia and their effects on the activity and enantioselectitivity of the enzyme evaluated using as model reactions the transesterification between n-octanol or 6-methyl-5-hepten-2-ol with vinyl acetate. The lipase co-lyophilized with sugars showed an activity which was up to 4.7-fold higher (at a sugar/lipase ratio >or= 20) than that observed without sugar. Analogously, lipase enantioselectivity, expressed as the enantiomeric ratio, increased up to 2.8-fold in the presence of sugars. The conformation of the lipase was investigated by means of Fourier transform infrared spectroscopy (FT/IR) in water and as lyophilized powder. The infrared spectra of lyophilized lipase in the presence and, even more so, in the absence of sugars were different from that of the enzyme in water. In particular, the band at around 1,654/cm, typically assigned to alpha-helix, was less intense in the lyophilized samples. Nevertheless, the enzyme in the presence of sugars showed a decrease of the bands at 1,614-1,620/cm and at 1,680-1,695/cm that indicates a lower content of intermolecular beta-sheets (typical of protein aggregates). Additionally the increase of the component at 1,546/cm in the amide II region is consistent with a hydrogen bond pattern of the enzyme more similar to that shown in water. These results suggest that although sugars are not able to fully preserve the native secondary structure, they might contribute to reduce the conformational changes caused by protein/protein interactions. These factors in combinations with others (e.g., ability to reduce deleterious interactions between the enzyme and inert supports) make sugars (both mono- and disaccharides) an interesting class of additives for improving the performance of biocatalysts in organic solvents.     

The point mutation A34F causes dimerization of GB1

The immunoglobulin-binding domain B1 of streptococcal protein G (GB1), a very stable, small, single-domain protein, is one of the most extensively used models in the area of protein folding and design. Variants derived from a library of randomized hydrophobic core residues previously revealed alternative folds, namely a completely intertwined tetramer (Frank et al., Nat Struct Biol 2002;9:877-885) and a domain-swapped dimer (Byeon et al., J Mol Biol 2003;333:141-152). Here, we report the NMR structure of the single amino acid mutant Ala-34-Phe which exists as side-by-side dimer. The dimer dissociation constant is 27 +/- 4 microM. The dimer interface comprises two structural elements: First, the beta-sheets of the two monomers pair in an antiparallel arrangement, thereby forming an eight-stranded beta-sheet. Second, the alpha-helix is shortened, ending in a loop that engages in intermolecular contacts. The largest difference between the monomer unit in the A34F dimer and the monomeric wild-type GB1 is the dissolution of the C-terminal half of the alpha-helix associated with a pronounced slow conformational motion of the interface loop. This involves a large movement of the Tyr-33 side chain that swings out from the monomer to engage in dimer contacts.     

Thermal denaturation of globular proteins. Fourier transform-infrared studies of the amide III spectral region.

Fourier transform-infrared (FT-IR) spectra are reported for the amide III spectral region of the native and thermally denatured forms of chymotrypsinogen, ribonuclease, bovine serum albumin, and lysozyme. Chymotrypsinogen denatures into structures containing substantial contributions from beta-sheets, while lysozyme and bovine serum albumin show increased amounts of random-coil forms. The changes observed for ribonuclease are quite small. Bovine serum albumin shows at least six bands in the 1,260-1,320 cm-1 region which undergo large intensity changes upon thermal denaturation, and hence are assignable to alpha-helical amide III modes. The large number of observed bands suggests that slight variations in helical geometry, symmetry, or interactions result in changed amide III frequencies, so that simple correlations between narrow frequency ranges and secondary structures may not be applicable for this mode. A widened frequency range is suggested as diagnostic for helical structures.