Thermal Unfolding Transition Research Articles

A helix initiation signal in T4 lysozyme identified by polyalanine mutagenesis

To better understand the relation between sequence and structure, and in an attempt to simplify the protein folding problem, a series of alanine substitutions was introduced into bacteriophage T4 lysozyme. In contrast to previous studies in this system, which were restricted to single α-helices, the present analysis included a helix–turn–helix region, a loop–helix region, and two α-helices that were well separated in the three-dimensional structure. It was shown previously that T4 lysozyme is very tolerant of alanine substitutions within α-helices, especially at solvent-exposed sites. The present study shows that the protein is also tolerant of such substitutions in turn and loop regions, although less than in helices. The results confirm that the structural information in the amino acid sequence is highly redundant. For example, the protein with the sequence 127AAAAAALAAAAWAAA 141 folds normally, has melting temperature only 0.8 °C lower than wildtype, and has a crystal structure that is also very similar to wildtype. Polyalanine substitutions within turns or loops can, however, lead to differences in structure and in folding. In one example the triple substitution K35A/S36A/P37A caused this region of the molecule to change to a more helical conformation. In a second case the mutant with the sequence 34AAAAALAAAKAALAAA 49, which spans a loop–helix region, had a dramatically altered thermal unfolding transition, suggesting that this region may tend to form a single, uninterrupted, helix. Substitution of Ala38 in the above construct with aspartic acid caused the unfolding to be more like wildtype, suggesting that residue 38, which is at a helix-capping position in the wildtype structure, provides an initiation signal that is essential in the polyalanine mutant for the correct formation of α-helix 39–50. In a typical protein, the information that codes for the 3D structure is presumably distributed over many amino acids. The present results suggest that in simplified sequences the key folding information may be restricted to a subset of critical residues, and so be more readily accessible to experimental analysis.

Influence of the hydrophobic face width on the degree of association of coiled–coil proteins

A very simplistic computational model for the simulation of the association of dimeric coiled–coil proteins has been extended to permit the formation of higher order quaternary structures. The main parameter of the new model is ω, the width of the potential well representing the attraction among hydrophobic residues. For values of ω ranging from 0.5 to 3.0 Å, our Monte Carlo simulations predict the formation of trimers at low temperatures. The different structures (monomers, dimers, and trimers) which populate the simulated system at various temperatures allow us to discuss the optimum value of ω from the point of view of the thermal unfolding transition. On the other hand, the detailed analysis of the trimer structure provides a different point of view about the performance of the model, when compared with the structural information experimentally available for trimeric coiled–coils.

Biphasic reductive unfolding of ribonuclease A is temperature dependent.

The kinetics of the reversible thermal unfolding, irreversible thermal unfolding, and reductive unfolding processes of bovine pancreatic ribonuclease A (RNase A) were investigated in NaCl/Pi solutions. Image parameters including Shannon entropy, Hamming distance, mutual information and correlation coefficient were used in the analysis of the CD and 1D NMR spectra. The irreversible thermal unfolding transition of RNase A was not a cooperative process, pretransitional structure changes occur before the main thermal denaturation. Different dithiothreitol (dithiothreitolred) concentration dependencies were observed between 303 and 313 K during denaturation induced by a small amount of reductive reagent. The protein selectively follows a major unfolding kinetics pathway with the selectivity can be altered by temperature and reductive reagent concentration. Two possible explanations of the selectivity mechanism were discussed.

Complementation of buried lysine and surface polar residues in a designed heterodimeric coiled coil.

The coiled coil is an attractive target for protein design. The helices of coiled coils are characterized by a heptad repeat of residues denoted a to g. Residues at positions a and d form the interhelical interface and are usually hydrophobic. An established strategy to confer structural uniqueness to two-stranded coiled coils is the use of buried polar Asn residues at position a, which imparts dimerization and conformational specificity at the expense of stability. Here we show that polar interactions involving buried position-a Lys residues that can interact favorably only with surface e' or g' Glu residues also impart structural uniqueness to a designed heterodimeric coiled coil with the nativelike properties of sigmoidal thermal and urea-induced unfolding transitions, slow hydrogen exchange and lack of ANS binding. The position-a Lys residues do not, however, confer a single preference for helix orientation, likely reflecting the ability of Lys at position a to from favorable interactions with g' or e' Glu residues in the parallel and antiparallel orientations, respectively. The Lys-Glu polar interaction is less destabilizing than the Asn-Asn a-->a' interaction, presumably reflecting a higher desolvation penalty associated with the completely buried polar position-a groups. Our results extend the range of approaches for two-stranded coiled-coil design and illustrate the role of complementing polar groups associated with buried and surface positions of proteins in protein folding and design.

Three‐way multivariate curve resolution applied to speciation of acid–base and thermal unfolding transitions of an alternating polynucleotide

Analytical speciation of acid-base equilibria and thermal unfolding transitions of an alternating random polynucleotide containing cytosine and hypoxanthine, poly(C, I), is studied. The results are compared with those obtained previously for single-stranded polynucleotides, poly(I) and poly(C), and for the double-stranded poly(I). poly(C), to examine the influence of the secondary structure on the acid-base properties of bases. This study is based on monitoring acid-base titrations and thermal unfolding experiments by molecular absorption, CD, and molecular fluorescence spectroscopies. Experimental data were analyzed by a novel chemometric approach based on a recently developed three-way Multivariate Curve Resolution method, which allowed the simultaneous analysis of data from several spectroscopies. This procedure improves the resolution of the concentration profiles and pure spectra for the species and conformations present in folding-unfolding and acid-base equilibria. The results from acid-base studies showed the existence of only three species in the pH range 2-12 at 37 degrees C and 0.15M ionic strength. No cooperative effects were detected from the resolved concentration profiles, showing that equilibria concerning alternating polynucleotides like poly(C, I) are simpler than those involving poly(I). poly(C). Thermal unfolding experiments at neutral pH confirmed the existence of two transitions and one intermediate conformation. This intermediate conformation could only be detected and resolved without ambiguities when molecular absorption and CD spectral data were analyzed simultaneously.

Temperature-induced changes in copper centers and protein conformation of two fungal laccases from Coriolus hirsutus and Coriolus zonatus

The paper reports on two fungal laccases from Coriolus hirsutus and Coriolus zonatus and their type-2 copper-depleted derivatives. Temperature-induced changes of the copper centers were characterized by optical and electron paramagnetic resonance (EPR) spectroscopy, and the overall protein stability by differential scanning microcalorimetry. The intact enzymes showed highly cooperative thermal unfolding transitions at about 90°C. Type-2 copper depletion led to uncoupling of the domains characterized by a different melting pattern which resolved three subtransitions. Melting curves monitored optically at 290, 340 and 610 nm showed additional transitions below thermal unfolding temperature. EPR spectra of the intact laccases showed the disintegration of the trinuclear copper cluster accompanied by loss of one of the copper ions and disappearance of the strong antiferromagnetic coupling in the type-3 site at 70°C and above 70°C. The copper centers of type-2 copper-depleted laccase showed reduced thermotolerance.

Design, Engineering, and Production of Human Recombinant T Cell Receptor Ligands Derived from Human Leukocyte Antigen DR2

Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen-presenting cells that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells. Susceptibility to multiple sclerosis is associated with certain MHC class II haplotypes, including human leukocyte antigen (HLA) DR2. Two DRB chains, DRB5*0101 and DRB1*1501, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501). Both isotypes can present an immunodominant peptide of myelin basic protein (MBP-(84-102)) to MBP-specific T cells from multiple sclerosis patients. We have previously demonstrated that the peptide binding/T cell recognition domains of rat MHC class II (alpha1 and beta1 domains) could be expressed as a single exon for structural and functional characterization; Burrows, G. G., Chang, J. W., Bächinger, H.-P., Bourdette, D. N., Wegmann, K. W., Offner, H., and Vandenbark A. A. (1999) Protein Eng. 12, 771-778; Burrows, G. G., Adlard, K. L., Bebo, B. F., Jr., Chang, J. W., Tenditnyy, K., Vandenbark, A. A., and Offner, H. (2000) J. Immunol. 164, 6366-6371). Single-chain human recombinant T cell receptor ligands (RTLs) of approximately 200 amino acid residues derived from HLA-DR2b were designed using the same principles and have been produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides. Structural characterization using circular dichroism predicted that these molecules retained the antiparallel beta-sheet platform and antiparallel alpha-helices observed in the native HLA-DR2 heterodimer. The proteins exhibited a cooperative two-state thermal unfolding transition, and DR2-derived RTLs with a covalently linked MBP peptide (MBP-(85-99)) showed increased stability to thermal unfolding relative to the empty DR2-derived RTLs. These novel molecules represent a new class of small soluble ligands for modulating the behavior of T cells and provide a platform technology for developing potent and selective human diagnostic and therapeutic agents for treatment of autoimmune disease.

Three-way multivariate curve resolution applied to speciation of acid-base and thermal unfolding transitions of an alternating polynucleotide

Analytical speciation of acid-base equilibria and thermal unfolding transitions of an alternating random polynucleotide containing cytosine and hypoxanthine, poly(C, I), is studied. The results are compared with those obtained previously for single-stranded polynucleotides, poly(I) and poly(C), and for the double-stranded poly(I). poly(C), to examine the influence of the secondary structure on the acid-base properties of bases. This study is based on monitoring acid-base titrations and thermal unfolding experiments by molecular absorption, CD, and molecular fluorescence spectroscopies. Experimental data were analyzed by a novel chemometric approach based on a recently developed three-way Multivariate Curve Resolution method, which allowed the simultaneous analysis of data from several spectroscopies. This procedure improves the resolution of the concentration profiles and pure spectra for the species and conformations present in folding-unfolding and acid-base equilibria. The results from acid-base studies showed the existence of only three species in the pH range 2-12 at 37 degrees C and 0.15M ionic strength. No cooperative effects were detected from the resolved concentration profiles, showing that equilibria concerning alternating polynucleotides like poly(C, I) are simpler than those involving poly(I). poly(C). Thermal unfolding experiments at neutral pH confirmed the existence of two transitions and one intermediate conformation. This intermediate conformation could only be detected and resolved without ambiguities when molecular absorption and CD spectral data were analyzed simultaneously.

Local and long-range interactions in the thermal unfolding transition of bovine pancreatic ribonuclease A.

This research was undertaken to distinguish between local and global unfolding in the reversible thermal denaturation of bovine pancreatic ribonclease A (RNase A). Local unfolding was monitored by steady-state and time-resolved fluorescence of nine mutants in each of which a single tryptophan was substituted for a wild-type residue. Global unfolding was monitored by far-UV circular dichroism and UV absorbance. All the mutants (except F8W and D38W) exhibited high specific enzymatic activity, and their far-UV CD spectra were very close to that of wild-type RNase A, indicating that the tryptophan substitutions did not affect the structure of any of the mutants (excluding K1W and Y92W) under folding conditions at 20 degrees C. Like wild-type RNase A, the various mutants exhibited reversible cooperative thermal unfolding transitions at pH 5, with transition temperatures 2.5-11 degrees C lower than that of the wild-type transition, as detected by far-UV CD or UV absorbance. Even at 80 degrees C, well above the cooperative transition of all the RNase A mutants, a considerable amount of secondary and tertiary structure was maintained. These studies suggest the following two-stage mechanism for the thermal unfolding transition of RNase A as the temperature is increased. First, at temperatures lower than those of the main cooperative transition, long-range interactions within the major hydrophobic core are weakened, e.g., those involving residues Phe-8 (in the N-terminal helix) and Lys-104 and Tyr-115 (in the C-terminal beta-hairpin motif). The structure of the chain-reversal loop (residues 91-95) relaxes in the same temperature range. Second, the subsequent higher-temperature cooperative unfolding transition is associated with a loss of secondary structure and additional changes in the tertiary contacts of the major hydrophobic core, e.g., those involving residues Tyr-73, Tyr-76, and Asp-38 on the other side of the molecule. The hydrophobic interactions of the C-terminal loop of the protein are enhanced by high temperature, and perhaps are responsible for the preservation of the local structural environment of Trp-124 at temperatures slightly above the major cooperative transition. The results shed new light on the thermal unfolding transitions, generally supporting the thermal unfolding hypothesis of Burgess and Scheraga, as modified by Matheson and Scheraga.

MJ1647, an open reading frame in the genome of the hyperthermophile Methanococcus jannaschii, encodes a very thermostable archaeal histone with a C-terminal extension.

All archaeal histones studied to date have similar lengths, 66 to 69 amino acid residues that form three alpha-helices separated by two beta-strand loop regions which together constitute a histone fold. In contrast, the eukaryal nucleosome core histones are larger, 102 to 135 residues in length, with N-terminal and C-terminal extensions flanking the histone fold that participate in gene regulation and higher-order chromatin assembly. In the Methanococcus jannaschii genome, MJ1647 was annotated as an open reading frame predicted to encode an archaeal histone with an approximately 27-amino-acid C-terminal extension, and we here document the DNA binding and assembly properties and thermodynamic stability parameters of the recombinant product of MJ1647 synthesized in Escherichia coli with (rMJ1647) and without (rMJ1647delta) the C-terminal extension. The presence of the C-terminal extension did not prevent homodimer formation or inhibit DNA binding, but the complexes formed by rMJ1647, presumably archaeal nucleosomes containing a (rMJ1647)4 tetramer, were apparently less stable than those formed by (rMJ1647delta)4. The presence of the C-terminal extension increased the thermostability of rMJ1647 when compared with rMJ1647delta in 0.2 M KCl at pH 4 but not in the absence of KCl at pH 1. Based on thermal unfolding transitions, rMJ1647 and rHAfB generated by expression of AF0337 cloned from the genome of the related hyperthermophile Archaeoglobus fulgidus in E. coli were found to have higher thermodynamic stabilities than all previously studied archaeal histones.

The single mutation Phe173 --> Ala induces a molten globule-like state in murine interleukin-6.

A series of three aromatic to alanine mutants of recombinant murine interleukin-6 lacking the 22 N-terminal residues (DeltaN22mIL-6) were constructed to investigate the role of these residues in the structure and function of mIL-6. While Y78A and Y97A have activities similar to that of DeltaN22mIL-6, F173A lacks biological activity. F173A retains high levels of secondary structure, as determined by far-UV circular dichroism (CD), but has substantially reduced levels of tertiary structure, as determined by near-UV CD and (1)H NMR spectroscopy. F173A also binds the hydrophobic dye 1-anilino-8-naphthalenesulfonic acid (ANS) over a range of pH values and exhibits noncooperative equilibrium unfolding (as judged by the noncoincidence of monophasic unfolding transitions monitored by far-UV CD and lambda(max), with midpoints of unfolding at 2.6 +/- 0. 1 and 3.5 +/- 0.3 M urea, respectively, and the lack of an observable thermal unfolding transition). These are all properties of molten globule states, suggesting that the loss of activity of F173A results from the disruption of the fine structure of the protein, rather than from the loss of a side chain that is important for ligand-receptor interactions. Surprisingly, under some conditions, this loosened conformation is no more susceptible to proteolytic attack than the parent protein. By analogy with human IL-6, Phe173 in DeltaN22mIL-6 makes multiple interhelical interactions, the removal of which appear to be sufficient to induce a molten globule-like conformation.

Role of free Cys121 in stabilization of bovine beta-lactoglobulin B.

Mixed disulfide derivatives of bovine beta-lactoglobulin (BLG) were studied by circular dichroism (CD), gel-permeation HPLC and high-sensitivity differential scanning calorimetry (HS-DSC). It was shown that modification of Cys121 with mercaptopropionic acid and mercaptoethanol does not affect the secondary structure of BLG, but results instead in tertiary and quaternary structure changes. At neutral pH, the equilibrium dimer<==>monomer of modified beta-lactoglobulin is shifted towards monomeric form. In contrast to native BLG, thermal denaturation of modified beta-lactoglobulin is fully reversible in neutral and acidic pH as demonstrated by CD and HS-DSC measurements. Modification of Cys121 results in a significant decrease of transition temperature (-6 degrees C) and enthalpy (-106 kJ/mol) at pH 2.05 while unfolding heat capacity increment remains unchanged. Thermal unfolding transitions of native and modified beta-lactoglobulin at pH 2.05 are well approximated by a two-state model suggesting that no intermediate states appear after modification. The difference in Gibbs energy of denaturation between native and modified beta-lactoglobulin, 8.5 kJ/mol at 37 degrees C and pH 2.05, does not depend on the nature of the introduced group (charged or neutral). Computer analysis of possible interactions involving Cys121 in a three-dimensional structure of beta-lactoglobulin revealed that the thiol group is too far away from neighboring residues to form side-chain hydrogen bonds. This suggests that the sulfhydryl group of Cys121 may contribute to the maintenance of BLG tertiary structure via water mediated H-bonding.

The spectroscopic analysis, inhibition and binding studies demonstrate the equivalence of Erythrina caffra trypsin inhibitor and the recombinant substitution variant recSerETI

A recombinant substitution mutant (recSerETI) of the Erythrina caffra trypsin inhibitor, with the N-terminal valine residue substituted by serine, was produced in E. coli and compared to the wildtype protein (wtETI) with respect to physicochemical and functional properties. The spectral properties, including UV absorbance, fluorescence emission and circular dichroism, were indistinguishable. Furthermore, the inhibitory activities of the two proteins regarding the inhibition of trypsin, chymotrypsin, tissue plasmininogen activator (t-PA) and reteplase (BM 06.022, t-PA deletion variant comprising the kringle 2 and the protease domains, isolated from transformed E. coli cells) and the affinity of the immobilized inhibitors for reteplase were closely similar. Five repetitive cycles of guanidinium chloride (GdmCl) -induced denaturation–renaturation yield the native mutant protein with its inhibitory activity fully restored. The only difference between the wildtype and the mutant protein refers to the intrinsic stability. Comparing the pH- and GdmCl-dependent transitions, as well as the thermal denaturation, recSerETI exhibits decreased stability compared to the wildtype protein. The pH range of stability is shifted from pH 1–9.5, for wtETI, to pH 2–9, for recSerETI; similarly the GdmCl-induced denaturation is found to occur at a GdmCl half concentration of 3.7 M instead of 4.5 M; in both cases the renaturation exhibits strong hysteresis. The mid-point of the thermal unfolding transition of the mutant protein is at ∼65°C, as compared to ∼75°C for the wildtype protein.

From coiled coils to small globular proteins: design of a native-like three-helix bundle.

A monomolecular native-like three-helix bundle has been designed in an iterative process, beginning with a peptide that noncooperatively assembled into an antiparallel three-helix bundle. Three versions of the protein were designed in which specific interactions were incrementally added. The hydrodynamic and spectroscopic properties of the proteins were examined by size exclusion chromatography, sedimentation equilibrium, fluorescence spectroscopy, and NMR. The thermodynamics of folding were evaluated by monitoring the thermal and guanidine-induced unfolding transitions using far UV circular dichroism spectroscopy. The attainment of a unique, native-like state was achieved through the introduction of: (1) helix capping interactions; (2) electrostatic interactions between partially exposed charged residues; (3) a diverse collection of apolar side chains within the hydrophobic core.

Cooperative folding of a protein mini domain: the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex

The peripheral subunit-binding domain from the dihydrolipoamide acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus is stably folded, despite its short sequence of only 43 amino acid residues. A 41 residue peptide derived from this domain, psbd41, undergoes a cooperative thermal unfolding transition with a tm of 54°C. This three-helix protein is monomeric as judged by ultracentrifugation and concentration-dependent CD measurements. Peptides corresponding to the individual helices are largely unstructured both alone and in combination, indicating that the unusual stability of this protein does not arise solely from unusually stable α-helices. Chemical denaturation by guanidine hydrochloride is also cooperative with a ΔGH2O of 3.1 kcal mol−1 at pH 8.0 and 25°C. The chemical denaturation is broad with an m-value of 760 cal mol−1 M−1. psbd41 contains a buried aspartate residue at position 34 that may provide stability and specificity to the fold. A mutant peptide, psbd41Asn was synthesized in which the buried aspartate residue was mutated to asparagine. This peptide still folds cooperatively and it is monomeric, but is much less thermostable than the wild-type with a tm of only 31°C. Chemical denaturations at 4°C give an m-value of 740 cal mol−1 M−1, similar to the wild-type, but the stability ΔGH2O is only 1.4 kcal mol−1. Both the wild-type and the mutant unfold at extremes of pH, but at 4°C psbd41Asn is folded over a narrower pH range than the wild-type. Although the mutant unfolds cooperatively by thermal and by chemical denaturation, its NMR spectrum is significantly broader than that of the wild-type and it binds ANS. These results show that Asp34 is vital for the stability and specificity of this structure, the second smallest natural sequence known to fold in the absence of disulfide bonds or metal or ligand-binding sites.

Structure and stability of the N-terminal domain of the ribosomal protein L9: evidence for rapid two-state folding.

The N-terminal domain, residues 1-56, of the ribosomal protein L9 has been chemically synthesized. The isolated domain is monomeric as judged by analytical ultracentrifugation and concentration-dependent CD. Complete 1H chemical shift assignments were obtained using standard methods. 2D-NMR experiments show that the isolated domain adopts the same structure as seen in the full-length protein. It consists of a three-stranded antiparallel beta-sheet sandwiched between two helixes. Thermal and urea unfolding transitions are cooperative, and the unfolding curves generated from different experimental techniques, 1D-NMR, far-UV CD, near-UV CD, and fluorescence, are superimposable. These results suggest that the protein folds by a two-state mechanism. The thermal midpoint of folding is 77 +/- 2 degrees C at pD 8.0, and the domain has a delta G degree folding = 2.8 +/- 0.8 kcal/mol at 40 degrees C, pH 7.0. Near the thermal midpoint of the unfolding transition, the 1D-NMR peaks are significantly broadened, indicating that folding is occurring on the intermediate exchange time scale. The rate of folding was determined by fitting the NMR spectra to a two-state chemical exchange model. Similar folding rates were measured for Phe 5, located in the first beta-strand, and for Tyr 25, located in the short helix between strands two and three. The domain folds extremely rapidly with a folding rate constant of 2000 s-1 near the midpoint of the equilibrium thermal unfolding transition.

Measurement of intrinsic fluorescence to probe the conformational flexibility and thermodynamic stability of a single tryptophan protein entrapped in a sol–gel derived glass matrix

In this study techniques based on measurement of intrinsic tryptophan (Trp) fluorescence were used to monitor the stability and conformational flexibility of the single tryptophan protein monellin which was entrapped in sol–gel derived glass monoliths formed from tetraethylorthosilicate. The monoliths were aged either in buffer (wet-aged) or in air (dry-aged). The steady-state fluorescence spectra, steady-state anisotropy and acrylamide quenching behaviour of the single Trp at position 3 were monitored during chemical denaturation with guanidine hydrochloride, and during thermal denaturation. These studies indicated that there were no significant improvements in either chemical or thermal stability when the protein was present in wet-aged monoliths. However, the long-term stability of the protein was improved six-fold when such monoliths were stored at 4 °C. The encapsulation of monellin into dry-aged monoliths caused the thermal unfolding transition to broaden and shift upward by 10 °C, and caused the long-term stability to improve by 12-fold (compared to solution). Chemical stability studies also showed a broader transition for the unfolding of the protein in dry-aged monoliths, and suggested that the protein was present in a distribution of environments, each with a slightly different thermodynamic stability. The steady-state fluorescence responses obtained during denaturation and the accessibility of native and denatured protein to quencher provided clear evidence that the entrapped protein had a smaller range of conformational motions compared to the protein in solution, and that the entrapped protein was not able to unfold completely. The restriction of conformational motion, along with the increased structural order of the internal environment of the monoliths, play a role in the improvements in thermal and long-term stability that were observed.

Granulocyte-colony stimulating factor maintains a thermally stable, compact, partially folded structure at pH2.

At acidic pH many proteins exist in a partially unfolded form, called the "A" state. This is defined as a flexible, expanded structure with well-defined, usually native-like secondary structure, but no unique tertiary structure, and showing no cooperativity during thermal-induced denaturation. Granulocyte-colony stimulating factor (G-CSF), a four-helix bundle cytokine, maintains both thermal stability and tertiary structure at pH 2.0. We therefore examined the conformation and thermal unfolding of G-CSF at pH 2.0, 4.0 and 7.0 using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). The secondary structure of the molecule remains highly helical as the pH is lowered from 7.0 to 2.0. The tertiary structure of the protein is slightly different at each pH value, but even at pH 2.0 G-CSF maintains a regular three-dimensional structure. The structure is hydrodynamically compact at these different pH values, with no increase in Stoke's radius even at pH 2.0. The thermal-induced denaturation of G-CSF was determined by monitoring changes in the CD or FTIR spectra. At pH 2.0 the temperature at which thermal-induced denaturation begins is higher than it is at pH 4.0 or 7.0, the thermal unfolding transition remains cooperative and some alpha-helical structure persists even at 86 degrees C. At pH 4.0 and 7.0, secondary and tertiary structures disappear simultaneously during thermal denaturation, whereas at pH 2.0 small changes in the far-UV CD region begin to occur first, followed by the simultaneous cooperative loss of tertiary structure and much of the remaining secondary structure. The structure of G-CSF at pH 2.0 is thus revealed as compact, with a unique, three-dimensional structure, highly helical secondary structure, and most importantly, a cooperative thermal unfolding transition. G-CSF at acid pH thus does not adopt the "A" state.

Dynamic structure/function relationships in the alpha-chymotrypsin deactivation process by heat and pH.

The activity decay of alpha-chymotrypsin due to temperature and pH has been related to the associated structural changes of the protein that could be studied with the help of melting temperature which was determined by using ultraviolet absorption spectroscopy, and fluorescence spectra measurements. The kinetic behaviour in activity loss of alpha-chymotrypsin followed a two-step deactivation model, involving an intermediate state. The increase in temperature and pH showed a clear deactivation effect, reducing exponentially the half-life of the enzyme. At pH 7.0, this two-step deactivation process was also observed with both the maximum fluorescence intensity (I(max)) and emission wavelength (lambda(max)). The series-type kinetic model allowed to establish a clear correlation between the activity and the fluorescence spectral normalized data: the intermediate state of the enzyme occurred at an identical deactivation denaturation level (alpha1), and a proportionality between the decay rate constants was observed. As a function of the incubation temperature, another correlation was observed between the alpha1 profile, initial lambda(max) and thermal unfolding transition, allowing to identify the intermediate state of the kinetic model as that obtained at the melting temperature (43.9 degrees C).

The Crystal Structure of a Hyperthermophilic Archaeal TATA-box Binding Protein

This study analyzes the three-dimensional structure of the TATA-box binding protein (TBP) from the hyperthermophilic archaea Pyrococcus woesei. The crystal structure of P. woeseiTBP ( PwTBP) was solved at 2.2 Å by X-ray diffraction and as expected from sequence homology (36% to 41% identical to eukaryotic TBPs) its overall structure is very similar to eukaryotic TBPs. The thermal unfolding transition temperature of this protein was measured by differential scanning calorimetry to be 101°C, which is more than 40°C higher than that of yeast TBP. Preliminary titration calorimetry data show that the affinity of PwTBP for its DNA target, unlike its eukaryotic counterparts, is enhanced by increasing the temperature and salt concentration. The structure reveals possible explanations for this thermostability and these unusual DNA binding properties. The crystal structure of this hyperthermostable protein was compared to its mesophilic homologs and analyzed for differences in the native structure that may contribute to thermostability. Differences found were: (1) a disulfide bond not found in mesophilic counterparts; (2) an increased number of surface electrostatic interactions; (3) more compact protein packing. The presumed DNA binding surface of PwTBP, like its eukaryotic counterparts, is hydrophobic but the electrostatic profile surrounding the protein is relatively neutral compared to the asymmetric positive potential that surrounds eukaryotic TBPs. The total reliance on a hydrophobic interface with DNA may explain the enhanced affinity of PwTBP for its DNA promoter at higher temperatures and increased salt concentration.