Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding.

Denaturant m values, the dependence of the free energy of unfolding on denaturant concentration, have been collected for a large set of proteins. The m value correlates very strongly with the amount of protein surface exposed to solvent upon unfolding, with linear correlation coefficients of R = 0.84 for urea and R = 0.87 for guanidine hydrochloride. These correlations improve to R = 0.90 when the effect of disulfide bonds on the accessible area of the unfolded protein is included. A similar dependence on accessible surface area has been found previously for the heat capacity change (delta Cp), which is confirmed here for our set of proteins. Denaturant m values and heat capacity changes also correlate well with each other. For proteins that undergo a simple two-state unfolding mechanism, the amount of surface exposed to solvent upon unfolding is a main structural determinant for both m values and delta Cp.     

Shape and accessible surface area of globular proteins.

The accessible surface area of a protein has been reported to be proportional to the $\text{2}\text{3}$ power of the real volume. When this relationship was compared to the area-volume relationship of simple geometric shapes, it was concluded that shape is not important in determining the accessible surface area and that spheres are good models for protein subunits. However, since the accessible surface area and the real volume are derived from different geometric figures, the relation between the two cannot be directly compared with the area-volume relationships of simple geometric shapes. When the volume of the geometric figure used to calculate the accessible surface area is estimated, the accessible area is proportional to the 0·77 power of that volume. Only this value can be directly compared with similar values for simple geometric figures. Indeed this comparison shows that protein molecules must be more aspherical at larger molecular weights, so shape is important in determining the area of a protein and spheres are not always good models for protein subunits. When the texture of the protein surface is considered, this comparison shows that protein molecules must be more aspherical or more textured at larger molecular weights.     

Thermodynamics of barnase unfolding.

The thermodynamics of barnase denaturation has been studied calorimetrically over a broad range of temperature and pH. It is shown that in acidic solutions the heat denaturation of barnase is well approximated by a 2-state transition. The heat denaturation of barnase proceeds with a significant increase of heat capacity, which determines the temperature dependencies of the enthalpy and entropy of its denaturation. The partial specific heat capacity of denatured barnase is very close to that expected for the completely unfolded protein. The specific denaturation enthalpy value extrapolated to 130 degrees C is also close to the value expected for the full unfolding. Therefore, the calorimetrically determined thermodynamic characteristics of barnase denaturation can be considered as characteristics of its complete unfolding and can be correlated with structural features--the number of hydrogen bonds, extent of van der Waals contacts, and the surface areas of polar and nonpolar groups. Using this information and thermodynamic information on transfer of protein groups into water, the contribution of various factors to the stabilization of the native structure of barnase has been estimated. The main contributors to the stabilization of the native state of barnase appear to be intramolecular hydrogen bonds. The contributions of van der Waals interactions between nonpolar groups and those of hydration effects of these groups are not as large if considered separately, but the combination of these 2 factors, known as hydrophobic interactions, is of the same order of magnitude as the contribution of hydrogen bonding.     

Complete sequence-specific 1H nuclear magnetic resonance assignments for the alpha-amylase polypeptide inhibitor tendamistat from Streptomyces tendae

The 1H nuclear magnetic resonance (n.m.r.) spectrum of the alpha-amylase inhibitor Tendamistat was completely assigned with the use of phase-sensitive homonuclear two-dimensional n.m.r. The assignments include the non-labile protons of the 74 amino acid residues as well as the labile protons which exchange sufficiently slowly to be observed in H2O solution. The proton chemical shifts are listed at 50 degrees C and pH 3.2, which coincides with the conditions used for the determination of the three-dimensional structure of Tendamistat.     

Determination of the complete three-dimensional structure of the alpha-amylase inhibitor tendamistat in aqueous solution by nuclear magnetic resonance and distance geometry

The complete three-dimensional structure of the alpha-amylase inhibitor Tendamistat in aqueous solution was determined by 1H nuclear magnetic resonance and distance geometry calculations using the program DISMAN. Compared to an earlier, preliminary determination of the polypeptide backbone conformation, stereo-specific assignments were obtained for 41 of the 89 prochiral groups in the protein, and a much more extensive set of experimental constraints was collected, including 842 distance constraints from nuclear Overhauser effects and over 100 supplementary constraints from spin-spin coupling constants and the identification of intramolecular hydrogen bonds. The complete protein molecule, including the amino acid side-chains is characterized by a group of nine structures corresponding to the results of the nine DISMAN calculations with minimal residual error functions. The average of the pairwise minimal root-mean-square distances among these nine structures is 0.85 A for the polypeptide backbone, and 1.52 A for all the heavy atoms. The procedures used for the structure determination are described and a detailed analysis is presented of correlations between the experimental input data and the precision of the structure determination.     

An empirical relationship between rotational correlation time and solvent accessible surface area

Structure–dynamics interrelationships are important in understanding protein function. We have explored the empirical relationship between rotational correlation times (τ_c and the solvent accessible surface areas (SASA) of 75 proteins with known structures. The theoretical correlation between SASA and τ_c through the equation SASA = K_rτ_c ^(2/3) is also considered. SASA was determined from the structure, τ_c ^calc was determined from diffusion tensor calculations, and τ_c ^expt was determined from NMR backbone¹³ C or ¹⁵N relaxation rate measurements. The theoretical and experimental values of τ_c correlate with SASA with regression analyses values of K_r as 1696 and 1896 m²s^-(2/3), respectively, and with corresponding correlation coefficients of 0.92 and 0.70.     

An empirical relationship between rotational correlation time and solvent accessible surface area

Structure–dynamics interrelationships are important in understanding protein function. We have explored the empirical relationship between rotational correlation times (_c and the solvent accessible surface areas (SASA) of 75 proteins with known structures. The theoretical correlation between SASA and _c through the equation SASA = K_rc ^(2/3) is also considered. SASA was determined from the structure, _c ^calc was determined from diffusion tensor calculations, and _c ^expt was determined from NMR backbone¹³ C or ¹⁵N relaxation rate measurements. The theoretical and experimental values of _c correlate with SASA with regression analyses values of K_r as 1696 and 1896 m²s^-(2/3), respectively, and with corresponding correlation coefficients of 0.92 and 0.70.     

The primary structure of alpha-amylase inhibitor Z-2685 from Streptomyces parvullus FH-1641. Sequence homology between inhibitor and alpha-amylase

The native and oxidized alpha-amylase inhibitor Z-2685, isolated from the culture medium of Streptomyces parvullus FH-1641, and its overlapping cleavage products were degraded by the automatic Edman technique. Digestion was carried out with pepsin, thermolysin and trypsin. The alpha-amylase inhibitor is a polypeptide consisting of 76 amino acids with a molecular mass of 8 129 Da. With the exception of methionine and lysine, all naturally occurring amino acids are present. It is interesting that identical regions exist, in particular the sequence Trp-Arg-Tyr common to all four known microbial inhibitor sequences. We believe that the side chains of these three amino acids are important for interacting with the alpha-amylase molecule. Computer alignment enabled us to show a possible binding region in the alpha-amylase molecule which might react with the inhibitors. Furthermore, homology exists to mammalian alpha-amylases. This result is explained by the assumption that the inhibitor evolved from a duplication of the original gene of the enzyme.     

Thermodynamics of apocytochrome b5 unfolding.

Apocytochrome b5 from rabbit liver was studied by scanning calorimetry, limited proteolysis, circular dichroism, second derivative spectroscopy, and size exclusion chromatography. The protein is able to undergo a reversible two-state thermal transition. However, transition temperature, denaturational enthalpy, and heat capacity change are reduced compared with the holoprotein. Apocytochrome b5 stability in terms of Gibbs energy change at protein unfolding (delta G) amounts to delta G = 7 +/- 1 kJ/mol at 25 degrees C (pH 7.4) compared with delta G = 25 kJ/mol for the holoprotein. Apocytochrome b5 is a compact, native-like protein. According to the spectral data, the cooperative structure is mainly based in the core region formed by residues 1-35 and 79-90. This finding is in full agreement with NMR data (Moore, C.D. & Lecomte, J.T.J., 1993, Biochemistry 32, 199-207).     

Thermodynamics of maltose binding protein unfolding.

The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. It is used widely as a carrier protein for the production of recombinant fusion proteins. The melting of recombinant MBP was studied by differential scanning and titration calorimetry and fluorescence spectroscopy under different solvent conditions. MBP exhibits a single peak of heat absorption with a delta(Hcal)/delta(HvH) ratio in the range of 1.3-1.5, suggesting that the protein comprises two strongly interacting thermodynamic domains. Binding of maltose resulted in elevation of the Tm by 8-15 degrees C, depending of pH. The presence of ligand at neutral pH, in addition to shifting the melting process to higher temperature, caused it to become more cooperative. The delta(Hcal)/delta(HvH) ratio decreased to unity, indicating that the two domains melt together in a single two-state transition. This ligand-induced merging of the two domains appears to occur only at neutral pH, because at low pH maltose simply stabilized MBP and did not cause a decrease of the delta(Hcal)/delta(HvH) ratio. Binding of maltose to MBP is characterized by very low enthalpy changes, approximately -1 kcal/mol. The melting of MBP is accompanied by an exceptionally large change in heat capacity. 0.16 cal/K-g, which is consistent with the high amount of nonpolar surface--0.72 A2/g--that becomes accessible to solvent in the unfolded state. The high value of delta Cp determines a very steep delta G versus T profile for this protein and predicts that cold denaturation should occur above freezing temperatures. Evidence for this was provided by changes in fluorescence intensity upon cooling the protein. A sigmoidal cooperative transition with a midpoint near 5 degrees C was observed when MBP was cooled at low pH. Analysis of the melting of several fusion proteins containing MBP illustrated the feasibility of assessing the folding integrity of recombinant products prior to separating them from the MBP carrier protein.     

Secondary structure of the alpha-amylase polypeptide inhibitor tendamistat from Streptomyces tendae determined in solution by 1H nuclear magnetic resonance

Complete sequence-specific 1H nuclear magnetic resonance assignments were obtained for the backbone hydrogen atoms in Tendamistat, a protein with 74 residues. From NOESY observation of 1H-1H short distance constraints, measurements of the spin-spin couplings 3JHN alpha and a qualitative identification of slowly exchanging amide protons, two antiparallel beta-sheets containing three and four strands, respectively, were identified. The peptide segments outside the beta-sheets do not form regular secondary structure. Preliminary data were obtained on the relative spatial arrangements of the two beta-sheets.     

Thermodynamics of the temperature-induced unfolding of globular proteins.

The heat capacity, enthalpy, entropy, and Gibbs energy changes for the temperature-induced unfolding of 11 globular proteins of known three-dimensional structure have been obtained by microcalorimetric measurements. Their experimental values are compared to those we calculate from the change in solvent-accessible surface area between the native proteins and the extended polypeptide chain. We use proportionality coefficients for the transfer (hydration) of aliphatic, aromatic, and polar groups from gas phase to aqueous solution, we estimate vibrational effects, and we discuss the temperature dependence of each constituent of the thermodynamic functions. At 25 degrees C, stabilization of the native state of a globular protein is largely due to two favorable terms: the entropy of non-polar group hydration and the enthalpy of interactions within the protein. They compensate the unfavorable entropy change associated with these interactions (conformational entropy) and with vibrational effects. Due to the large heat capacity of nonpolar group hydration, its stabilizing contribution decreases quickly at higher temperatures, and the two unfavorable entropy terms take over, leading to temperature-induced unfolding.     

Contribution of hydration and non-covalent interactions to the heat capacity effect on protein unfolding.

The heat capacity change upon protein unfolding has been analysed using the heat capacity data for the model compounds' transfer into water, corrected for volume effects. It has been shown that in the unfolding, the heat capacity increment is contributed to by the effect of hydration of the non-polar groups, which is positive and decreases with temperature increase, and by the effect of hydration of the polar groups, which is negative and decreases in magnitude as temperature increases. The sum of these two effects is very close to the total heat capacity increment of protein unfolding at room temperature but is likely to deviate from it at higher temperatures. Therefore, the expected heat capacity effect caused by the increase of configurational freedom of the polypeptide chain upon unfolding seems to be compensated for by some other effect, perhaps associated with fluctuation of the native protein structure.     

Relationship between chemical shift value and accessible surface area for all amino acid atoms

Background  

Chemical shifts obtained from NMR experiments are an important tool in determining secondary, even tertiary, protein structure. The main repository for chemical shift data is the BioMagResBank, which provides NMR-STAR files with this type of information. However, it is not trivial to link this information to available coordinate data from the PDB for non-backbone atoms due to atom and chain naming differences, as well as sequence numbering changes.     

Kinetics and energetics of the binding between barley alpha-amylase/subtilisin inhibitor and barley alpha-amylase 2 analyzed by surface plasmon resonance and isothermal titration calorimetry

The kinetics and energetics of the binding between barley alpha-amylase/subtilisin inhibitor (BASI) or BASI mutants and barley alpha-amylase 2 (AMY2) were determined using surface plasmon resonance and isothermal titration calorimetry (ITC). Binding kinetics were in accordance with a 1:1 binding model. At pH 5.5, [Ca(2+)] = 5 mM, and 25 degrees C, the k(on) and k(off) values were 8.3 x 10(+4) M(-1) s(-1) and 26.0 x 10(-4) s(-1), respectively, corresponding to a K(D) of 31 nM. K(D) was dependent on pH, and while k(off) decreased 16-fold upon increasing pH from 5.5 to 8.0, k(on) was barely affected. The crystal structure of AMY2-BASI shows a fully hydrated Ca(2+) at the protein interface, and at pH 6.5 increase of [Ca(2+)] in the 2 microM to 5 mM range raised the affinity 30-fold mainly due to reduced k(off). The K(D) was weakly temperature-dependent in the interval from 5 to 35 degrees C as k(on) and k(off) were only increasing 4- and 12-fold, respectively. A small salt dependence of k(on) and k(off) suggested a minor role for global electrostatic forces in the binding and dissociation steps. Substitution of a positively charged side chain in the mutant K140L within the AMY2 inhibitory site of BASI accordingly did not change k(on), whereas k(off) increased 13-fold. ITC showed that the formation of the AMY2-BASI complex is characterized by a large exothermic heat (Delta H = -69 +/- 7 kJ mol(-1)), a K(D) of 25 nM (27 degrees C, pH 5.5), and an unfavorable change in entropy (-T Delta S = 26 +/- 7 kJ mol(-1)). Calculations based on the thermodynamic data indicated minimal structural changes during complex formation.     

A chimera-like alpha-amylase inhibitor suggesting the evolution of Phaseolus vulgaris alpha-amylase inhibitor

White kidney bean (Phaseolus vulgaris) contains two kinds of alpha-amylase inhibitors, one heat-stable (alpha AI-s) and one heat-labile (alpha AI-u). alpha AI-s has recently been revealed to be a tetrameric complex, alpha(2)beta(2), with two active sites [Kasahara et al. (1996) J. Biochem. 120, 177-183]. The present study was undertaken to reveal the molecular features of alpha AI-u, which is composed of three kinds of subunits, alpha, beta, and gamma. The gamma-subunit, in contrast to the alpha- and beta-subunits that are indistinguishable from the alpha- and beta-subunits of alpha AI-s, was found to correspond to a subunit of an alpha-amylase inhibitor-like protein, which has been identified as an inactive, evolutionary intermediate between arcelin and the alpha-amylase inhibitor in a P. vulgaris defense protein family. The polypeptide molecular weight of alpha AI-u determined by the light-scattering technique, together with the polypeptide molecular weights of the subunits, suggests that alpha AI-u is a trimeric complex, alpha beta gamma. The inhibition of alpha AI-u by increasing amounts of porcine pancreatic alpha-amylase (PPA) indicates that an inactive 1:1 complex is formed between alpha AI-u and PPA. Molecular weight estimation of the complex by the light-scattering technique confirmed that it is a complex of alpha AI-u with one PPA molecule. Thus it seems probable that alpha AI-u is an evolutionary intermediate of the P. vulgaris alpha-amylase inhibitor.     

Heat capacity of proteins. II. Partial molar heat capacity of the unfolded polypeptide chain of proteins: protein unfolding effects

Using the heat capacity values for amino acid side-chains and the peptide unit determined in the accompanying paper, we calculated the partial heat capacities of the unfolded state for four proteins (apomyoglobin, apocytochrome c, ribonuclease A, lysozyme) in aqueous solution in the temperature range from 5 to 125 degrees C, with an assumption that the constituent amino acid residues contribute additively to the integral heat capacity of a polypeptide chain. These ideal heat capacity functions of the extended polypeptide chains were compared with the calorimetrically determined heat capacity functions of the heat and acid-denatured proteins. The average deviation of the experimental functions from the calculated ideal ones in the whole studied temperature range does not exceed the experimental error (5%). Therefore, the heat-denatured state of a protein, in solutions with acidic pH preventing aggregation, approximates well the completely unfolded state of this macromolecule. The heat capacity change caused by hydration of amino acid residues upon protein unfolding was also determined and it was shown that this is the major contributor to the observed heat capacity effect of unfolding. Its value is different for different proteins and correlates well with the surface area of non-polar groups exposed upon unfolding. The heat capacity effect due to the configurational freedom gain by the polypeptide chain was found to contribute only a small part of the overall heat capacity change on unfolding.     

Thermodynamics and kinetics of the thermal unfolding of plastocyanin

The thermal denaturation of plastocyanin in aqueous solution was investigated by means of DSC, ESR and absorbance techniques, with the aim of determining the thermodynamic stability of the protein and of characterizing the thermally induced conformational changes of its active site. The DSC and absorbance experiments indicated an irreversible and kinetically controlled denaturation path. The extrapolation of the heat capacity and optical data at infinite scan rate made it possible to calculate the kinetic and thermodynamic parameters associated with the denaturation steps. The denaturation pathway proposed, and the parameters found from the calorimetric data, were checked by computer simulation using an equation containing the information necessary to describe the denaturation process in detail. ESR and absorbance measurements have shown that structural changes of the copper environment occur during the protein denaturation. In particular, the geometry of the copper-ligand atoms changes from being tetrahedral to square planar and the disruption of the active site precedes the global protein denaturation. The thermodynamic enthalpic change, the half-width transition temperature, and the value of ΔCp, were used to calculate the thermodynamic stability, ΔG, of the reversible process over the entire temperature range of denaturation. The low thermal stability found for plastocyanin, is discussed in connection with structural factors stabilizing the native state of a protein. Received: 17 July 1997 / Revised version: 22 November 1997 / Accepted: 15 January 1998