Protein Stability

Caspase-2, the most evolutionarily conserved member in the human caspase family, may play important roles in stress-induced apoptosis, cell cycle regulation, and tumor suppression. In biochemical assays, caspase-2 uniquely prefers a pentapeptide (such as VDVAD) rather than a tetrapeptide, as required for efficient cleavage by other caspases. We investigated the molecular basis for pentapeptide specificity using peptide analog inhibitors and substrates that vary at the P5 position. We determined the crystal structures of apo caspase-2, caspase-2 in complex with peptide inhibitors VDVAD-CHO, ADVAD-CHO, and DVAD-CHO, and a T380A mutant of caspase-2 in complex with VDVAD-CHO. Two residues, Thr-380 and Tyr-420, are identified to be critical for the P5 residue recognition; mutation of the two residues reduces the catalytic efficiency by about 4- and 40-fold, respectively. The structures also provide a series of snapshots of caspase-2 in different catalytic states, shedding light on the mechanism of capase-2 activation, substrate binding, and catalysis. By comparing the apo and inhibited caspase-2 structures, we propose that the disruption of a non-conserved salt bridge between Glu-217 and the invariant Arg-378 is important for the activation of caspase-2. These findings broaden our understanding of caspase-2 substrate specificity and catalysis.

The polypeptide alpha3, which was synthesized by us to produce an amphipathic helix structure, contains the regular three times repeated sequence (LETLAKA)(3), and alpha3 forms a fibrous assembly. To clarify how the side chains of amino acid residues affect the formation of alpha helix, Leu residues, which are located in the hydrophobic surface of an amphipathic helix, were replaced by other hydrophobic aliphatic amino acid residues systematically, and the characters of the resulting polypeptides were studied. According to the circular dichroism (CD) spectra, the Ile-substituted polypeptides formed alpha helix like alpha3. However, their helix formation ability was weaker than that of alpha3 under some conditions. The Val-substituted polypeptides formed alpha helix only under restricted condition. The Ala-substituted polypeptides did not form alpha helix under any condition. Thus, it is clear that the order of the alpha helix formation ability is as follows: Leu >or= Ile > Val > Ala. The formation of alpha helix was confirmed by Fourier Transform Infrared (FTIR) spectra. Through electron microscopic observation, it was clarified that the formation of the alpha helix structure correlates with the formation of a fibrous assembly. The amphipathic alpha helix structure would be stabilized by the formation of the fibrous assembly.

Protein IL-1β plays a key role in the innate immunity and development of the host reaction to microbial invasion and tissue injury. In structure, it has been reported to contain a hydrophobic cavity in protein interior, however the importance has not been clarified. We intend to realize how protein stability correlates with hydrophobicity on inside of cavity. The structures of chicken IL-1β and the variant, Y157F, have been reported to be nearly identical. The replacement of phenylalanine at residue 157 makes no structural difference in its side chain orientation. Two structures own highly similar hydrophobic cavities in both size and shape. A visible difference is an existing hydrogen bond between tyrosine side chain hydroxyl group and I133 carbonyl group in native IL-1β, where mutant Y157F lacks the hydrogen bonding. Surprisingly, melting temperature of Y157F is 10-degree higher than native IL-1β, reflected by a circular dichroism measurement. To resolve the contradiction of that hydrogen bond usually stabilizes macromolecular folding, we probe protein backbone dynamics by NMR. NMR H/D exchange experiment reveals that contact between sheets β11 and β13 is weakened in the presence of hydrogen bond between I133 CO and Y157 OH, meanwhile the corresponding region is relatively stable in Y157F. We purpose that Y157 OH group introduces an unfavorable energy in the hydrophobic cavity because of its polarity, thus hydroxyl sequesters itself on forming a hydrogen bond with the proximate residue, I133. The structural element β11, where I133 is located at, therefore exerts a force to move toward to the cavity (or Y157) and away from the paired -sheet, β13. The effect might destabilize the pair-wise  sheets and further perturbs the overall protein stability.

Factor XIII deficiency.

Many germ line antibodies have asparagine residues at specific sites to achieve specific antigen recognition. To study the role of asparagine residues in the stabilization of antigen-antibody complexes, we examined the interaction between hen egg white lysozyme (HEL) and the corresponding HyHEL-10 variable domain fragment (Fv). We introduced Ala and Asp substitutions into the Fv side chains of L-Asn-31, L-Asn-32, and L-Asn-92, which interact directly with residues in HEL via hydrogen bonding in the wild-type Fv-HEL complex, and we investigated the interactions between these mutant antibodies and HEL. Isothermal titration calorimetric analysis showed that all the mutations decreased the negative enthalpy change and decreased the association constants of the interaction. Structural analyses showed that the effects of the mutations on the structure of the complex could be compensated for by conformational changes and/or by gains in other interactions. Consequently, the contribution of two hydrogen bonds was minor, and their abolition by mutation resulted in only a slight decrease in the affinity of the antibody for its antigen. By comparison, the other two hydrogen bonds buried at the interfacial area had large enthalpic advantage, despite entropic loss that was perhaps due to stiffening of the interface by the bonds, and were crucial to the strength of the interaction. Deletion of these strong hydrogen bonds could not be compensated for by other structural changes. Our results suggest that asparagine can provide the two functional groups for strong hydrogen bond formation, and their contribution to the antigen-antibody interaction can be attributed to their limited flexibility and accessibility at the complex interface.

The study of the thermodynamic properties and cooperativity involved in three centered hydrogen (THB) bond formation using aromatic ortho-A substituted amides, oxalamates and bis- oxalamides (A = H, OMe, F, CH2OH, NO2, COCH3) as model molecules is reported. ∆H° and ∆S° associated with disruption of intramolecular hydrogen bonding by solvent were estimated using temperature dependence data of the N-H chemical shift. The results suggest that the influence of the A group is more important when electron-withdrawing, increasing both the enthalpy and entropy with an important contribution from conformational changes. The data allowed the estimation of the Ph=NH + rotational barrier of 14.0 kJ mol -1 in the amide and 16.7- 18.0 kJ mol -1 in oxalyl moiety. Correlations between ∆H° and ∆S° with NH temperature gradients predicted an enthalpy change of 18.7(1.0) and 24.4(1.7) kJ mol -1 for the energy required to break a full THB bond (A⋅⋅⋅H⋅⋅⋅O=C) and entropy differences between the non hydrogen bonded and hydrogen bonded state of 42.0(4.7) and 61.9(11) J mol -1 K -1 in oxalamate and bis-oxalamide series, respectively, in agreement with the participation of cooperative effects.

The lovastatin hydrolase PcEST from the fungus Penicillium chrysogenum exhibits enormous potential for industrial-scale applications in single-step production of monacolin J, the key precursor for synthesis of the cholesterol-lowering drug simvastatin. This enzyme specifically and efficiently catalyzes the conversion of lovastatin to monacolin J but cannot hydrolyze simvastatin. Understanding the catalytic mechanism and the structure–function relationship of PcEST is therefore important for further lovastatin hydrolase screening, engineering, and commercial applications. Here, we solved four X-ray crystal structures, including apo PcEST (2.3 Å), PcEST in complex with monacolin J (2.48 Å), PcEST complexed with the substrate analog simvastatin (2.4 Å), and an inactivated PcEST variant (S57A) with the lovastatin substrate (2.3 Å). Structure-based biochemical analyses and mutagenesis assays revealed that the Ser57 (nucleophile)–Tyr170 (general base)–Lys60 (general acid) catalytic triad, the hydrogen-bond network (Trp344 and Tyr127) around the active site, and the specific substrate-binding tunnel together determine efficient and specific lovastatin hydrolysis by PcEST. Moreover, steric effects on nucleophilic attack caused by the 2′,2-dimethybutyryl group of simvastatin resulted in no activity of PcEST on simvastatin. On the basis of structural comparisons, we propose several indicators to define lovastatin esterases. Furthermore, using structure-guided enzyme engineering, we developed a PcEST variant, D106A, having improved solubility and thermostability, suggesting a promising application of this variant in industrial processes. To our knowledge, this is the first report describing the mechanism and structure–function relationship of lovastatin hydrolase and providing insights that may guide rapid screening and engineering of additional lovastatin esterase variants. The lovastatin hydrolase PcEST from the fungus Penicillium chrysogenum exhibits enormous potential for industrial-scale applications in single-step production of monacolin J, the key precursor for synthesis of the cholesterol-lowering drug simvastatin. This enzyme specifically and efficiently catalyzes the conversion of lovastatin to monacolin J but cannot hydrolyze simvastatin. Understanding the catalytic mechanism and the structure–function relationship of PcEST is therefore important for further lovastatin hydrolase screening, engineering, and commercial applications. Here, we solved four X-ray crystal structures, including apo PcEST (2.3 Å), PcEST in complex with monacolin J (2.48 Å), PcEST complexed with the substrate analog simvastatin (2.4 Å), and an inactivated PcEST variant (S57A) with the lovastatin substrate (2.3 Å). Structure-based biochemical analyses and mutagenesis assays revealed that the Ser57 (nucleophile)–Tyr170 (general base)–Lys60 (general acid) catalytic triad, the hydrogen-bond network (Trp344 and Tyr127) around the active site, and the specific substrate-binding tunnel together determine efficient and specific lovastatin hydrolysis by PcEST. Moreover, steric effects on nucleophilic attack caused by the 2′,2-dimethybutyryl group of simvastatin resulted in no activity of PcEST on simvastatin. On the basis of structural comparisons, we propose several indicators to define lovastatin esterases. Furthermore, using structure-guided enzyme engineering, we developed a PcEST variant, D106A, having improved solubility and thermostability, suggesting a promising application of this variant in industrial processes. To our knowledge, this is the first report describing the mechanism and structure–function relationship of lovastatin hydrolase and providing insights that may guide rapid screening and engineering of additional lovastatin esterase variants.

PDZ domains1 are modular protein interaction domains that play a role in protein targeting and protein complex assembly. Once termed Discs-large homology regions (DHRs) or GLGF repeats (after a conserved Gly-Leu-Gly-Phe sequence found within the domain), these domains of ∼90 amino acids are now primarily known by an acronym of the first three PDZ-containing proteins identified: the postsynaptic protein PSD-95/SAP90, theDrosophila septate junction protein Discs-large, and the tight junction protein ZO-1.

Chirality transfer, induction, and circularly polarized luminescence (CPL) using supramolecular hosts, such as macrocycles and cages, have been explored for wide-ranging applications in chiral reco...

Side chains of amino acid residues are the determining factor that distinguishes proteins from other unstable chain polymers. In simple models they are often represented implicitly (e.g., by spin states) or simplified as one atom. Here we study side chain effects using two-dimensional square lattice and three-dimensional tetrahedral lattice models, with explicitly constructed side chains formed by two atoms of different chirality and flexibility. We distinguish effects due to chirality and effects due to side chain flexibilities, since residues in proteins are L residues, and their side chains adopt different rotameric states. For short chains, we enumerate exhaustively all possible conformations. For long chains, we sample effectively rare events such as compact conformations and obtain complete pictures of ensemble properties of conformations of these models at all compactness region. This is made possible by using sequential Monte Carlo techniques based on chain growth method. Our results show that both chirality and reduced side chain flexibility lower the folding entropy significantly for globally compact conformations, suggesting that they are important properties of residues to ensure fast folding and stable native structure. This corresponds well with our finding that natural amino acid residues have reduced effective flexibility, as evidenced by statistical analysis of rotamer libraries and side chain rotatable bonds. We further develop a method calculating the exact side chain entropy for a given backbone structure. We show that simple rotamer counting underestimates side chain entropy significantly for both extended and near maximally compact conformations. We find that side chain entropy does not always correlate well with main chain packing. With explicit side chains, extended backbones do not have the largest side chain entropy. Among compact backbones with maximum side chain entropy, helical structures emerge as the dominating configurations. Our results suggest that side chain entropy may be an important factor contributing to the formation of alpha helices for compact conformations.

The conformational models of the active site of adenosine deaminase (ADA) and its complexes in the basic state with adenosine and 13 isosteric analogues of the aza, deaza, and azadeaza series were constructed. The optimization of the conformational energy of the active site and the nucleoside bound with it in the complex was achieved in the force field of the whole enzyme (the 1ADD structure was used) within the molecular mechanics model using the AMBER 99 potentials. The stable conformational states of each of the complexes, as well as the optimal conformation of the ADA in the absence of ligand, were determined. It was proved that the conformational state that is close to the structure of the ADA complex with 1-deazaadenosine (1ADD) known from the X-ray study corresponds to one of the local minima of the potential surface. Another, a significantly deeper minimum was determined; it differs from the first minimum by the mutual orientation of side chains of amino acid residues. A similar conformational state is optimal for the ADA active site in the absence of the bound ligand. A qualitative correlation exists between the values of potential energies of the complexes in this conformation and the enzymatic activity of ADA toward the corresponding nucleosides. The dynamics of conformational conversions of the active site after the binding of substrate or its analogues, as well as the possibility of the estimation of the inhibitory properties of nucleosides on the basis of calculations, are discussed.

Peptidyl-prolyl isomerase (PPIase) activity is exhibited by many proteins belonging to the PPIase family. However, the catalytic mechanism of this activity remains to be completely elucidated. Here, we selected human FK506-binding protein 12 (FKBP12) as the model PPIase and investigated the nature of amino acid residues essential for the activity. The crystal structures of several complexes of PPIase with short peptides revealed that the residues Asp37, Arg42, Phe46, Val55, Trp59, and Tyr82 in the substrate-binding cavity of FKBP12 appear to play key roles in the PPIase activity. Each of these six residues was substituted by 20 common amino acid residues. The activity of each mutant protein was measured using a peptide analog by the chymotrypsin digestion assay and then compared with wild-type FKBP12. It was found that site-specific interactions by the side chains of amino acid residues constituting the substrate-binding cavity were not essential for the PPIase activity, although the 37th, 55th, and 82nd amino acid residues significantly contributed to the activity. This suggests that the PPIase activity requires only the hydrophobic cavity that captures the Pro-containing peptide.

Human calcitonin (HCT) is a thyroid polypeptide hormone that has 32 amino acid residues. The HCT is well known for an amyloid forming peptide and its inhibition of bone mineralization of calcium-phosphorous as osteoporosis. The HCT peptide aggregates to form insoluble fibril toward diseases. In the fibril formation of HCT, the local conformation changes from an α-helix in a monomeric HCT to a beta-sheet structure in a fibril. The role of the amino acid residues for fibrillation is unclear; therefore, we simulated to determine the interaction energies between amino acid residues for stability of fibril using molecular dynamics simulation.

Protein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., alpha-helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic-aromatic contacts. The Met8 --> Leu mutation in CMTI-I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rearrangement of a hydrophobic cluster formed by side chains of residues 8, Ile6, and Leu17 that leads to partial breaking of a hydrogen bond formed by the amide group of Leu17 with water and to a reduction of a hydrophobic surface buried within the cluster. The mutation perturbs also the protein folding. In aerobic conditions the reduced wild-type protein folds effectively into its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is that conservative mutations of hydrophobic residues can destabilize a protein structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobic clusters formed by such residues can lead to local changes in protein hydration, and consequently, can affect considerably protein stability and folding process.

β-Hairpin is an essential secondary structure unit of protein. Understanding the formation mechanism of the β hairpin and kinetic stability helps to gain insight into the formation of protein secondary structure as well as the mechanism of protein folding. Replica exchange molecular dynamics (REMD) approach is applied to investigate the folding mechanism of Trpzip4 β-hairpin. The REMD method is more efficient than conventional MD in searching for a representative set of the low energy minima in the existence of a large energy gap between the native state and any of the other possible state. The potential energy, backbone RMSD and solvent-accessible surface area (SASA) present descending trends in the process of REMD at 288 K. Methyl groups (most in threonines) show hydrophobic interactions with SASA decrements. The hydropho- bic interactions and intra hydrogen bond forming drive the folding and maintain the low energy structure. Two low energy structures, β-hairpin and helix-coil conformation, are sampled by REMD. In β-hairpin structure, the hydroxyl group (OH) of Thr49 at β turn is accessible to form hydrogen bonds with carboxyl group (COO - ) or C=O group of Asp46; while the hy- droxyl group (OH) of Thr51 is inclined to interact with water molecule with methyl orienting inwards. As a result, the strong interactions between Asp46 and Thr49 cause a bend at β turn. In helix-coil conformation, hydrophobic interactions play be- tween indole ring and methyl group. One of the zip-in paths of Trpzip4 indicates that, the forming of hydrogen bonds at β turn affects the whole folding process of β hairpin. The forming of hydrogen bonds at β turn takes place first, since it has advantages of distance and strong interactions in both side chains and backbone. Total solvent-accessible surface area de- creases significantly as it folds to the β hairpin structure. REMD method could sample large phase space for the low energy minima, and present scenarios of potential energy distribution on phase space with related variables as hydrophobic SASA, backbone RMSD, and hydrogen bonds for specific conformation. This work sheds some lights on the understanding of β hairpin folding. Keywords β-hairpin; folding; replica exchange molecular dynamics; hydrogen bond; hydrophobic interaction