Sequence-specific polypeptoids: A diverse family of heteropolymers with stable secondary structure

We have synthesized and characterized a family of structured oligo-N-substituted-glycines (peptoids) up to 36 residues in length by using an efficient solid-phase protocol to incorporate chemically diverse side chains in a sequence-specific fashion. We investigated polypeptoids containing side chains with a chiral center adjacent to the main chain nitrogen. Some of these sequences have stable secondary structure, despite the achirality of the polymer backbone and its lack of hydrogen bond donors. In both aqueous and organic solvents, peptoid oligomers as short as five residues give rise to CD spectra that strongly resemble those of peptide α-helices. Differential scanning calorimetry and CD measurements show that polypeptoid secondary structure is highly stable and that unfolding is reversible and cooperative. Thermodynamic parameters obtained for unfolding are similar to those obtained for the α-helix to coil transitions of peptides. This class of biomimetic polymers may enable the design of self-assembling macromolecules with novel structures and functions.

A model for the structure of the P domains in the subtilisin-like prohormone convertases

The proprotein convertases are a family of at least seven calcium-dependent endoproteases that process a wide variety of precursor proteins in the secretory pathway. All members of this family possess an N-terminal proregion, a subtilisin-like catalytic module, and an additional downstream well-conserved region of ≈150 amino acid residues, the P domain, which is not found in any other subtilase. The pro and catalytic domains cannot be expressed in the absence of the P domains; their thermodynamic instability may be attributable to the presence of large numbers of negatively charged Glu and Asp side chains in the substrate binding region for recognition of multibasic residue cleavage sites. Based on secondary structure predictions, we here propose that the P domains consist of 8-stranded β-barrels with well-organized inner hydrophobic cores, and therefore are independently folded components of the proprotein convertases. We hypothesize further that the P domains are integrated through strong hydrophobic interactions with the catalytic domains, conferring structural stability and regulating the properties and activity of the convertases. A molecular model of these interdomain interactions is proposed in this report.

Peptide interfacial adsorption is kinetically limited by the thermodynamic stability of self association

We present a study of the adsorption of two peptides at the octane–water interface. The first peptide, Lac21, exists in mixed monomer–tetramer equilibrium in bulk solution with an appreciable monomer concentration. The second peptide, Lac28, exists as a tetramer in solution, with minimal exposed hydrophobic surface. A kinetic limitation to interfacial adsorption exists for Lac28 at moderate to high surface coverage that is not observed for Lac21. We estimate the potential energy barrier for Lac28 adsorption to be 42 kJ/mol and show that this is comparable to the expected free energy barrier for tetramer dissociation. This finding suggests that, at moderate to high surface coverage, adsorption is kinetically limited by the availability of interfacially active monomeric “domains” in the subinterfacial region. We also show how the commonly used empirical equation for protein adsorption dynamics can be used to estimate the potential energy barrier for adsorption. Such an approach is shown to be consistent with a formal description of diffusion–adsorption, provided a large potential energy barrier exists. This work demonstrates that the dynamics of interfacial adsorption depend on protein thermodynamic stability, and hence structure, in a quantifiable way.

Thermodynamic basis of the enhanced specificity of structured DNA probes

Molecular beacons are DNA probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary nucleic acids, they undergo a conformational transition that switches on their fluorescence. These probes recognize their targets with higher specificity than probes that cannot form a hairpin stem, and they easily discriminate targets that differ from one another by only a single nucleotide. Our results show that molecular beacons can exist in three different states: bound to a target, free in the form of a hairpin structure, and free in the form of a random coil. Thermodynamic analysis of the transitions between these states reveals that enhanced specificity is a general feature of conformationally constrained probes.

NMR investigation of ferricytochrome c unfolding: Detection of an equilibrium unfolding intermediate and residual structure in the denatured state

Horse ferricytochrome c (cyt c) undergoes exchange of one of its axial heme ligands (Met-80) for one or more non-native ligands under denaturing conditions. We have used 1H NMR spectroscopy to detect two conformations of paramagnetic cyt c with non-native heme ligation through a range of urea concentrations. One non-native form is an equilibrium unfolding intermediate observed under partially denaturing conditions and is attributed to replacement of Met-80 with one or more Lys side chains. The second non-native form, in which the native Met ligand is replaced by a His, is observed under strongly denaturing conditions. Thermodynamic analysis of these data indicates a relatively small ΔG (17 kJ/mol) for the transition from native to the Lys-ligated intermediate and a significantly larger ΔG (47 kJ/mol) for the transition from native to the His-ligated species. Although CD and fluorescence data indicate that the equilibrium unfolding of cyt c is a two-state process, these NMR results implicate an intermediate with His-Lys ligation.

NTDB: Thermodynamic Database for Nucleic Acids

A new thermodynamic database for normal and modified nucleic acids has been developed. This Thermodynamic Database for Nucleic Acids (NTDB) includes sequence, structure and thermodynamic information as well as experimental methods and conditions. In this release, there are 1851 sequences containing both normal and modified nucleic acids. A user-friendly web-based interface has been developed to allow data searching under different conditions. Useful thermodynamic tools for the study of nucleic acids have been collected and linked for easy usage. NTDB is available at http://ntdb.chem.cuhk.edu.hk.

The thermodynamic origin of the stability of a thermophilic ribozyme

Understanding the mechanism of thermodynamic stability of an RNA structure has significant implications for the function and design of RNA. We investigated the equilibrium folding of a thermophilic ribozyme and its mesophilic homologue by using hydroxyl radical protection, small-angle x-ray scattering, and circular dichroism. Both RNAs require Mg2+ to fold to their native structures that are very similar. The stability is measured as a function of Mg2+ and urea concentrations at different temperatures. The enhanced stability of the thermophilic ribozyme primarily is derived from a tremendous increase in the amount of structure formed in the ultimate folding transition. This increase in structure formation and cooperativity arises because the penultimate and the ultimate folding transitions in the mesophilic ribozyme become linked into a single transition in the folding of the thermophilic ribozyme. Therefore, the starting point, or reference state, for the transition to the native, functional thermophilic ribozyme is significantly less structured. The shift in the reference state, and the resulting increase in folding cooperativity, is likely due to the stabilization of selected native interactions that only form in the ultimate transition. This mechanism of using a less structured intermediate and increased cooperativity to achieve higher functional stability for tertiary RNAs is fundamentally different from that commonly proposed to explain the increased stability of thermophilic proteins.

Interplay of structure and disorder in cochaperonin mobile loops.

Protein-protein interactions typically are characterized by highly specific interfaces that mediate binding with precisely tuned affinities. Binding of the Escherichia coli cochaperonin GroES to chaperonin GroEL is mediated, at least in part, by a mobile polypeptide loop in GroES that becomes immobilized in the GroEL/GroES/nucleotide complex. The bacteriophage T4 cochaperonin Gp31 possesses a similar highly flexible polypeptide loop in a region of the protein that shows low, but significant, amino acid similarity with GroES and other cochaperonins. When bound to GroEL, a synthetic peptide representing the mobile loop of either GroES or Gp31 adopts a characteristic bulged hairpin conformation as determined by transferred nuclear Overhauser effects in NMR spectra. Thermodynamic considerations suggest that flexible disorder in the cochaperonin mobile loops moderates their affinity for GroEL to facilitate cycles of chaperonin-mediated protein folding.

Structure of the ripple phase in lecithin bilayers.

The phases of the x-ray form factors are derived for the ripple (Pbeta') thermodynamic phase in the lecithin bilayer system. By combining these phases with experimental intensity data, the electron density map of the ripple phase of dimyristoyl-phosphatidylcholine is constructed. The phases are derived by fitting the intensity data to two-dimensional electron density models, which are created by convolving an asymmetric triangular ripple profile with a transbilayer electron density profile. The robustness of the model method is indicated by the result that many different models of the transbilayer profile yield essentially the same phases, except for the weaker, purely ripple (0,k) peaks. Even with this residual ambiguity, the ripple profile is well determined, resulting in 19 angstroms for the ripple amplitude and 10 degrees and 26 degrees for the slopes of the major and the minor sides, respectively. Estimates for the bilayer head-head spacings show that the major side of the ripple is consistent with gel-like structure, and the minor side appears to be thinner with lower electron density.