Inside of the burst-phase intermediate of a protein folding. Hydration of hydrophobic groups.

The thermal effect of the formation of the "burst-phase" folding intermediate has been studied using a titration calorimeter. It is shown that, unlike the total thermal effect of native structure formation, it can be both positive and negative depending on the temperature. The reasons for this paradoxical behavior are analyzed. A conclusion is drawn about the leading role of dehydration of non-polar groups in the first stage of folding.

Thermodynamic invariants of gel to the liquid-crystal 1,2-diacylphosphatidylcholines transition.

The bilayer phase transitions from the ripple gel phase (P'(ß)) to the liquid-crystal phase (L(α)) of a series of 1,2-diacylphosphatidylcholines containing a linear saturated acyl chain (C=14-19) have been studied by high-pressure scanning microcalorimetry. It has been shown that at ambient pressure, the transition temperature increases non-linearly depending on the acyl chain length. Pressure stabilizes the gel phase of lipids in a similar way; the pressure derivatives of the logarithm transition temperature as function of pressure are identical for all lipids. Based on the results obtained it has been concluded that the ratio γ of volume to enthalpy increments upon transitions in 1,2-diacylphosphatidylcholines is not dependent on the acyl chain length. When pressure grows, this ratio decreases drastically remaining identical for all lipids studied. Besides it has been demonstrated that increments of coefficients of thermal expansibility and isothermal compressibility are also rigidly bound to each other. Semi-empirical equations permitting to estimate volume parameters of the gel-to-liquid transition for 1,2-diacylphosphatidylcholines are given. The reasons for invariance of γ are discussed.

Dependence on acyl chain length of energy and volume parameters of the gel to liquid-crystalline transition of 1,2-diacylphosphatidylcholines. Theoretical consideration.

The effect of acyl chain length on energy and volume parameters of gel to liquid-crystal transitions in phospholipids is analyzed. It is demonstrated that simple structural and thermodynamic considerations allow predicting some thermodynamic and volume characteristics of transitions and their dependencies on the acyl chains length.

Synthetic RGD-containing alpha-helical coiled coil peptides promote integrin-dependent cell adhesion.

Integrin receptors are the main mediators of cell adhesion to the extracellular matrix. They bind to their ligands by interacting with short amino acid sequences, such as the RGD sequence. Soluble, small RGD-based peptides have been used to block integrin-binding to ligands, thereby interfering with cell adhesion, migration and survival, while substrate-immobilized RGD sequences have been used to enhance cell binding to artificial surfaces. This approach has several important medical applications, e.g. in suppression of tumor angiogenesis or stimulation of bone formation around implants. However, the relatively weak affinity of short RGD-containing peptides often results in incomplete integrin inhibition or ineffective ligation. In this work, we designed and synthesized several new multivalent RGD-containing molecules and tested their ability to inhibit or to promote integrin-dependent cell adhesion when used in solution or immobilized on substrates, respectively. These molecules consist of an oligomeric structure formed by alpha-helical coiled coil peptides fused at their amino-terminal ends with an RGD-containing fragment. When immobilized on a substrate, these peptides specifically promoted integrin alphaVbeta3-dependent cell adhesion, but when used in solution, they blocked alphaVbeta3-dependent cell adhesion to the natural substrates fibronectin and vitronectin. One of the peptides was nearly 10-fold more efficient than fibronectin or vitronectin in promoting cell adhesion, and almost 100-fold more efficient than a linear RGD tripeptide in blocking adhesion. These results indicate that alpha-helical coiled coil peptides carrying an amino-terminal RGD motif can be used as soluble antagonists or surface-immobilized agonists to efficiently inhibit or promote integrin alphaVbeta3-mediated cell adhesion, respectively.

Organization of designed nanofibrils assembled from alpha-helical peptides as determined by electron microscopy.

Self-assembling peptides present attractive platforms for engineering materials with controlled nanostructures. Recently, an alpha-helical fibril forming peptide (alphaFFP) was designed that self-assembles into nanofibrils at acid pH. Circular dichroism spectroscopy, electron-microscopy and x-ray fibre diffraction data showed that the most likely structure of alphaFFP fibrils is a five-stranded coiled coil rope. In the present study, scanning transmission electron microscopy (STEM) was used to improve our understanding of the alphaFFP fibril structure. The measurements of fibril mass per length suggest that there are ten alpha-helices in transverse sections of the fibrils. Based on the known data, it is proposed that a predominant fibrillar structure of alphaFFP is a dimer of alpha-helical five stranded protofilaments wrapped around a common axis. It is shown that these structures have an axial dimension of 58 +/- 16 nm and a width of 4 +/- 1 nm. A small number of thin fibrils is also observed in the negative stained preparation and STEM images. The thin fibrils may correspond to the single protofilament.

Kinetics and thermodynamics of the unfolding and refolding of the three-stranded alpha-helical coiled coil, Lpp-56.

Temperature-induced reversible unfolding and refolding of the three-stranded alpha-helical coiled coil, Lpp-56, were studied by kinetic and thermodynamic methods, using CD spectroscopy, dynamic light scattering, and scanning calorimetry. It was found that both unfolding and refolding reactions of this protein in neutral solution in the presence of 100 mM NaCl are characterized by unusually slow kinetics, which permits detailed investigation of the mechanism of these reactions. Kinetic analyses show that the unfolding of this coiled coil represents a single-stage first-order reaction, while the refolding represents a single-stage third-order reaction. The activation enthalpy and entropy for unfolding do not depend noticeably on temperature and are both significantly greater than those for the folding reaction, which show a significant dependence on temperature. The activation heat capacity change for the unfolding reaction is close to zero, while it is quite significant for the folding reaction. The correlation between the activation and structural parameters obtained for the Lpp-56 coiled coil suggests that interhelical van der Waals interactions are disrupted in the transition state, which is nevertheless still compact, and water has not yet penetrated into the interface; the transition from the transient state to the unfolded state results in hydration of exposed apolar groups of the interface and the disruption of helices. The low propensity for the Lpp-56 strands to fold and associate is caused by the high number of charged groups at neutral pH. On one hand, these charges give rise to considerable repulsive forces destabilizing the helical conformation of the strands. On the other hand, they align the folded helices in parallel and in register so that the apolar sides face each other, and the oppositely charged groups may form salt links, which are important for the formation of the trimeric coiled coil. A decrease in pH, which eliminates the salt links, dramatically decreases the stability of Lpp-56; its structure becomes less rigid and unfolds much faster.

Compact globular structure of Thermus thermophilus ribosomal protein S1 in solution: sedimentation and calorimetric study.

Ribosomal protein S1 of Thermus thermophilus overexpressed in Escherichia coli cells has been isolated and subjected to studies by analytical sedimentation and differential scanning microcalorimetry techniques. It has been demonstrated that the protein of 60 kDa sediments at s020,w = 4.6 S and has the diffusion coefficient D020,w = 6.7 x 10(-7) cm2/s in 25 mm HEPES-NaOH buffer, pH 7.5 (similarly to bovine serum albumin of 66 kDa that sediments at s0 20,w = 4.4 S and D020,w =6.0 x 10(-7) cm2/s), indicating its compact globular conformation under these conditions. The microcalorimetry study has shown the presence of a cooperative tertiary structure melting at 90 degrees C, but with several (probably three) independent cooperative domains. In the presence of 100 mm NaCl the protein becomes more asymmetric (s020,w = 3.1 S) but does not lose its cooperativity and thermostability, this suggesting just the weakening of interdomain ionic interactions. The compact globular conformation of protein S1 seems to be most likely within the ribosome.