Some useful ideas for multistate protein design: Effect of amino acid substitutions on the multistate proteins stability and the rate of protein structure formation.

The design of new protein variants is usually confined to slightly "fixing" an already existing protein, adapting it to certain conditions or to a new substrate. This is relatively easy to do if the fragment of the protein to be affected, such as the active site of the protein, is known. But what if you need to "fix" the stability of a protein or the rate of its native or intermediate state formation? Having studied a large number of protein mutant forms, we have established the effect of various amino acid substitutions on the energy landscape of the protein. As a result, we have revealed a number of patterns to help researchers identify amino acid residues that determine the folding rate and the stability of globular proteins states and design a mutant form of a protein with desired properties.

Verification of the Stabilized Protein Design Based on the Prediction of Intrinsically Disordered Regions: Ribosomal Proteins L1.

In our previous papers, we proposed the idea that programs predicting intrinsically disordered regions in amino acid sequences can be used for finding weakened sites in proteins. The regions predicted by such programs are suitable targets for the introduction of protein-stabilizing mutations. However, for each specific protein, it remains unclear what determines protein stabilization - the amino acid sequence (and accordingly, prediction of weakened sites) or the 3D structure. To answer this question, it is necessary to study two proteins with similar structures but different amino acid sequences and, consequently, different predictions of weakened regions. By introducing identical mutations into identical elements of the two proteins, we will be able to reveal whether predictions of the weakened sites or the 3D protein structure are the key factors in the protein stability increase. Here, we have chosen ribosomal proteins L1 from the halophilic archaeon Haloarcula marismortui (HmaL1) and extremophilic bacterium Aquifex aeolicus (AaeL1). These proteins are identical in their structure but different in amino acid sequences. A disulfide bond introduced into the region predicted as the structured one in AaeL1 did not lead to the increase in the protein melting temperature. At the same time, a disulfide bond introduced into the same region in HmaL1 that was predicted as a weakened one, resulted in the increase in the protein melting temperature by approximately 10°C.

Optical and magnetic properties of the ground state of Cr3+ doping ions in REM3(BO3)4 single crystals.

New data about the state of Cr3+ doping ions in a single crystal of YGa3(BO3)4 have been obtained by studying different methods. Using electron paramagnetic resonance, it was found that the Cr3+ ions substitute the trivalent gallium ions. The obtained spin Hamiltonian parameters of the Cr3+ ions in the YGa3(BO3)4 single crystal (g = 1.9743 ± 0.0004; D = -0.465 ± 0.001 cm-1; E = -0.013 ± 0.001 cm-1) were analyzed and compared with those in TmAl3(BO3)4, EuAl3(BO3)4, and YAl3(BO3)4. The deviation of the Z-axis in the spectrum from the crystallographic axis С3 is 1,7 degrees in YGa3(BO3)4. In situ X-ray diffraction was used to study the structural and elastic properties of huntite-type borates in the temperature range of RT-1073 K. In the radioluminescence (RL) spectra, the Cr3+-related emission bands were observed in the red wavelength range, and the presence of other defect-related bands was also registered in some cases. Thermally stimulated luminescence (TSL) glow curves were acquired over a wide temperature range, and the trap depths of the most prominent bands were calculated. The 11B NMR spectra show that two nonequivalent spectral components exist for BO3 structural elements.

[Artificial Cysteine Bridges on the Surface of Green Fluorescent Protein Affect Hydration of Its Transition and Intermediate States].

Studying the effect of cysteine bridges on different energy levels of multistage folding proteins will enable a better understanding of the process of folding and functioning of globular proteins. In particular, it will create prospects for directed change in the stability and rate of protein folding. In this work, using the method of differential scanning microcalorimetry, we have studied the effect of three cysteine bridges introduced in different structural elements of the green fluorescent protein on the denaturation enthalpies, activation energies, and heat-capacity increments when this protein passes from native to intermediate and transition states. The studies have allowed us to confirm that, with this protein denaturation, the process hardly damages the structure initially, but then changes occur in the protein structure in the region of 4-6 beta sheets. The cysteine bridge introduced in this region decreases the hydration of the second transition state and increases the hydration of the second intermediate state during the thermal denaturation of the green fluorescent protein.

Can the fluorescence of green fluorescent protein chromophore be related directly to the nativity of protein structure?

In studies of green fluorescence protein (GFP) or other proteins with the use of GFP as a marker, the fluorescence of GFP is for the most part related directly to the nativity of its structure. Naturally, such a relation does exist since the chromophore of this protein is formed autocatalytically only just after GFP acquires its native structure. However, the fluorescence method may not yield reliable information on protein structure when studying renaturation and denaturation of this protein (with the formed chromophore). Using proteolysis, denaturant gradient gel electrophoresis and circular dichroism, we demonstrate herein that at major disturbances of the native structure of protein GFP-cycle3 the intensity of fluorescence of its chromophore can change insignificantly. In other words, the chromophore fluorescence does not reliably mirror alterations in protein structure. Since the main conclusions of this study are especially qualitative, it can be suggested that during renaturation/denaturation of wild-type GFP and its "multicolored" mutants their fluorescence is also not always associated with the changes in the structure of these proteins.

Shift of fibril-forming ability of the designed alpha-helical coiled-coil peptides into the physiological pH region.

Recently, we designed a short alpha-helical fibril-forming peptide (alphaFFP) that can form alpha-helical nanofibrils at acid pH. The non-physiological conditions of the fibril formation hamper biomedical application of alphaFFP. It was hypothesized that electrostatic repulsion between glutamic acid residues present at positions (g) of the alphaFFP coiled-coil sequence prevent the fibrillogenesis at neutral pH, while their protonation below pH 5.5 triggers axial growth of the fibril. To test this hypothesis, we synthesized alphaFFPs where all glutamic acid residues were substituted by glutamines or serines. The electron microscopy study confirmed that the modified alphaFFPs form nanofibrils in a wider range of pH (2.5-11). Circular dichroism spectroscopy, sedimentation, diffusion and differential scanning calorimetry showed that the fibrils are alpha-helical and have elongated and highly stable cooperative tertiary structures. This work leads to a better understanding of interactions that control the fibrillogenesis of the alphaFFPs and opens opportunities for their biomedical application.

De novo design of fibrils made of short alpha-helical coiled coil peptides.

BACKGROUND: The alpha-helical coiled coil structures formed by 25-50 residues long peptides are recognized as one of Nature's favorite ways of creating an oligomerization motif. Known de novo designed and natural coiled coils use the lateral dimension for oligomerization but not the axial one. Previous attempts to design alpha-helical peptides with a potential for axial growth led to fibrous aggregates which have an unexpectedly big and irregular thickness. These facts encouraged us to design a coiled coil peptide which self-assembles into soluble oligomers with a fixed lateral dimension and whose alpha-helices associate in a staggered manner and trigger axial growth of the coiled coil. Designing the coiled coil with a large number of subunits, we also pursue the practical goal of obtaining a valuable scaffold for the construction of multivalent fusion proteins. RESULTS: The designed 34-residue peptide self-assembles into long fibrils at slightly acid pH and into spherical aggregates at neutral pH. The fibrillogenesis is completely reversible upon pH change. The fibrils were characterized using circular dichroism spectroscopy, sedimentation diffusion, electron microscopy, differential scanning calorimetry and X-ray fiber diffraction. The peptide was deliberately engineered to adopt the structure of a five-stranded coiled coil rope with adjacent alpha-helices, staggered along the fibril axis. As shown experimentally, the most likely structure matches the predicted five-stranded arrangement. CONCLUSIONS: The fact that the peptide assembles in an expected fibril arrangement demonstrates the credibility of our conception of design. The discovery of a short peptide with fibril-forming ability and stimulus-sensitive behavior opens new opportunities for a number of applications.

Structure and stability of recombinant protein depend on the extra N-terminal methionine residue: S6 permutein from direct and fusion expression systems.

Two permuted variants of S6 ribosomal protein were obtained in direct and fusion expression systems, respectively. The product of direct expression contained the extra N-terminal methionine residue. The structural properties and conformational stability of these permuteins were compared using 1-D (1)H-NMR, circular dichroism, intrinsic fluorescence, differential scanning calorimetry and resistance to urea-induced unfolding. A pronounced difference in all the parameters studied has been demonstrated. This means that the structure of recombinant protein can be sensitive to peculiarities of the expression and purification procedures, leading particularly to the presence or absence of the Met at the first position in the target protein sequence.