Anatoly S. Glukhov

SS-Stabilizing Proteins Rationally: Intrinsic Disorder-Based Design of Stabilizing Disulphide Bridges in GFP.

Abstract The most attractive and methodologically convenient way to enhance protein stability is via the introduction of disulphide bond(s). However, the effect of the artificially introduced SS-bond on protein stability is often quite unpredictable. This raises the question of how to choose the protein sites in an intelligent manner, so that the ‘fastening’ of these sites by the SS-bond(s) would provide maximal protein stability. We hypothesize that the successful design of a stabilizing SS-bond requires finding highly mobile protein regions. Using GFP as an illustrative example, we demonstrate that the knowledge of the peculiarities of the intramolecular hydrophobic interactions, combined with the understanding of the local intrinsic disorder propensities (that can be evaluated by various disorder predictors, e.g., PONDRFIT), is sufficient to find the candidate sites for the introduction of stabilizing SS-bridge(s). In fact, our analysis revealed that the insertion of the engineered SS-bridge between two highly flexible regions of GFP noticeably increased the conformational stability of this protein toward the thermal and chemical unfolding. Therefore, our study represents a novel approach for the rational design of stabilizing disulphide bridges in proteins.

Genomic analysis of Pseudomonas putida phage tf with localized single-strand DNA interruptions.

The complete sequence of the 46,267 bp genome of the lytic bacteriophage tf specific to Pseudomonas putida PpG1 has been determined. The phage genome has two sets of convergently transcribed genes and 186 bp long direct terminal repeats. The overall genomic architecture of the tf phage is similar to that of the previously described Pseudomonas aeruginosa phages PaP3, LUZ24 and phiMR299-2, and 39 out of the 72 products of predicted tf open reading frames have orthologs in these phages. Accordingly, tf was classified as belonging to the LUZ24-like bacteriophage group. However, taking into account very low homology levels between tf DNA and that of the other phages, tf should be considered as an evolutionary divergent member of the group. Two distinguishing features not reported for other members of the group were found in the tf genome. Firstly, a unique end structure – a blunt right end and a 4-nucleotide 3′-protruding left end – was observed. Secondly, 14 single-chain interruptions (nicks) were found in the top strand of the tf DNA. All nicks were mapped within a consensus sequence 5′-TACT/RTGMC-3′. Two nicks were analyzed in detail and were shown to be present in more than 90% of the phage population. Although localized nicks were previously found only in the DNA of T5-like and phiKMV-like phages, it seems increasingly likely that this enigmatic structural feature is common to various other bacteriophages.

Sequential melting of two hydrophobic clusters within the green fluorescent protein GFP-cycle3.

The analysis of the three-dimensional structure of green fluorescent protein (GFP-cycle3) revealed the presence of two well-defined hydrophobic clusters located on the opposite sides of the GFP β-can that might contribute to the formation of partially folded intermediate(s) during GFP unfolding. The microcalorimetric analysis of the nonequilibrium melting of GFP-cycle3 and its two mutants, I14A and I161A, revealed that due to the sequential melting of the mentioned hydrophobic clusters, the temperature-induced denaturation of this protein most likely occurs in three stages. The first and second stages involve melting of a smaller hydrophobic cluster formed around the residue I161, whereas a larger hydrophobic cluster (formed around the residues I14) is melted only at the last GFP-cycle3 denaturation step or remains rather structured even in the denatured state.

Multi-State Proteins: Approach Allowing Experimental Determination of the Formation Order of Structure Elements in the Green Fluorescent Protein

The most complex problem in studying multi-state protein folding is the determination of the sequence of formation of protein intermediate states. A far more complex issue is to determine at what stages of protein folding its various parts (secondary structure elements) develop. The structure and properties of different intermediate states depend in particular on these parts. An experimental approach, named μ-analysis, which allows understanding the order of formation of structural elements upon folding of a multi-state protein was used in this study. In this approach the same elements of the protein secondary structure are “tested” by substitutions of single hydrophobic amino acids and by incorporation of cysteine bridges. Single substitutions of hydrophobic amino acids contribute to yielding information on the late stages of protein folding while incorporation of ss-bridges allows obtaining data on the initial stages of folding. As a result of such an μ-analysis, we have determined the order of formation of beta-hairpins upon folding of the green fluorescent protein.

High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid.

Bacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of newly dissociated pb10 to the 120 hexamers of the capsid protein. In extreme conditions, pb10 protects the phage from releasing its genome. We conclude that pb10 may function to reinforce the capsid thus favouring phage survival in harsh environments.

Independent of Their Localization in Protein the Hydrophobic Amino Acid Residues Have No Effect on the Molten Globule State of Apomyoglobin and the Disulfide Bond on the Surface of Apomyoglobin Stabilizes This Intermediate State

At present it is unclear which interactions in proteins reveal the presence of intermediate states, their stability and formation rate. In this study, we have investigated the effect of substitutions of hydrophobic amino acid residues in the hydrophobic core of protein and on its surface on a molten globule type intermediate state of apomyoglobin. It has been found that independent of their localization in protein, substitutions of hydrophobic amino acid residues do not affect the stability of the molten globule state of apomyoglobin. It has been shown also that introduction of a disulfide bond on the protein surface can stabilize the molten globule state. However in the case of apomyoglobin, stabilization of the intermediate state leads to relative destabilization of the native state of apomyoglobin. The result obtained allows us not only to conclude which mutations can have an effect on the intermediate state of the molten globule type, but also explains why the introduction of a disulfide bond (which seems to “strengthen” the protein) can result in destabilization of the protein native state of apomyoglobin.

Bacteriophage T5 Mutants Carrying Deletions in tRNA Gene Region

A new series of heat-stable (st) mutants of bacteriophage T5, which contains deletions in the tRNA gene region, has been isolated. An accurate mapping of the deletion boundaries for more than 30 mutants of phage T5 has been carried out. As a result of the analysis of nucleotide sequences flanking the deleted regions in wild-type phage DNA, it has been shown that they all contain short, direct repeats of different lengths (2–35 nucleotide residues), and that only one repetition is retained in the mutant phage DNA. On the basis of the obtained results, it was suggested that deletion mutants of the phage T5 are formed as a result of illegal recombination occurring with the participation of short repeats in DNA (SHDIR). Based on the example of two mutants, it has been shown that the resistance to thermal inactivation depends on the size of the deleted region.

An approach for the assessment of the order of disruption of the elements of protein structure upon protein unfolding: A study of carbonic anhydrase B

An experimental approach named μ-analysis has been developed in order to elucidate the sequence of the loss of ordered structure by elements of a protein during the denaturation of the molecule. This approach is applicable for the analysis of proteins that fold (unfold) in a multistep process that involve the formation (destruction) of a range of intermediate states. The concept of the approach consists in systematic analysis of mutagenized forms of the protein with point substitutions of hydrophobic amino-acid residues and additional cysteine bridges. Importantly, the substitutions of the amino-acid residues must be localized to the same structural elements of the protein. Point substitutions of hydrophobic amino-acid residues mainly provide information on the structural elements of the protein that are disrupted at the final stages of protein denaturation. The addition of cysteine bridges to the surface of the protein molecule allows investigation of structural elements of the protein that are the first to unfold upon protein denaturation. Calorimetric studies of non-equilibrium melting of bovine carbonic anhydrase B yielded information on the rate constants of the unfolding of ten mutant forms of the protein. The analysis of the effects of mutations on the rates of different stages of protein unfolding allowed for elucidation of the order of disruption of structural elements of carbonic anhydrase B upon thermal denaturation.