Design of stable circular permutants of the GroEL chaperone apical domain.

Enhancing protein stability holds paramount significance in biotechnology, therapeutics, and the food industry. Circular permutations offer a distinctive avenue for manipulating protein stability while keeping intra-protein interactions intact. Amidst the creation of circular permutants, determining the optimal placement of the new N- and C-termini stands as a pivotal, albeit largely unexplored, endeavor. In this study, we employed PONDR-FIT's predictions of disorder propensity to guide the design of circular permutants for the GroEL apical domain (residues 191-345). Our underlying hypothesis posited that a higher predicted disorder value would correspond to reduced stability in the circular permutants, owing to the increased likelihood of fluctuations in the novel N- and C-termini. To substantiate this hypothesis, we engineered six circular permutants, positioning glycines within the loops as locations for the new N- and C-termini. We demonstrated the validity of our hypothesis along the set of the designed circular permutants, as supported by measurements of melting temperatures by circular dichroism and differential scanning microcalorimetry. Consequently, we propose a novel computational methodology that rationalizes the design of circular permutants with projected stability. Video Abstract.

Utilizing Extraepitopic Amino Acid Substitutions to Define Changes in the Accessibility of Conformational Epitopes of the Bacillus cereus HlyII C-Terminal Domain.

Hemolysin II (HlyII)-one of the pathogenic factors of Bacillus cereus, a pore-forming ß-barrel toxin-possesses a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD), which plays an important role in the functioning of the toxin. Our previous work described a monoclonal antibody (HlyIIC-20), capable of strain-specific inhibition of hemolysis caused by HlyII, and demonstrated the dependence of the efficiency of hemolysis on the presence of proline at position 324 in HlyII outside the conformational antigenic determinant. In this work, we studied 16 mutant forms of HlyIICTD. Each of the mutations, obtained via multiple site-directed mutagenesis leading to the replacement of amino acid residues lying on the surface of the 3D structure of HlyIICTD, led to a decrease in the interaction of HlyIIC-20 with the mutant form of the protein. Changes in epitope structure confirm the high conformational mobility of HlyIICTD required for the functioning of HlyII. Comparison of the effect of the introduced mutations on the effectiveness of interactions between HlyIICTD and HlyIIC-20 and a control antibody recognizing a non-overlapping epitope enabled the identification of the amino acid residues N339 and K340, included in the conformational antigenic determinant recognized by HlyIIC-20.

Relationship between Changes in the Protein Folding Pathway and the Process of Amyloid Formation: The Case of Bovine Carbonic Anhydrase II.

Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile, destabilized) state is increased. It implies the assumption that some structural intermediates are more convenient for amyloid formation than the others. Hence, if a mutation affects the protein folding pathway, one should expect that this mutation could affect the rate of amyloid formation as well. In the current work, we have compared the effects of amino acid substitutions of bovine carbonic anhydrase II on its unfolding pathway and on its ability to form amyloids at acidic pH and an elevated temperature. Wild-type protein and four mutant forms (L78A, L139A, I208A, and M239A) were studied. We analyzed the change of the protein unfolding pathway by the time-resolved fluorescence technique and the process of amyloid formation by thioflavin T fluorescence assay and electron microscopy. It was revealed that I208A substitution accelerates amyloid formation and affects the structure of the late (molten globule-like)-intermediate state of carbonic anhydrase, whereas the other mutations slow down the growth of amyloids and have either no effect on the unfolding pathway (L78A, L139A) or alter the conformational states arising at the early unfolding stage (M239A).

Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy.

Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea. Intrinsic steady-state and time-resolved fluorescence spectra and circular dichroism spectra were used to determine the stages of the protein unfolding. Molecular dynamics methods were applied to find the differences in the surroundings of tryptophans in both luciferases. We found that the unfolding pathway is the same for the studied luciferases. However, the results obtained indicate more stable tertiary and secondary structures of P. leiognathi luciferase as compared to enzyme from V. harveyi during the last stage of denaturation, including the unfolding of individual subunits. The distinctions in fluorescence of the two proteins are associated with differences in the structure of the C-terminal domain of α-subunits, which causes different quenching of tryptophan emissions. The time-resolved fluorescence technique proved to be a more effective method for studying protein unfolding than steady-state methods.

Substitutions of Amino Acids with Large Number of Contacts in the Native State Have no Effect on the Rates of Protein Folding.

Various effects of amino acid substitutions on properties of globular proteins have been described in a large number of research papers. Nevertheless, no definite "rule" has been formulated as of yet that could be used by experimentalists to introduce desirable changes in the properties of proteins. Herein we attempt to establish such a "rule". To this end, a hypothesis is proposed on the effects of substitutions of hydrophobic residues with large number of contacts on free energies of different states of a globular protein. The hypothesis states: Substitutions of hydrophobic residues engaged in a large number of residue-residue contacts would not change the folding rate of a protein but could affect its unfolding rate. This hypothesis was verified by both theoretical and experimental analyses, generating a general rule that can facilitate the work of experimentalists on constructing mutant forms of proteins.

The major mRNP protein YB-1: structural and association properties in solution.

YB-1 is a major mRNP protein participating in the regulation of transcription and translation of a wide range of eukaryotic genes in many organisms probably due to its influence on mRNA packing into mRNPs. While the functional properties of YB-1 are extensively studied, little is known about its structural properties. In the present work we focused on studying its secondary structure, rigidity of its tertiary structure, compactness, and oligomerization in vitro by using far UV-CD, DSC, one-dimensional (1)H NMR, SAXS, sedimentation and FPLC. It was shown that only the cold shock domain within the entire YB-1 chain has a well-packed tertiary structure undergoing cooperative heat and cold denaturation transitions. In contrast, the rest of the YB-1 molecule is not rigidly packed and consists of PP II-like helical secondary structure elements and coil-like regions. At the same time, the overall dimension of the protein molecule is unexpectedly small. The polypeptide chains of YB-1 have a high tendency to form oligomers at neutral pH, while the extent and structural organization of the oligomers depend on protein concentration and ionic strength varying from compact monomeric units up to high molecular weight oligomers. These oligomers in solution are unstable and dissociate upon protein concentration decrease.

Correction: Finkelstein et al. How Can Ice Emerge at 0 °C? Biomolecules 2022, 12, 981.

We regret to state that our article "How Can Ice Emerge at 0 °C?" [...].

Physics of Ice Nucleation and Antinucleation: Action of Ice-Binding Proteins.

Ice-binding proteins are crucial for the adaptation of various organisms to low temperatures. Some of these, called antifreeze proteins, are usually thought to inhibit growth and/or recrystallization of ice crystals. However, prior to these events, ice must somehow appear in the organism, either coming from outside or forming inside it through the nucleation process. Unlike most other works, our paper is focused on ice nucleation and not on the behavior of the already-nucleated ice, its growth, etc. The nucleation kinetics is studied both theoretically and experimentally. In the theoretical section, special attention is paid to surfaces that bind ice stronger than water and thus can be "ice nucleators", potent or relatively weak; but without them, ice cannot be nucleated in any way in calm water at temperatures above -30 °C. For experimental studies, we used: (i) the ice-binding protein mIBP83, which is a previously constructed mutant of a spruce budworm Choristoneura fumiferana antifreeze protein, and (ii) a hyperactive ice-binding antifreeze protein, RmAFP1, from a longhorn beetle Rhagium mordax. We have shown that RmAFP1 (but not mIBP83) definitely decreased the ice nucleation temperature of water in test tubes (where ice originates at much higher temperatures than in bulk water and thus the process is affected by some ice-nucleating surfaces) and, most importantly, that both of the studied ice-binding proteins significantly decreased the ice nucleation temperature that had been significantly raised in the presence of potent ice nucleators (CuO powder and ice-nucleating bacteria Pseudomonas syringae). Additional experiments on human cells have shown that mIBP83 is concentrated in some cell regions of the cooled cells. Thus, the ice-binding protein interacts not only with ice, but also with other sites that act or potentially may act as ice nucleators. Such ice-preventing interaction may be the crucial biological task of ice-binding proteins.

The influence of Pseudomonas syringae on water freezing and ice melting.

Pseudomonas syringae is a widely spread plant pathogen known to have ice-nucleating proteins that serve as crystallization sites promoting ice growth at near-zero temperatures. Three temperatures that characterize water freezing and ice melting are (i) the freezing point of water, (ii) the temperature of coexistence of ice and water, and (iii) the melting point of ice. Here we show the influence of different concentrations of P. syringae on these three parameters. P. syringae appears to affect both the freezing point of water and the temperature of the coexistence of ice and water. Additionally, we propose a research technique for studying the freezing/melting process that is simple and requires no complex equipment.

How Can Ice Emerge at 0 °C?

The classical nucleation theory shows that bulk water freezing does not occur at temperatures above ≈ -30 °C, and that at higher temperatures ice nucleation requires the presence of some ice-binding surfaces. The temperature and rate of ice nucleation depend on the size and level of complementarity between the atomic structure of these surfaces and various H-bond-rich/depleted crystal planes. In our experiments, the ice nucleation temperature was within a range from -8 °C to -15 °C for buffer and water in plastic test tubes. Upon the addition of ice-initiating substances (i.e., conventional AgI or CuO investigated here), ice appeared in a range from -3 °C to -7 °C, and in the presence of the ice-nucleating bacterium Pseudomonas syringae from -1 °C to -2 °C. The addition of an antifreeze protein inhibited the action of the tested ice-initiating agents.

Recruitment of TNF ligands to lipid rafts is mediated by their physical association with caveolin-1.

Activities of the tumour necrosis factor (TNF) family members are associated with their targeting to lipid rafts, specialised regions of the plasma membrane. Herein, we investigated the physical association of TNF and its family members cluster of differentiation 40 ligand (CD40L) and tumour necrosis factor-related apoptosis-inducing ligand with caveolin-1, a lipid raft resident protein. We discovered that the intracellular domains of TNF and CD40L interact with caveolin-1, and the membrane proximal region of TNF is required for the binding of caveolin-1 domains. Full-length TNF can form a complex with caveolin-1 in membrane rafts of HeLa cells, and caveolin-1 knockdown leads to impaired TNF transport to rafts. These findings provide the first evidence of a direct interaction between TNF, CD40L and caveolin-1 and suggest that caveolin-1 may be responsible for recruiting TNF to lipid rafts.

Design and Analysis of a Mutant form of the Ice-Binding Protein from Choristoneura fumiferana.

Ice-binding proteins are expressed in the cells of some cold adapted organisms, helping them to survive at extremely low temperatures. One of the problems in studying such proteins is the difficulty of their isolation and purification. For example, eight cysteine residues in the cfAF (antifreeze protein from the eastern spruce budworm Choristoneura fumiferana) form intermolecular bridges during the overexpression of this protein. This impedes the process of the protein purification dramatically. To overcome this issue, in this work, we designed a mutant form of the ice-binding protein cfAFP, which is much easier to isolate that the wild-type protein. The mutant form named mIBP83 did not lose the ability to bind to ice surface. Besides, observation of the processes of freezing and melting of ice in the presence of mIBP83 showed that this protein affects the process of ice melting, increasing its melting temperature, and does not decrease the water freezing temperature.

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 ß-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.

Folding intermediate and folding nucleus for I-->N and U-->I-->N transitions in apomyoglobin: contributions by conserved and nonconserved residues.

Kinetic investigation on the wild-type apomyoglobin and its 12 mutants with substitutions of hydrophobic residues by Ala was performed using stopped-flow fluorescence. Characteristics of the kinetic intermediate I and the folding nucleus were derived solely from kinetic data, namely, the slow-phase folding rate constants and the burst-phase amplitudes of Trp fluorescence intensity. This allowed us to pioneer the phi-analysis for apomyoglobin. As shown, these mutations drastically destabilized the native state N and produced minor (for conserved residues of G, H helices) or even negligible (for nonconserved residues of B, C, D, E helices) destabilizing effect on the state I. On the other hand, conserved residues of A, G, H helices made a smaller contribution to stability of the folding nucleus at the rate-limiting I-->N transition than nonconserved residues of B, D, E helices. Thus, conserved side chains of the A-, G-, H-residues become involved in the folding nucleus before crossing the main barrier, whereas nonconserved side chains of the B-, D-, E-residues join the nucleus in the course of the I-->N transition.

Loops linking secondary structure elements affect the stability of the molten globule intermediate state of apomyoglobin.

Apomyoglobin is a widely used model for studying the molecular mechanisms of globular protein folding. This work aimed to analyze the effects of rigidity and length of loops linking protein secondary structure elements on the stability of the molten globule intermediate state. For this purpose, we studied folding/unfolding of mutant apomyoglobin forms with substitutions of loop-located proline residues to glycine and with loop extension by three or six glycine residues. The kinetic and equilibrium experiments performed gave an opportunity to calculate free energies of different apomyoglobin states. Our analysis revealed that the mutations introduced into the apomyoglobin loops have a noticeable effect on the stability of the intermediate state compared to the unfolded state.

Intrinsic disorder-based design of stable globular proteins.

Directed stabilization of globular proteins via substitution of a minimal number of amino acid residues is one of the most complicated experimental tasks. In this work, we have successfully used algorithms for the evaluation of intrinsic disorder predisposition (such as PONDR® FIT and IsUnstruct) as tools for searching for the weakened regions in structured globular proteins. We have shown that the weakened regions found by these programs as regions with highest levels of predicted intrinsic disorder predisposition are a suitable target for introduction of stabilizing mutations.

Experimental approach to study the effect of mutations on the protein folding pathway.

Is it possible to compare the physicochemical properties of a wild-type protein and its mutant form under the same conditions? Provided the mutation has destabilized the protein, it may be more correct to compare the mutant protein under native conditions to the wild-type protein destabilized with a small amount of the denaturant. In general, is it appropriate to compare the properties of proteins destabilized by different treatments: mutations, pH, temperature, and denaturants like urea? These issues have compelled us to search for methods and ways of presentation of experimental results that would allow a comparison of mutant forms of proteins under different conditions and lead to conclusions on the effect of mutations on the protein folding/unfolding pathway. We have studied equilibrium unfolding of wild-type bovine carbonic anhydrase II (BCA II) and its six mutant forms using different urea concentrations. BCA II has been already studied in detail and is a good model object for validating new techniques. In this case, time-resolved fluorescence spectroscopy was chosen as the basic research method. The main features of this experimental method allowed us to compare different stages of unfolding of studied proteins and prove experimentally that a single substitution of the amino acid in three mutant forms of BCA II affected the native state of the protein but did not change its unfolding pathway. On the contrary, the inserted disulfide bridge in three other mutant forms of BCA II affected the protein unfolding pathway. An important result of this research is that we have validated the new approach allowing investigation of the effect of mutations on the folding of globular proteins, because in this way it is possible to compare proteins in the same structural states rather than under identical conditions.

Intrinsic Disorder-Based Design of Stable Globular Proteins.

Directed stabilization of globular proteins via substitution of a minimal number of amino acid residues is one of the most complicated experimental tasks. This work summarizes our research on the effect of amino acid substitutions on the protein stability utilizing the outputs of the analysis of intrinsic disorder predisposition of target proteins. This allowed us to formulate the basis of one of the possible approaches to the stabilization of globular proteins. The idea is quite simple. To stabilize a protein as a whole, one needs to find its "weakest spot" and stabilize it, but the question is how this weak spot can be found in a query protein. Our approach is based on the utilization of the computational tools for the per-residue evaluation of intrinsic disorder predisposition to search for the "weakest spot" of a query protein (i.e., the region(s) with the highest local predisposition for intrinsic disorder). When such "weakest spot" is found, it can be stabilized through a limited number of point mutations by introducing order-promoting residues at hot spots, thereby increasing structural stability of a protein as a whole. Using this approach, we were able to obtain stable mutant forms of several globular proteins, such as Gαo, GFP, ribosome protein L1, and circular permutant of apical domain of GroEL.

Two HlyIIR dimers bind to a long perfect inverted repeat in the operator of the hemolysin II gene from Bacillus cereus.

HlyIIR is a negative transcriptional regulator of hemolysin II gene from B. cereus. It binds to a long DNA perfect inverted repeat (44bp) located upstream the hlyII gene. Here we show that HlyIIR is dimeric in solution and in bacterial cells. No protein-protein interactions between dimers and no significant modification of target DNA conformation upon complex formation were observed. Two HlyIIR dimers were found to bind to native operator independently with Kd level in the nanomolar range. The minimal HlyIIR binding site was identified as a half of the long DNA perfect inverted repeat.

Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily.

BACKGROUND: The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases) exhibit a common PD-(D/E)XK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI), and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally. RESULTS: Using protein fold-recognition methods we generated a new alignment between R.Eco29kI and I-TevI, which suggested a reassignment of one of the putative catalytic residues. A theoretical model of R.Eco29kI was constructed to illustrate its predicted three-dimensional fold and organization of the active site, comprising amino acid residues Y49, Y76, R104, H108, E142, and N154. A series of mutants was constructed to generate amino acid substitutions of selected residues (Y49A, R104A, H108F, E142A and N154L) and the mutant proteins were examined for their ability to bind the DNA containing the Eco29kI site 5'-CCGCGG-3' and to catalyze the cleavage reaction. Experimental data reveal that residues Y49, R104, E142, H108, and N154 are important for the nuclease activity of R.Eco29kI, while H108 and N154 are also important for specific DNA binding by this enzyme. CONCLUSION: Substitutions of residues Y49, R104, H108, E142 and N154 predicted by the model to be a part of the active site lead to mutant proteins with strong defects in the REase activity. These results are in very good agreement with the structural model presented in this work and with our prediction that R.Eco29kI belongs to the GIY-YIG superfamily of nucleases. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD-(D/E)XK or HNH superfamilies of nucleases, and is instead a member of the unrelated GIY-YIG superfamily.