Analysis of the amino acid effect on protein folding by atom pair contacts

New atomic pair contacts with considering the coordinates of each atom in a residue are introduced here. We analyze the ability of all the 20 amino acid residues to form long-range and short-range contacts by calculating the average numbers of short- and long-range contacts between different amino acid pairs. It is concluded that Phe-Phe, Leu-Phe and Leu-Leu have a high tendency to form contacts. The relative ability to form atom pair contact does not depend on the limiting value of R_C. The average number of contacts per residue, which is the scale of the relative ability to form contacts for the 20 amino acid residue types, is also calculated. The result shows that hydrophobic residues with large numbers of long-range contacts more easily form long-range contacts, while the hydrophilic ones form long-range contacts less often. Linear regression analysis by a new method of counting contacts concludes that either contact order (CO) or total contact distance (TCD) parameter has a significant correlation with the logarithms of folding rates. The relative deviations between the experimentally observed and the two parameters CO and TCD are smaller than that with previous methods. Moreover, the values of CO_λ-μ and TCD_λ-μ between λ-type and μ-type amino acids are investigated. Comparisons between the Fauchere-Pliska hydrophobicity scale and the average number of contacts per residue formed are also made. The new knowledge of atomic pair contacts can help us understand the importance of amino acid residue type and its sequence in globular structure of the protein in detail.     

Analysis of structural statistical properties of proteases and nonproteases

The different structural properties of proteases and nonproteases are well investigated in this paper. The average percentage of residues having a number of long-range contacts greater than or equal to N_L(≥N_L) for the proteases is larger than that for the nonproteases. The average number of long-range contacts per residue in four secondary structure μ (μ=H, E, T, and N) for the proteases is also larger than that for the nonproteases. We calculate the average contact order (CO) per protein, the average long-range order (LRO) per protein, and the average total contact distance (TCD) per protein, and find that the average value of LRO for the nonproteases is smaller than that for the proteases. However, both proteases and nonproteases have the same average values of CO and TCD. The average number of long-range contacts per residue C_L for the proteases is larger than that for the nonproteases, however, the average number of short-range contacts per residue C_S for the proteases is smaller than that for the nonproteases. It is also shown that the square of radius of gyration for the proteases is relatively smaller than for the nonproteases. This finding implies that proteases are more compact than nonproteases. In protein molecule, each residue has a different ability to form contacts, and in general the number of residues having a small number of contacts is greater than that having a large number of contacts. Here we have concluded that the probability P(n) of amino acid residues having n pairs of contacts in all residues fits a good Gaussian distribution, and there has the same form of Gaussian distribution for 20 amino acid residues. The most probable number of contacts, n_C, for the proteases is greater than that for the nonproteases for 20 amino acid residues and one has a good correlation with the Fauchere-Pliska hydrophobicity (FPH) scale. Finally we discuss the relative contribution of amino acid residues involved in cation-π interactions. The higher fraction of cation-π interactions observed in the proteases is found to be reflection of the more general, more frequent occurrence of these interactions in these proteins. All these findings would be helpful for us to understand structural differences between the proteases and other proteins.     

Folding of Right- and Left-Handed Three-Helix Proteins

We are the first to investigate the relationship between protein handedness and the rate of protein folding. Our findings demonstrate that small three-helix, left-handed proteins are less densely packed and should result in faster folding than that of right-handed, three-helix proteins. At the same time, right-handed, three-helix proteins have higher mechanical stability than the left-handed proteins. Moreover, from our analysis we have revealed that bacterial three-helix proteins have some advantages in packing over eukaryotic right-handed, three-helix proteins, which should result in faster folding.     

Experimental evidence for the correlation of bond distances in peptide groups detected in ultrahigh-resolution protein structures

The structural analysis of a deamidated derivative of ribonucleaseA, determined at 0.87 Å resolution, reveals a highly significantnegative correlation between CN and CO bond distances in peptidegroups. This trend, i.e. the CO bond lengthens when the CN bondshortens, is also found in seven out of eight protein structures,determined at ultrahigh resolution (<0.95 Å). In fiveof them the linear correlation is statistically significantat the 95% confidence level. The present findings are consistentwith the traditional view of amide resonance and, although alreadyfound in small peptide structures, they represent a new andimportant result. In fact, in a protein structure the fine detailsof the peptide geometry are only marginally affected by thecrystal field and they are mostly produced by intramolecularand solvent interactions. The analysis of very high-resolutionprotein structures can reveal subtle information about localelectronic features of proteins which may be critical to folding,function or ligand binding.     

Protein Folding and Misfolding on Surfaces

Protein folding, misfolding and aggregation, as well as the way misfolded and aggregated proteins affects cell viability are emerging as key themes in molecular and structural biology and in molecular medicine. Recent advances in the knowledge of the biophysical basis of protein folding have led to propose the energy landscape theory which provides a consistent framework to better understand how a protein folds rapidly and efficiently to the compact, biologically active structure. The increased knowledge on protein folding has highlighted its strict relation to protein misfolding and aggregation, either process being in close competition with the other, both relying on the same physicochemical basis. The theory has also provided information to better understand the structural and environmental factors affecting protein folding resulting in protein misfolding and aggregation into ordered or disordered polymeric assemblies. Among these, particular importance is given to the effects of surfaces. The latter, in some cases make possible rapid and efficient protein folding but most often recruit proteins/peptides increasing their local concentration thus favouring misfolding and accelerating the rate of nucleation. It is also emerging that surfaces can modify the path of protein misfolding and aggregation generating oligomers and polymers structurally different from those arising in the bulk solution and endowed with different physical properties and cytotoxicities.     

Identification of Two Early Folding Stage Prion Non-Local Contacts Suggested to Serve as Key Steps in Directing the Final Fold to Be Either Native or Pathogenic

The initial steps of the folding pathway of the C-terminal domain of the murine prion protein mPrP(90–231) are predicted based on the sequential collapse model (SCM). A non-local dominant contact is found to form between the connecting region between helix 1 and β-sheet 1 and the C-terminal region of helix 3. This non-local contact nucleates the most populated molten globule-like intermediate along the folding pathway. A less stable early non-local contact between segments 120–124 and 179–183, located in the middle of helix 2, promotes the formation of a less populated molten globule-like intermediate. The formation of the dominant non-local contact constitutes an example of the postulated Nature’s Shortcut to the prion protein collapse into the native structure. The possible role of the less populated molten globule-like intermediate is explored as the potential initiation point for the folding for three pathogenic mutants (T182A, I214V, and Q211P in mouse prion numbering) of the prion protein.     

Folding core predictions from network models of proteins

Two different computational methods are employed to predict protein folding nuclei from native state structures, one based on an elastic network (EN) model and the other on a constraint network model of freely rotating rods. Three sets of folding cores are predicted with these models, and their correlation against the slow exchange folding cores identified by native state hydrogen-deuterium exchange (HX) experiments is used to test each method. These three folding core predictions rely on differences in the underlying models and relative importance of global or local motions for protein unfolding/folding reactions. For non-specific residue interactions, we use the Gaussian Network Model (GNM) to identify folding cores in the limits of two classes of motions, shortly referred to as global and local. The global mode minima from GNM represent the residues with the greatest potential for coordinating collective motions and are explored as potential folding nuclei. Additionally, the fast mode peaks that have previously been labeled as the kinetically hot residues are identified as a second folding core set dependent on local interactions. Finally, a third folding core set is defined by the most stable residues in a simulated thermal denaturation procedure of the FIRST software. This method uses an all-atomic analysis of the rigidity and flexibility of protein structures, which includes specific hydrophobic, polar and charged interactions. Comparison of the three folding core sets to HX data indicate that the fast mode peak residues determined by the GNM and the rigid folding cores of FIRST provide statistically significant enhancements over random correlation. The role of specific interactions in protein folding is also investigated by contrasting the differences between these two network-based computational methods.     

Sequence-structure specificity--how does an inverse folding approach work?

The inverse folding approach is a powerful tool in protein structure prediction when the native state of a sequence adopts one of the known protein folds. This is because some proteins show strong sequence- structure specificity in inverse folding experiments that allow gaps and insertions in the sequence-structure alignment. In those cases when structures similar to their native folds are included in the structure database, the z-scores (which measure the sequence-structure specificity) of these folds are well separated from those of other alternative structures. In this paper, we seek to understand the origin of this sequence-structure specificity and to identify how the specificity arises on passing from a short peptide chain to the entire protein sequence. To accomplish this objective, a simplified version of inverse folding, gapless inverse folding, is performed using sequence fragments of different sizes from 53 proteins. The results indicate that usually a significant portion of the entire protein sequence is necessary to show sequence-structure specificity, but there are regions in the sequence that begin to show this specificity at relatively short fragment size (15-20 residues). An island picture, in which the regions in the sequence that recognize their own native structure grow from some seed fragments, is observed as the fragment size increases. Usually, more similar structures to the native states are found in the top-scoring structural fragments in these high-specificity regions.      

Conservation of mechanism, variation of rate: folding kinetics of three homologous four-helix bundle proteins

The amino acid sequence of a protein determines both its final folded structure and the folding mechanism by which this structure is attained. The differences in folding behaviour between homologous proteins provide direct insights into the factors that influence both thermodynamic and kinetic properties. Here, we present a comprehensive thermodynamic and kinetic analysis of three homologous homodimeric four-helix bundle proteins. Previous studies with one member of this family, Rop, revealed that both its folding and unfolding behaviour were interesting and unusual: Rop folds (k(0)(f) = 29 s(-1)) and unfolds (k(0)(u) = 6 x 10(-7) s(-1)) extremely slowly for a protein of its size that contains neither prolines nor disulphides in its folded structure. The homologues we discuss have significantly different stabilities and rates of folding and unfolding. However, the rate of protein folding directly correlates with stability for these homologous proteins: proteins with higher stability fold faster. Moreover, in spite of possessing differing thermodynamic and kinetic properties, the proteins all share a similar folding and unfolding mechanism. We discuss the properties of these naturally occurring Rop homologues in relation to previously characterized designed variants of Rop.     

Ribosome: The Structure–Function Relation and a New Paradigm to the Protein Folding Problem

The rate of protein synthesis is about seven and fifteen amino acids per second, in the eukaryotic and the bacterial ribosome, respectively. Hence, a few minutes is required to synthesize a polypeptide of an average length. This is much longer than the time needed for the hydrophobic collapse (folding) to take place. So a polypeptide gets enough time to form its local secondary to tertiary structures cotranslationally and put such segments in proper order while in association with the ribosome, unless something prevents its entire length from folding. As reported earlier, ribosomes from prokaryotes, eukaryotes, and mitochondria act as molds for protein folding, and each mold has a set of recognition sites for all proteins. More specifically, the mold is the peptidyl transferase center (PTC), a part of the large RNA of the large ribosomal subunit. Specific amino acids from different random coil regions in a protein interact with specific nucleotides in the PTC, which brings the entire length of the protein into the small space of the PTC mold. The mold thus helps to stabilize the entropy-driven collapsed state of the polypeptide. The process also divides the protein into small segments; each segment is connected at two ends with two nucleotides and can fold in the ribosomal environment. The segments dissociate in such a sequence that the organization proceeds hierarchically from the core of the globular protein radially towards the outer surface. Then the protein dissociates from the ribosome in a “folding competent state” which does the final fine tuning in folding outside the ribosome. While the ribosomal contact and release are over in 1–2 minutes in vitro, the fine tuning takes about 5–10 minutes. Release from the ribosome needs no added energy factor from outside, like ATP.     

Resistance of recombinant proteins to proteolysis during folding and in the folded state

Protein purification often involves the use of denaturing agents for solubilization. During refolding, following removal of the denaturants, the proteins of interest are exposed to proteases present in the expression system. Here the resistance of selected recombinant proteins to three widely used proteolytic enzymes, trypsin (EC 3.4.21.4), proteinase K (EC 3.4.21.14) and endoproteinase Glu-C (EC 3.4.21.19), was investigated during folding and in the folded state. Target proteins and protease mixtures were denatured in 8 mol dm^?3 urea and the proteins were allowed to refold by removal of the urea by dialysis. The proteolytic products were analyzed by sodium dodecyl sulfate–polyacrylamide gels and the protein digestion during folding was compared with the digestion under similar conditions in physiological buffer. Depending on the folding state of the proteins, the proteases had different effects on the substates. During folding, certain recombinant proteins were more efficiently digested by trypsin and, in particular, by proteinase K in comparison with digestion in the folded state, while other protein substrates were more resistant to proteolytic degradation in a denatured or partially denatured state than their folded counterparts. Incubation of most substrate proteins with endoproteinase Glu-C yielded kinetics of digestion that were essentially similar for both partially folded and unfolded substrates. The results reported may be useful for protection of sensitive proteins and in studies of protein folding mechanisms.     

Zebrafish Motile Cilia as a Model for Primary Ciliary Dyskinesia

Zebrafish is a vertebrate teleost widely used in many areas of research. As embryos, they develop quickly and provide unique opportunities for research studies owing to their transparency for at least 48 h post fertilization. Zebrafish have many ciliated organs that include primary cilia as well as motile cilia. Using zebrafish as an animal model helps to better understand human diseases such as Primary Ciliary Dyskinesia (PCD), an autosomal recessive disorder that affects cilia motility, currently associated with more than 50 genes. The aim of this study was to validate zebrafish motile cilia, both in mono and multiciliated cells, as organelles for PCD research. For this purpose, we obtained systematic high-resolution data in both the olfactory pit (OP) and the left–right organizer (LRO), a superficial organ and a deep organ embedded in the tail of the embryo, respectively. For the analysis of their axonemal ciliary structure, we used conventional transmission electron microscopy (TEM) and electron tomography (ET). We characterised the wild-type OP cilia and showed, for the first time in zebrafish, the presence of motile cilia (9 + 2) in the periphery of the pit and the presence of immotile cilia (still 9 + 2), with absent outer dynein arms, in the centre of the pit. In addition, we reported that a central pair of microtubules in the LRO motile cilia is common in zebrafish, contrary to mouse embryos, but it is not observed in all LRO cilia from the same embryo. We further showed that the outer dynein arms of the microtubular doublet of both the OP and LRO cilia are structurally similar in dimensions to the human respiratory cilia at the resolution of TEM and ET. We conclude that zebrafish is a good model organism for PCD research but investigators need to be aware of the specific physical differences to correctly interpret their results.     

Uncovering the Properties of Energy-Weighted Conformation Space Networks with a Hydrophobic-Hydrophilic Model

The conformation spaces generated by short hydrophobic-hydrophilic (HP) lattice chains are mapped to conformation space networks (CSNs). The vertices (nodes) of the network are the conformations and the links are the transitions between them. It has been found that these networks have “small-world” properties without considering the interaction energy of the monomers in the chain, i. e. the hydrophobic or hydrophilic amino acids inside the chain. When the weight based on the interaction energy of the monomers in the chain is added to the CSNs, it is found that the weighted networks show the “scale-free” characteristic. In addition, it reveals that there is a connection between the scale-free property of the weighted CSN and the folding dynamics of the chain by investigating the relationship between the scale-free structure of the weighted CSN and the noted parameter Z score. Moreover, the modular (community) structure of weighted CSNs is also studied. These results are helpful to understand the topological properties of the CSN and the underlying free-energy landscapes.     

Endoplasmic Reticulum Stress and Unfolded Protein Response Signaling in Plants

Plants are sensitive to a variety of stresses that cause various diseases throughout their life cycle. However, they have the ability to cope with these stresses using different defense mechanisms. The endoplasmic reticulum (ER) is an important subcellular organelle, primarily recognized as a checkpoint for protein folding. It plays an essential role in ensuring the proper folding and maturation of newly secreted and transmembrane proteins. Different processes are activated when around one-third of newly synthesized proteins enter the ER in the eukaryote cells, such as glycosylation, folding, and/or the assembling of these proteins into protein complexes. However, protein folding in the ER is an error-prone process whereby various stresses easily interfere, leading to the accumulation of unfolded/misfolded proteins and causing ER stress. The unfolded protein response (UPR) is a process that involves sensing ER stress. Many strategies have been developed to reduce ER stress, such as UPR, ER-associated degradation (ERAD), and autophagy. Here, we discuss the ER, ER stress, UPR signaling and various strategies for reducing ER stress in plants. In addition, the UPR signaling in plant development and different stresses have been discussed.     

Prediction of contact maps with neural networks and correlated mutations

Contact maps of proteins are predicted with neural network-basedmethods, using as input codings of increasing complexity includingevolutionary information, sequence conservation, correlatedmutations and predicted secondary structures. Neural networksare trained on a data set comprising the contact maps of 173non-homologous proteins as computed from their well resolvedthree-dimensional structures. Proteins are selected from theProtein Data Bank database provided that they align with atleast 15 similar sequences in the corresponding families. Thepredictors are trained to learn the association rules betweenthe covalent structure of each protein and its contact map witha standard back propagation algorithm and tested on the sameprotein set with a cross-validation procedure. Our results indicatethat the method can assign protein contacts with an averageaccuracy of 0.21 and with an improvement over a random predictorof a factor >6, which is higher than that previously obtainedwith methods only based either on neural networks or on correlatedmutations. Furthermore, filtering the network outputs with aprocedure based on the residue coordination numbers, the accuracyof predictions increases up to 0.25 for all the proteins, withan 8-fold deviation from a random predictor. These scores arethe highest reported so far for predicting protein contact maps.     

Generation and analysis of proline mutants in protein G

The pyrrolidine ring of the amino acid proline reduces the conformational freedom of the protein backbone in its unfolded form and thus enhances protein stability. The strategy of inserting proline into regions of the protein where it does not perturb the structure has been utilized to stabilize many different proteins including enzymes. However, most of these efforts have been based on trial and error, rather than rational design. Here, we try to understand proline's effect on protein stability by introducing proline mutations into various regions of the B1 domain of Streptococcal protein G. We also applied the Optimization of Rotamers By Iterative Techniques computational protein design program, using two different solvation models, to determine the extent to which it could predict the stabilizing and destabilizing effects of prolines. Use of a surface area dependent solvation model resulted in a modest correlation between the experimental free energy of folding and computed energies; on the other hand, use of a Gaussian solvent exclusion model led to significant positive correlation. Including a backbone conformational entropy term to the computational energies increases the statistical significance of the correlation between the experimental stabilities and both solvation models.     

Scoring function based on weighted residue network

Molecular docking is an important method for the research of protein-protein interaction and recognition. A protein can be considered as a network when the residues are treated as its nodes. With the contact energy between residues as link weight, a weighted residue network is constructed in this paper. Two weighted parameters (strength and weighted average nearest neighbors' degree) are introduced into this model at the same time. The stability of a protein is characterized by its strength. The global topological properties of the protein-protein complex are reflected by the weighted average nearest neighbors' degree. Based on this weighted network model and these two parameters, a new docking scoring function is proposed in this paper. The scoring and ranking for 42 systems' bound and unbounded docking results are performed with this new scoring function. Comparing the results obtained from this new scoring function with that from the pair potentials scoring function, we found that this new scoring function has a similar performance to the pair potentials on some items, and this new scoring function can get a better success rate. The calculation of this new scoring function is easy, and the result of its scoring and ranking is acceptable. This work can help us better understand the mechanisms of protein-protein interactions and recognition.     

Stability constraints and protein evolution: the role of chain length, composition and disulfide bonds

Stability of the native state is an essential requirement in protein evolution and design. Here we investigated the interplay between chain length and stability constraints using a simple model of protein folding and a statistical study of the Protein Data Bank. We distinguish two types of stability of the native state: with respect to the unfolded state (unfolding stability) and with respect to misfolded configurations (misfolding stability). Several contributions to stability are evaluated and their correlations are disentangled through principal components analysis, with the following main results. (1) We show that longer proteins can fulfil more easily the requirements of unfolding and misfolding stability, because they have a higher number of native interactions per residue. Consistently, in longer proteins native interactions are weaker and they are less optimized with respect to non-native interactions. (2) Stability against misfolding is negatively correlated with the strength of native interactions, which is related to hydrophobicity. Hence there is a trade-off between unfolding and misfolding stability. This trade-off is influenced by protein length: less hydrophobic sequences are observed in very long proteins. (3) The number of disulfide bonds is positively correlated with the deficit of free energy stabilizing the native state. Chain length and the number of disulfide bonds per residue are negatively correlated in proteins with short chains and uncorrelated in proteins with long chains. (4) The number of salt bridges per residue and per native contact increases with chain length. We interpret these observations as an indication that the constraints imposed by unfolding stability are less demanding in long proteins and they are further reduced by the competing requirement for stability against misfolding. In particular, disulfide bonds appear to be positively selected in short proteins, whereas they evolve in an effectively neutral way in long proteins.