Unperturbed dimensions of crosslinked histones evaluated using random-flight statistics and rotational isomeric state theory

Unperturbed dimensions have been computed via rotational isomeric state theory for approximately 700 dimers obtained from histones 2A, 2B, and 4. Dimers result from a crosslink joining either lysyl or tyrosyl residues in different histones. Sets of statistical weights were chosen that yield either a low helicity or a helical content near 40%. These extremes correspond to helicities of histones in the commonly used acid/urea and sodium dodecyl sulfate systems, respectively. Many features of the rotational isomeric state results can be sucessfully reproduced by much simpler expressions based on random-flight statistics. For example, the two methods are about equally effective in predicting how the radius of gyration of a given type of crosslinked dimer should vary from that of the analogous linear polypeptide chain. This result is in marked contrast to that attained using crosslinked, partially helical homopolypeptides. The unexpected success of random-flight statistics with partially helical crosslinked histones is due to the suppression of long helical segments by helix-breaking amino acid residues. Random-flight statistics should be applicable to other crosslinked, partially helical proteins if their amino acid sequences contain the usual representation of helix-breaking amino acid residues.     

Information contained in the amino acid sequence of theα1(I)-chain of collagen and its consequences upon the formation of the triple helix,of fibrils and crosslinks

The molecule of type I collagen from skin consists of two alpha1(I)-chains and one alpha2-chain. The sequence of the entire alpha1-chain comprising 1052 residues is summarily presented and discussed. Apart from the 279 residues of alpha1(I)-CB8 whose sequence has been established for rat skin collagen, all sequences have been determined for calf skin collagen. In order to facilitate sequence analysis, the alpha1-chain was cleaved into defined fragments by cyanogen bromide or hydroxylamine or limited collagenase digestion. Most of the sequence was established by automated stepwise Edman degradation. The alpha1-chain contains two basically different types of sequences: the triple helical region of 1011 amino acid residues in which every third position is occupied by glycine and the N- and C-terminal regions not displaying this type of regularity. Both of these non-triple helical regions carry oxidizable lysine or hydroxylysine residues as functional sites for the intermolecular crosslink formation. Implications of the amino acid sequence for the stability of the triple helix and the fibril as well as for formation of crosslinks are discussed. Evaluation of the sequence in connection with electron microscopical investigations yielded the parameters of the axial arrangement of the molecules within the fibrils. Axial stagger of the molecules by a distance D = 670 angstrom = 233 amino acid residues results in maximal interaction of polar sequence regions of adjacent molecules and similarly of regions of hydrophobic residues. Ordered aggregation of molecules into fibrils is, therefore, regulated by electrostatic and electrophobic forces. Possible loci of intermolecular crosslinks between the alpha1-chains of adjacent molecules may be deduced from the dimensions of the axial aggregation of molecules.     

Towards unambiguous assignment of methyl-containing residues by double and triple sensitivity-enhanced HCCmHm-TOCSY experiments

Chemical shift assignment of methyl-containing residues is essential in protein NMR spectroscopy, as these residues are abundant in protein interiors and provide the vast majority of long-range NOE connectivities for structure determination. These residues also constitute an integral part of hydrophobic cavities, the surroundings for many enzymatic reactions. Here we present a powerful strategy for the assignment of methyl-containing residues in a uniformly ¹³C/¹⁵N double labeled protein sample. The approach is based on novel four-dimensional HCCmHm-TOCSY experiments, two of them utilizing gradient selection and sensitivity enhancement in all three indirectly detected dimensions. Regardless of the number of dimensions, the proposed experiments can be executed using only one transient per FID, providing outstanding resolution and sensitivity. A complete assignment of the 51 methyl-containing residues in the 16 kDa Mus musculus coactosin was accomplished using a four-dimensional HCCmHm-TOCSY spectrum recorded in 16 hours.     

N-glycosylation at noncanonical Asn-X-Cys sequences in plant cells

The vesicular transport pathway in plant cells is often used for higher accumulation of recombinant proteins. In the endoplasmic reticulum, which acts as a gateway to the vesicular transport pathway, N-glycosylation occurs on specific Asn residues. This N-glycosylation in recombinant proteins must be carefully regulated as it can impact their enzymatic activity, half lives in serum when injected, structural stability, etc. In eukaryotic cells, including plant cells, N-glycans were found to be attached to Asn residues in Asn-X-Ser/Thr (X ≠ Pro) sequences. However, recently, N-glycosylations at noncanonical Asn-X-Cys sequences have been found in mammals and yeast. Our laboratory has discovered that N-glycans are attached to Asn residues at Asn-Thr-Cys sequences of double-repeated B subunit of Shiga toxin 2e produced in plant cells, the first reported case of N-glycosylation at a noncanonical Asn-X-Cys sequence in plant cells.     

Grouping of amino acids and recognition of protein structurally conserved regions by reduced alphabets of amino acids

Sequence alignment is a common method for finding protein structurally conserved/similar regions. However, sequence alignment is often not accurate if sequence identities between to-be-aligned sequences are less than 30%. This is because that for these sequences, different residues may play similar structural roles and they are incorrectly aligned during the sequence alignment using substitution matrix consisting of 20 types of residues. Based on the similarity of physicochemical features, residues can be clustered into a few groups. Using such simplified alphabets, the complexity of protein sequences is reduced and at the same time the key information encoded in the sequences remains. As a result, the accuracy of sequence alignment might be improved if the residues are properly clustered. Here, by using a database of aligned protein structures (DAPS), a new clustering method based on the substitution scores is proposed for the grouping of residues, and substitution matrices of residues at different levels of simplification are constructed. The validity of the reduced alphabets is confirmed by relative entropy analysis. The reduced alphabets are applied to recognition of protein structurally conserved/similar regions by sequence alignment. The results indicate that the accuracy or efficiency of sequence alignment can be improved with the optimal reduced alphabet with N around 9.     

Revealing the set of mutually correlated positions for the protein families of immunoglobulin fold

In this study, I explain the observation that a rather limited number of residues (about 10) establishes the immunoglobulin fold for the sequences of about 100 residues. Immunoglobulin fold proteins (IgF) comprise SCOP protein superfamilies with rather different functions and with less than 10% sequence identity; their alignment can be accomplished only taking into account the 3D structure. Therefore, I believe that discovering the additional common features of the sequences is necessary to explain the existence of a common fold for these SCOP superfamilies. We propose a method for analysis of pair-wise interconnections between residues of the multiple sequence alignment which helps us to reveal the set of mutually correlated positions, inherent to almost every superfamily of this protein fold. Hence, the set of constant positions (comprising the hydrophobic common core) and the set of variable but mutually correlated ones can serve as a basis of having the common 3D structure for rather distinct protein sequences.     

A Combinatorial Approach to Detect Coevolved Amino Acid Networks in Protein Families of Variable Divergence

Communication between distant sites often defines the biological role of a protein: amino acid long-range interactions are as important in binding specificity, allosteric regulation and conformational change as residues directly contacting the substrate. The maintaining of functional and structural coupling of long-range interacting residues requires coevolution of these residues. Networks of interaction between coevolved residues can be reconstructed, and from the networks, one can possibly derive insights into functional mechanisms for the protein family. We propose a combinatorial method for mapping conserved networks of amino acid interactions in a protein which is based on the analysis of a set of aligned sequences, the associated distance tree and the combinatorics of its subtrees. The degree of coevolution of all pairs of coevolved residues is identified numerically, and networks are reconstructed with a dedicated clustering algorithm. The method drops the constraints on high sequence divergence limiting the range of applicability of the statistical approaches previously proposed. We apply the method to four protein families where we show an accurate detection of functional networks and the possibility to treat sets of protein sequences of variable divergence.     

Evidence for cytosine methylation of non-symmetrical sequences in transgenic Petunia hybrida.

A considerable proportion of cytosine residues in plants are methylated at carbon 5. According to a well-accepted rule, cytosine methylation is confined to symmetrical sequences such as CpG and CpNpG, which provide the signal for faithful transmission of symmetrical methylation patterns by maintenance methylase. Using a genomic sequencing technique, we have analysed cytosine methylation patterns within a hypermethylated and a hypomethylated state of a transgene in Petunia hybrida. Examination of a part of the transgene promoter revealed that in both states m5C residues located within non-symmetrical sequences could be detected. Non-symmetrical C residues in the two states were methylated at frequencies of 5.9 and 31.9%, respectively. Methylation appeared to be distributed heterogeneously, but some DNA regions were more intensively methylated than others. Our results show that at least in a transgene, a heterogeneous methylation pattern, which does not depend on symmetry of target sequences, can be established and conserved.     

Contact interactions method: a new algorithm for protein folding simulations.

Computer simulations of simple exact lattice models are an aid in the study of protein folding process; they have sometimes resulted in predictions experimentally proved. The contact interactions (CI) method is here proposed as a new algorithm for the conformational search in the low-energy regions of protein chains modeled as copolymers of hydrophobic and polar monomers configured as self-avoiding walks on square or cubic lattices. It may be regarded as an extension of the standard Monte Carlo method improved by the concept of cooperativity deriving from nonlocal contact interactions. A major difference with respect to other algorithms is that criteria for the acceptance of new conformations generated during the simulations are not based on the energy of the entire molecule, but cooling factors associated with each residue define regions of the model protein with higher or lower mobility. Nine sequences of length ranging from 20 to 64 residues were used on the square lattice and 15 sequences of length ranging from 46 to 136 residues were used on the cubic lattice. The CI algorithm proved very efficient both in two and three dimensions, and allowed us to localize energy minima not localized by other searching algorithms described in the literature. Use of this algorithm is not limited to the conformational search, because it allows the exploration of thermodynamic and kinetic behavior of model protein chains.     

Molecular modeling indicates that homodimers form the basis for intermediate filament assembly from human and mouse epidermal keratins

Mammalian epidermal keratin molecules adopt rod-shaped conformations that aggregate to form cytoplasmic intermediate filaments. To investigate these keratin conformations and the basis for their patterns of molecular association, graphical methods were developed to relate known amino acid sequences to probable spacial configurations. The results support the predominantly α-helical conformation of keratin chains, interrupted by short non-α-helical linkages. However, it was found that many of the linkages have amino acid sequences typical of β-strand conformations. Space-filling atomic models revealed that the β-strand sequences would permit the formation of 2-chain and 4-chain cylindrical β-helices, fully shielding the hydrophobic amino acid chains that alternate with hydrophilic residues in these sequences. Because of the locations of the β-helical regions in human and mouse stratum corneum keratin chains, only homodimers of the keratins could interact efficiently to form 2-chain and 4-chain β-helices. Tetramers having the directions and degrees of overlap of constituent dimers that have been identified by previous investigators are also predicted from the interactions of β-helical motifs. Heterotetramers formed from dissimilar homodimers could combine, through additional β-helical structures, to form higher oligomers having the dimensions seen in electron microscopic studies. Previous results from chemical crosslinking studies can be interpreted to support the concept of homodimers rather than heterodimers as the basis for keratin filament assembly. © 1995 Wiley-Liss, Inc.     

Structure of alpha-alpha-hairpins with short connections.

This paper presents the results of detailed stereochemical analysis of structures and sequences of alpha-alpha-hairpins with short connections. It is shown that alpha-alpha-hairpins of each given type have very similar patterns of hydrophobic, hydrophilic and glycine residues in their amino acid sequences. These results can be used in the prediction of alpha-alpha-hairpin conformation as well as in protein design and engineering.     

Modeling alpha-helical transmembrane domains: the calculation and use of substitution tables for lipid-facing residues.

Amino acid substitution tables are calculated for residues in membrane proteins where the side chain is accessible to the lipid. The analysis is based upon the knowledge of the three-dimensional structures of two homologous bacterial photosynthetic reaction centers and alignments of their sequences with the sequences of related proteins. The patterns of residue substitutions show that the lipid-accessible residues are less conserved and have distinctly different substitution patterns from the inaccessible residues in water-soluble proteins. The observed substitutions obtained from sequence alignments of transmembrane regions (identified from, e.g., hydrophobicity analysis) can be compared with the patterns derived from the substitution tables to predict the accessibility of residues to the lipid. A Fourier transform method, similar to that used for the calculation of a hydrophobic moment, is used to detect periodicity in the predicted accessibility that is compatible with the presence of an alpha-helix. If the putative transmembrane region is identified as helical, then the buried and exposed faces can be discriminated. The presence of charged residues on the lipid-exposed face can help to identify the regions that are in contact with the polar environment on the borders of the bilayer, and the construction of a meaningful three-dimensional model is then possible. This method is tested on an alignment of bacteriorhodopsin and two related sequences for which there are structural data at near atomic resolution.     

The eight-cysteine motif, a versatile structure in plant proteins.

A number of protein sequences deduced from the molecular analysis of plant cDNA or genomic libraries can be grouped in relation to a defined number of cysteine residues located in distinct positions of their sequences. This is the case for a group of around 500 polypeptides from different species that contain a small domain (less than 100 amino acids residues) displaying a pattern of eight-cysteines in a specific order. The plant sequences containing this motif belong to proteins having different functions, ranging from storage, protection, enzyme inhibition and lipid transfer, to cell wall structure. The eight-cysteine motif (8CM) appears to be a structural scaffold of conserved helical regions connected by variable loops, as observed by three-dimensional structure analysis. It is proposed that the cysteine residues would form a network of disulfide bridges necessary, for the maintenance of the tertiary structure of the molecule together with the central helical core, while the variable loops would provide the sequences required for the specific functions of the proteins.     

Structural analysis of homologous repeated domains in alpha-actinin and spectrin

The amino acid sequences of chick and slime mould alpha-actinin each contain four repeats of approximately 122 residues. These repeats are homologous to the 18-22 repeats, each of approximately 106 residues, found in the alpha and beta subunits of spectrin and fodrin, and to the multiple repeats of approximately 110 residues found in the Duchenne muscular dystrophy protein (dystrophin). The repeats correspond to the elongated rod-like portion of these molecules. We present a multiple sequence alignment of 21 repeats from this superfamily (8 alpha-actinin and 13 spectrin/fodrin), based on optimal pairwise alignments, from which a characteristic consensus pattern of amino acid types is deduced. Trp 46 is invariant in all but one repeat, and physicochemical classes of amino acids are conserved at 25 other positions. Secondary structure prediction on both the alpha-actinin and spectrin repeats taken together with the distribution of proline residues in the sequences, strongly suggest that each repeated domain consists of a four-helix structure. Our predictions differ significantly from previous three-helix models based on analyses of fewer sequences. To determine possible interdomain regions, sites of limited proteolysis of the native chick alpha-actinin dimer were determined and located in the amino acid sequence. The majority of these sites were in corresponding positions in different repeats within a segment predicted as a long helix. We propose a model, consistent with the overall dimensions of the rod-like portions of the molecules, in which these long, probably interrupted helices, link adjacent domains.     

Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins.

Based on the recently determined X-ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their three-dimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved. The conservation in the three remaining sequences is somewhat lower. The conserved residues include the active site, disulfide bridges, salt bridges, and residues in the core of the proteins. Most invariant residues are located at the edges of secondary structural elements. A clear structural basis for the preservation of many of these residues can be determined from comparison of the two X-ray structures.     

Hybrid transfer RNA genes in phage T4

We describe the isolation and characterization of two unusual amber suppressor forms of T4 tRNALeu. The sequences of the suppressor tRNAs can be described as hybrids of wild-type tRNALeu and suppressor tRNAGln molecules: the chain lengths and majority of the nucleotide residues corresponded to tRNALeu, but CUA anticodons flanked by 2-14 residues were identical to tRNAGln. The uncertainty as to the exact number of flanking residues correlated with tRNAGln is due to the similarity of the two tRNA sequences in this region. No evidence was found for changes in other T4 tRNAs. We propose that genes for the hybrid tRNAs were produced by mispairing of DNAs at anticodon segments of tRNALeu and tRNAGln with a double crossover flanking those segments.     

Predicting reliable regions in protein alignments from sequence profiles

For applications such as comparative modelling one major issue is the reliability of sequence alignments. Reliable regions in alignments can be predicted using sub-optimal alignments of the same pair of sequences. Here we show that reliable regions in alignments can also be predicted from multiple sequence profile information alone.Alignments were created for a set of remotely related pairs of proteins using five different test methods. Structural alignments were used to assess the quality of the alignments and the aligned positions were scored using information from the observed frequencies of amino acid residues in sequence profiles pre-generated for each template structure. High-scoring regions of these profile-derived alignment scores were a good predictor of reliably aligned regions.These profile-derived alignment scores are easy to obtain and are applicable to any alignment method. They can be used to detect those regions of alignments that are reliably aligned and to help predict the quality of an alignment. For those residues within secondary structure elements, the regions predicted as reliably aligned agreed with the structural alignments for between 92% and 97.4% of the residues. In loop regions just under 92% of the residues predicted to be reliable agreed with the structural alignments. The percentage of residues predicted as reliable ranged from 32.1% for helix residues to 52.8% for strand residues.This information could also be used to help predict conserved binding sites from sequence alignments. Residues in the template that were identified as binding sites, that aligned to an identical amino acid residue and where the sequence alignment agreed with the structural alignment were in highly conserved, high scoring regions over 80% of the time. This suggests that many binding sites that are present in both target and template sequences are in sequence-conserved regions and that there is the possibility of translating reliability to binding site prediction.     

Estimation of amino acid residue substitution rates at local spatial regions and application in protein function inference: a Bayesian Monte Carlo approach

The amino acid sequences of proteins provide rich information for inferring distant phylogenetic relationships and for predicting protein functions. Estimating the rate matrix of residue substitutions from amino acid sequences is also important because the rate matrix can be used to develop scoring matrices for sequence alignment. Here we use a continuous time Markov process to model the substitution rates of residues and develop a Bayesian Markov chain Monte Carlo method for rate estimation. We validate our method using simulated artificial protein sequences. Because different local regions such as binding surfaces and the protein interior core experience different selection pressures due to functional or stability constraints, we use our method to estimate the substitution rates of local regions. Our results show that the substitution rates are very different for residues in the buried core and residues on the solvent-exposed surfaces. In addition, the rest of the proteins on the binding surfaces also have very different substitution rates from residues. Based on these findings, we further develop a method for protein function prediction by surface matching using scoring matrices derived from estimated substitution rates for residues located on the binding surfaces. We show with examples that our method is effective in identifying functionally related proteins that have overall low sequence identity, a task known to be very challenging.     

Grouping of amino acids and recognition of protein structurally conserved regions by reduced alphabets of amino acids

Sequence alignment is a common method for finding protein structurally conserved/similar regions. However, sequence alignment is often not accurate if sequence identities between to-be-aligned sequences are less than 30%. This is because that for these sequences, different residues may play similar structural roles and they are incorrectly aligned during the sequence alignment using substitution matrix consisting of 20 types of residues. Based on the similarity of physicochemical features, residues can be clustered into a few groups. Using such simplified alphabets, the complexity of protein sequences is reduced and at the same time the key information encoded in the sequences remains. As a result, the accuracy of sequence alignment might be improved if the residues are properly clustered. Here, by using a database of aligned protein structures (DAPS), a new clustering method based on the substitution scores is proposed for the grouping of residues, and substitution matrices of residues at different levels of simplification are constructed. The validity of the reduced alphabets is confirmed by relative entropy analysis. The reduced alphabets are applied to recognition of protein structurally conserved/similar regions by sequence alignment. The results indicate that the accuracy or efficiency of sequence alignment can be improved with the optimal reduced alphabet with N around 9. Supported by the National Natural Science Foundation of China (Grant Nos. 90403120, 10474041 and 10021001) and the Nonlinear Project (973) of the NSM