An AMBER/DYANA/MOLMOL Phosphorylated Amino Acid Library Set and Incorporation into NMR Structure Calculations

Protein structure determination using Nuclear Magnetic Resonance (NMR) requires the use of molecular dynamics programs that incorporate both NMR experimental and implicit atomic data. Atomic parameters for each amino acid type are encoded in libraries used by structure calculation programs such as DYANA and AMBER. However, only a few non-standard amino acid library sets are included in these programs or the molecular visualization program MOLMOL. Our laboratory is calculating the phosphorylated and non-phosphorylated states of peptides and proteins using NMR methods. To calculate chemically correct structures, we have extended the available molecular libraries for these programs to include the modified amino acids phosphoserine, phosphothreonine, and phosphotyrosine.     

Three-dimensional structure and determinants of stability of the iron-sulfur cluster scaffold protein IscU from Escherichia coli

The highly conserved protein, IscU, serves as the scaffold for iron-sulfur cluster (ISC) assembly in the ISC system common to bacteria and eukaryotic mitochondria. The apo-form of IscU from Escherichia coli has been shown to populate two slowly interconverting conformational states: one structured (S) and one dynamically disordered (D). Furthermore, single-site amino acid substitutions have been shown to shift the equilibrium between the metamorphic states. Here, we report three-dimensional structural models derived from NMR spectroscopy for the S-state of wild-type (WT) apo-IscU, determined under conditions where the protein was 80% in the S-state and 20% in the D-state, and for the S-state of apo-IscU(D39A), determined under conditions where the protein was ~95% in the S-state. We have used these structures in interpreting the effects of single site amino acid substitutions that alter %S = (100 × [S])/([S] + [D]). These include different residues at the same site, %S: D39V > D39L > D39A > D39G ≈ WT, and alanine substitutions at different sites, %S: N90A > S107A ≈ E111A > WT. Hydrophobic residues at residue 39 appear to stabilize the S-state by decreasing the flexibility of the loops that contain the conserved cysteine residues. The alanine substitutions at positions 90, 107, and 111, on the other hand, stabilize the protein without affecting the loop dynamics. In general, the stability of the S-state correlates with the compactness and thermal stability of the variant.     

Mutagenic mapping of helical structures in the transmembrane segments of the yeast alpha-factor receptor

The alpha-mating pheromone receptor encoded by the yeast STE2 gene is a G protein coupled receptor that initiates signaling via a MAP kinase pathway that prepares haploid cells for mating. To establish the range of allowed amino acid substitutions within transmembrane segments of this receptor, we conducted extensive random mutagenesis of receptors followed by screening for receptor function. A total of 157 amino acid positions in seven different mutagenic libraries corresponding to the seven predicted transmembrane segments were analyzed, yielding 390 alleles that retain at least 60 % of normal signaling function. These alleles contained a total of 576 unique amino acid substitutions, including 61 % of all the possible amino acid changes that can arise from single base substitutions. The receptor exhibits a surprising tolerance for amino acid substitutions. Every amino acid in the mutagenized regions of the transmembrane regions could be substituted by at least one other residue. Polar amino acids were tolerated in functional receptors at 115 different positions (73 % of the total). Hydrophobic amino acids were tolerated in functional receptors at all mutagenized positions. Substitutions introducing proline residues were recovered at 53 % of all positions where they could be brought about by single base changes. Residues with charged side-chains could also be tolerated at 53 % of all positions where they were accessible through single base changes. The spectrum of allowed amino acid substitutions was characterized in terms of the hydrophobicity, radius of gyration, and charge of the allowed substitutions and mapped onto alpha-helical structures. By comparing the patterns of allowed substitutions with the recently determined structure of rhodopsin, structural features indicative of helix-helix interactions can be discerned in spite of the extreme sequence divergence between these two proteins.     

Gene synthesis, expression, structures, and functional activities of site-specific mutants of ubiquitin

To study the structure and function of ubiquitin we have chemically synthesized a ubiquitin gene that encodes the amino acid sequence of animal ubiquitin, inserting a series of restriction enzyme sites that divide the gene into eight "mutagenesis modules." A series of site-specific mutations were constructed to selectively perturb various regions of the molecule. The mutant genes were expressed in a large quantity of Escherichia coli, and the modified proteins were purified. To determine the structural effects of the amino acid substitutions, the solution structure of ubiquitin was investigated by two-dimensional NMR and each of the mutant proteins were screened for structural perturbations. With one exception, virtually no changes were seen other than at the point of mutation. Functional studies of the mutant proteins with the ubiquitin-activating enzyme E1 and in the reticulocyte protein degradation assay were used to identify regions of the molecule important to ubiquitin's activity in intracellular proteolysis.     

Relationships between protein structure and dynamics from a database of NMR-derived backbone order parameters

The amplitude of protein backbone NH group motions on a time-scale faster than molecular tumbling may be determined by analysis of (15)N NMR relaxation data according to the Lipari-Szabo model free formalism. An internet-accessible database has been compiled containing 1855 order parameters from 20 independent NMR relaxation studies on proteins whose three-dimensional structures are known. A series of statistical analyses has been performed to identify relationships between the structural features and backbone dynamics of these proteins. Comparison of average order parameters for different amino acid types indicates that amino acids with small side-chains tend to have greater backbone flexibility than those with large side-chains. In addition, the motions of a given NH group are also related to the sizes of the neighboring amino acids in the primary sequence. The secondary structural environment appears to influence backbone dynamics relatively weakly, with only subtle differences between the order parameter distributions of loop structures and regular hydrogen bonded secondary structure elements. However, NH groups near helix termini are more mobile on average than those in the central regions of helices. Tertiary structure influences are also relatively weak but in the expected direction, with more exposed residues being more flexible on average than residues that are relatively inaccessible to solvent.     

Recent developments in structural proteomics for protein structure determination

The major challenges in structural proteomics include identifying all the proteins on the genome-wide scale, determining their structure-function relationships, and outlining the precise three-dimensional structures of the proteins. Protein structures are typically determined by experimental approaches such as X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. However, the knowledge of three-dimensional space by these techniques is still limited. Thus, computational methods such as comparative and de novo approaches and molecular dynamic simulations are intensively used as alternative tools to predict the three-dimensional structures and dynamic behavior of proteins. This review summarizes recent developments in structural proteomics for protein structure determination; including instrumental methods such as X-ray crystallography and NMR spectroscopy, and computational methods such as comparative and de novo structure prediction and molecular dynamics simulations.     

NMR Resonance Assignment Methodology: Characterizing Large Sparsely Labeled Glycoproteins

Characterization of proteins using NMR methods begins with assignment of resonances to specific residues. This is usually accomplished using sequential connectivities between nuclear pairs in proteins uniformly labeled with NMR active isotopes. This becomes impractical for larger proteins, and especially for proteins that are best expressed in mammalian cells, including glycoproteins. Here an alternate protocol for the assignment of NMR resonances of sparsely labeled proteins, namely, the ones labeled with a single amino acid type, or a limited subset of types, isotopically enriched with ¹⁵N or ¹³C, is described. The protocol is based on comparison of data collected using extensions of simple two-dimensional NMR experiments (correlated chemical shifts, nuclear Overhauser effects, residual dipolar couplings) to predictions from molecular dynamics trajectories that begin with known protein structures. Optimal pairing of predicted and experimental values is facilitated by a software package that employs a genetic algorithm, ASSIGN_SLP_MD. The approach is applied to the 36-kDa luminal domain of the sialyltransferase, rST6Gal1, in which all phenylalanines are labeled with ¹⁵N, and the results are validated by elimination of resonances via single-point mutations of selected phenylalanines to tyrosines. Assignment allows the use of previously published paramagnetic relaxation enhancements to evaluate placement of a substrate analog in the active site of this protein. The protocol will open the way to structural characterization of the many glycosylated and other proteins that are best expressed in mammalian cells.     

Variations of sequences and amino acid compositions of proteins that sustain their biological functions: An analysis of the cyclophilin family of proteins.

The sequences of the ubiquitous and phylogenetically diversified cyclophilin family of proteins were divided into six groups, namely, vertebrates, invertebrates, other metazoa, plants, fungi, and prokaryotes. These groups of sequences were aligned with the multiple sequence alignment program Clustal-W. The variations of amino acid substitutions and amino acid compositions for these six groups of cyclophilins were calculated using a novel suite of multiple-sequence alignment analysis routines. The cyclophilins from vertebrates can be divided for at least two distinct structural classes that differ from each other by a variable-length amino acid insert within the loop that links alpha-helix II and beta-strand III. A similar structural feature is also present in the other groups of cyclophilins, namely, those from invertebrates, other metazoa, plants, and fungi. The sequences of cyclophilins from fungi and prokaryotes are more diversified than those from vertebrates, and their alterations involve structures other than the amino acid inserts within the loops. Variations of the hydrophobicity and bulkiness of amino acid substitutions of the aligned sequences were calculated for each group of cyclophilins and for the alignment of all the sequences. The variations have clear asymmetry that may signify the need for modification of the physical properties of certain fragments of cyclophilins that are involved in interactions with various cellular components in the evolving environment.     

Accumulation of amino acid substitutions promotes irreversible structural changes in the hemagglutinin of human influenza AH3 virus during evolution

During protein evolution the amino acid substitutions accumulate with time. However, the effect of accumulation of the amino acid substitutions to structural changes has not been estimated well. We will propose that the discordance of amino acid substitution on the HA protein of influenza A virus is useful for the assessment of structural changes during evolution. Discordance value can be obtained from the experimental data of tolerance or intolerance by introducing site directed mutagenesis at the homologous positions of two HA proteins holding the same amino acid residues. The value of discordance correlated to the number of amino acid differences among proteins. In the H3HA discordance rate was calculated to be 0.45% per one amino acid change. Furthermore, discordance of amino acid substitutions suggests that tolerable amino acid substitutions in different order have a probability of promoting irreversible divergence of the HA protein to different subtypes.     

Comparison of the computed three-dimensional structures of oncogenic forms (bound to GDP) of theras-Gene-Encoded p21 protein with the structure of the normal (non-transforming) wild-type protein

Theras-oncogene-encoded p21 protein becomes oncogenic if amino acid substitutions occur at critical positions in the polypeptide chain. The most commonly found oncogenic forms contain Val in place of Gly 12 or Leu in place of Gln 61. To determine the effects of these substitutions on the three-dimensional structure of the whole p21 protein, we have performed molecular dynamics calculations on each of these three proteins bound to GDP and magnesium ion to compute the average structures of each of the three forms. Comparisons of the computed average structures shows that both oncogenic forms with Val 12 and Leu 61 differ substantially in structure from that of the wild type (containing Gly 12 and Gln 61) in discrete regions: residues 10–16, 32–47, 55–74, 85–89, 100–110, and 119–134. All of these regions occur in exposed loops, and several of them have already been found to be involved in the cellular functioning of the p21 protein. These regions have also previously been identified as the most flexible domains of the wild-type protein and have been bound to be the same ones that differ in conformation between transforming and nontransforming p21 mutant proteins neither of which binds nucleotide. The two oncogenic forms have similar conformations in their carboxyl-terminal domains, but differ in conformation at residues 32–47 and 55–74. The former region is known to be involved in the interaction with at least three downstream effector target proteins. Thus, differences in structure between the two oncogenic proteins may reflect different relative affinities of each oncogenic protein for each of these effector targets. The latter region, 55–74, is known to be a highly mobile segment of the protein. The results strongly suggest that critical oncogenic amino acid substitutions in the p21 protein cause changes in the structures of vital domains of this protein.     

Estimation of evolutionary distances from protein spatial structures

New equations are derived to estimate the number of amino acid substitutions per site between two homologous proteins from the root mean square (RMS) deviation between two spatial structures and from the fraction of identical residues between two sequences. The equations are based on evolutionary models, analyzing predominantly structural changes and not sequence changes. Evolution of spatial structure is treated as a diffusion in an elastic force field. Diffusion accounts for structural changes caused by amino acid substitutions, and elastic force reflects selection, which preserves protein fold. Obtained equations are supported by analysis of protein spatial structures. Received: 21 September 1995 / Accepted: 19 May 1997     

Fold change in evolution of protein structures

Typically, protein spatial structures are more conserved in evolution than amino acid sequences. However, the recent explosion of sequence and structure information accompanied by the development of powerful computational methods led to the accumulation of examples of homologous proteins with globally distinct structures. Significant sequence conservation, local structural resemblance, and functional similarity strongly indicate evolutionary relationships between these proteins despite pronounced structural differences at the fold level. Several mechanisms such as insertions/deletions/substitutions, circular permutations, and rearrangements in beta-sheet topologies account for the majority of detected structural irregularities. The existence of evolutionarily related proteins that possess different folds brings new challenges to the homology modeling techniques and the structure classification strategies and offers new opportunities for protein design in experimental studies.     

Amino acid substitutions in structurally related proteins. A pattern recognition approach. Determination of a new and efficient scoring matrix

Amino acid substitutions in evolutionarily related proteins have been studied from a structural point of view. We consider here that an amino acid al in a protein p1 has been replaced by the amino acid a2 in the structurally similar protein p2 if, after superposition of the p1 and p2 structures, the a1 and a2 C alpha atoms are no more than 1.2 A apart. Thirty-two proteins, grouped in 11 classes, have been analysed by this method. This produced 2860 amino acid pairs (substitutions), which were analysed by multi-dimensional statistical methods. The main results are as follows: (1) according to the observed exchangeability of amino acid side-chains, only four groups (strong clusters) could be delineated; (i) Ile and Val, (ii) Leu and Met, (iii) Lys, Arg and Gln, and (iv) Tyr and Phe. The other residues could not be classified. (2) The matrix of distances between amino acids, or scoring matrix, determined from this study, is different from any other published matrix. (3) Except for the distance matrices based on the chemical properties of amino acid side-chains, which can be grouped together, all other published matrices are different from one another. (4) The distance matrix determined in this study seems to be very efficient for aligning distantly related protein sequences.     

Temperature adaptation of lactate dehydrogenase. Structural, functional and genetic aspects

Comparison of the primary structures of thermophilic, mesophilic and psychrophilic lactate dehydrogenase (LDH) reveals a multitude of temperature-related amino acid substitutions. In the substitutions amino acid residues occurring preferentially in thermophilic, mesophilic (psychrophilic) LDH were found. On this basis, amino acid residues could be classified in an order from typical thermophilic (thermostabilizing) to typical mesophilic (thermolabilizing, increasing dynamics of the enzyme molecule) residues. The temperature-dependent ratio between thermostabilizing and thermolabilizing amino acid residues forms the basis for the specific structural and functional properties of thermophilic or mesophilic LDH. It is interesting that there appears to be a relationship between this order from thermophilic to mesophilic amino acid residues and the type of bases coding for these individual residues in the translation step of protein biosynthesis. Temperature-related amino acid substitutions are based on temperature-related base substitutions. A possible mechanism of temperature adaptation of LDH through alternative selection of thermophilic and mesophilic amino acid residues at the level of tRNA (anticodon)-mRNA (codon) interactions is discussed. These temperature-adaptation processes are evolutionary events in which the evolution and structure of the genetic code are involved.     

Amino acid substitutions in the C-terminal AAA+ module of Hsp104 prevent substrate recognition by disrupting oligomerization and cause high temperature inactivation

Hsp104 is an important determinant of thermotolerance in yeast and is an unusual molecular chaperone that specializes in the remodeling of aggregated proteins. The structural requirements for Hsp104-substrate interactions remain unclear. Upon mild heat shock Hsp104 formed cytosolic foci in live cells that indicated co-localization of the chaperone with aggregates of thermally denatured proteins. We generated random amino acid substitutions in the C-terminal 199 amino acid residues of a GFP-Hsp104 fusion protein, and we used a visual screen to identify mutants that remained diffusely distributed immediately after heat shock. Multiple amino acid substitutions were required for loss of heat-inducible redistribution, and this correlated with complete loss of nucleotide-dependent oligomerization. Based on the multiply substituted proteins, several single amino acid substitutions were generated by site-directed mutagenesis. The singly substituted proteins retained the ability to oligomerize and detect substrates. Intriguingly, some derivatives of Hsp104 functioned well in prion propagation and multiple stress tolerance but failed to protect yeast from extreme thermal stress. We demonstrate that these proteins co-aggregate in the presence of other thermolabile proteins during heat treatment both in vitro and in vivo suggesting a novel mechanism for uncoupling the function of Hsp104 in acute severe heat shock from its functions at moderate temperatures.     

Energy Landscapes of Protein Aggregation and Conformation Switching in Intrinsically Disordered Proteins

The protein folding problem was apparently solved recently by the advent of a deep learning method for protein structure prediction called AlphaFold. However, this program is not able to make predictions about the protein folding pathways. Moreover, it only treats about half of the human proteome, as the remaining proteins are intrinsically disordered or contain disordered regions. By definition these proteins differ from natively folded proteins and do not adopt a properly folded structure in solution. However these intrinsically disordered proteins (IDPs) also systematically differ in amino acid composition and uniquely often become folded upon binding to an interaction partner. These factors preclude solving IDP structures by current machine-learning methods like AlphaFold, which also cannot solve the protein aggregation problem, since this meta-folding process can give rise to different aggregate sizes and structures. An alternative computational method is provided by molecular dynamics simulations that already successfully explored the energy landscapes of IDP conformational switching and protein aggregation in multiple cases. These energy landscapes are very different from those of ‘simple’ protein folding, where one energy funnel leads to a unique protein structure. Instead, the energy landscapes of IDP conformational switching and protein aggregation feature a number of minima for different competing low-energy structures. In this review, I discuss the characteristics of these multifunneled energy landscapes in detail, illustrated by molecular dynamics simulations that elucidated the underlying conformational transitions and aggregation processes.     

Determination of the solution structures of domains II and III of protein G from Streptococcus by 1H nuclear magnetic resonance.

We have used 1H nuclear magnetic resonance spectroscopy to determine the solution structures of two small (61 and 64 residue) immunoglobulin G (IgG)-binding domains from protein G, a cell-surface protein from Streptococcus strain G148. The two domains differ in sequence by four amino acid substitutions, and differ in their affinity for some subclasses of IgG. The structure of domain II was determined using a total of 478 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints; that of domain III was determined using a total of 445 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints. A protocol which involved distance geometry, simulated annealing and restrained molecular dynamics was used to determine ensembles of 40 structures consistent with these restraints. The structures are found to consist of an alpha-helix packed against a four-stranded antiparallel-parallel-antiparallel beta-sheet. The structures of the two domains are compared to each other and to the reported structure of a similar domain from a protein G from a different strain of Streptococcus. We conclude that the difference in affinity of domains II and III for IgG is due to local changes in amino acid side-chains, rather than a more extensive change in conformation, suggesting that one or more of the residues which differ between them are directly involved in interaction with IgG.     

The influence of amino acid substitutions on the conformational energy of cytochrome c.

Conformational energies have been evaluated for each of the staggered side-chain conformations associated with the 261 amino acid substitutions known to occur among 60 eucaryotic species. At least 86% of these substitutions can be sterically accommodated (one at a time) within the structure of horse-heart cytochrome c resulting from conformational energy refinement. Simultaneous incorporation of all pertinent amino acid substitutions found in eight representative species into the refined horse-heart structure is also shown to be sterically possible, with few exceptions. In two cases (Pekin duck cytochrome with 10 substitutions and Samia cynthia cytochrome with 24 substitutions), all substitutions could be readily incorporated, and the total energies associated with their computed structures differed by less than 10 kcal/mol from that of horse-heart cytochrome c. In the cytochromes from rattlesnake (22 substitutions), tuna (18 substitutions), and Neurospora crassa (36 substitutions), tyrosine could not be substituted for phenylalanine at position 46, within the constraints of the calculations. However, when all of the remaining substitutions were incorporated into these three cytochromes, their computed conformational energies differed by less than 30 kcal/mol from that of horse-heart cytochrome c. Between two and four amino acid substitutions cause high energies in the cytochromes from human, baker's yeast, and cotton seed, but all of the remaining substitutions are consistent with a low energy conformation. These results suggest that the structures of homologous proteins may be even more similar than has previously been recognized. Substitutions of all possible amino acid types at the invariant positions (where all eucaryotic cytochromes c bear the same amino acid) have revealed some cases where different amino acids can be accommodated, thus demonstrating that the biological constraints on amino acid substitutions are often different from the purely steric constraints investigated in this work.     

Two-dimensional infrared spectroscopy: a promising new method for the time resolution of structures.

Recently, new methods for determining time-evolving structures using infrared analogs of NMR spectroscopy have been introduced that have outstanding potential in structural biology. Already, within the past two years, structures of dipeptides, tripeptides and pentapeptides have been determined on much faster timescales than the conformational dynamics. Also, two-dimensional infrared correlation spectra of some proteins and isotopically edited alanine-rich helices have been examined.     

Comparison of the computed three-dimensional structures of oncogenic forms (bound to GDP) of theras-Gene-Encoded p21 protein with the structure of the normal (non-transforming) wild-type protein

Theras-oncogene-encoded p21 protein becomes oncogenic if amino acid substitutions occur at critical positions in the polypeptide chain. The most commonly found oncogenic forms contain Val in place of Gly 12 or Leu in place of Gln 61. To determine the effects of these substitutions on the three-dimensional structure of the whole p21 protein, we have performed molecular dynamics calculations on each of these three proteins bound to GDP and magnesium ion to compute the average structures of each of the three forms. Comparisons of the computed average structures shows that both oncogenic forms with Val 12 and Leu 61 differ substantially in structure from that of the wild type (containing Gly 12 and Gln 61) in discrete regions: residues 10–16, 32–47, 55–74, 85–89, 100–110, and 119–134. All of these regions occur in exposed loops, and several of them have already been found to be involved in the cellular functioning of the p21 protein. These regions have also previously been identified as the most flexible domains of the wild-type protein and have been bound to be the same ones that differ in conformation between transforming and nontransforming p21 mutant proteins neither of which binds nucleotide. The two oncogenic forms have similar conformations in their carboxyl-terminal domains, but differ in conformation at residues 32–47 and 55–74. The former region is known to be involved in the interaction with at least three downstream effector target proteins. Thus, differences in structure between the two oncogenic proteins may reflect different relative affinities of each oncogenic protein for each of these effector targets. The latter region, 55–74, is known to be a highly mobile segment of the protein. The results strongly suggest that critical oncogenic amino acid substitutions in the p21 protein cause changes in the structures of vital domains of this protein.