Restricted Conformational Space Research Articles

Conformational flexibility of a free and TCR-bound pMHC-I protein investigated by long-term molecular dynamics simulations

BackgroundMajor histocompatibility complexes (MHCs) play a crucial role in the cell-mediated adaptive immune response as they present antigenic peptides (p) which are recognized by host T cells through a complex formation of the T cell receptor (TCR) with pMHC. In the present study, we report on changes in conformational flexibility within a pMHC molecule upon TCR binding by looking at molecular dynamics (MD) simulations of the free and the TCR-bound pMHC-I protein of the LC13-HLA-B*44:05-pEEYLQAFTY complex.ResultsWe performed long-term MD simulations with a total simulation time of 8 µs, employing 10 independent 400 ns replicas for the free and the TCR-bound pMHC system. Upon TCR ligation, we observed a reduced dynamic flexibility in the central residues of the peptide and the MHC α1-helix, altered occurrences of hydrogen bonds between the peptide and the MHC, a reduced conformational entropy of the peptide-binding groove, as well as a decreased solvent accessible surface area.ConclusionsIn summary, our results from 8 µs MD simulations indicate a restricted conformational space of the MHC peptide-binding groove upon TCR ligation and suggest a minimum simulation time of approximately 100 ns for biomolecules of comparable complexity to draw meaningful conclusions. Given the relatively long total simulation time, our results contribute to a more detailed view on conformational flexibility properties of the investigated free and TCR-bound pMHC-I system.

Total Synthesis of Natural Products Containing Enamine or Enol Ether Derivatives.

Enamine and enol ethers are nucleophilic functional groups that are well known to most chemists. When enamine or enol ethers are present in natural products, they are nearly exclusively found as derivatives having a direct connection to electron-withdrawing groups for stabilization, and the resulting larger entities, such as enamides or enol acylates, can be further extended or modified in the framework of natural products. The restricted conformational space that is associated with even simple enamine and enol ether derivatives can be a strong determinant of the overall molecular structure, and the more polarized derivatives can endow some natural products with electrophilic properties and thus facilitate covalent interactions with biological targets.In this Account, I describe our efforts (published since 2016) to prepare natural products from several different classes that all feature enamine or enol ether derivatives as key functionalities. Our choice of targets has been guided by a desire to illuminate unknown biological mechanisms associated with the compounds or, alternatively, to improve upon known biological activities that appear to be promising from a biomedical perspective. In the present text, however, the exclusive focus will be on the syntheses.First, I will discuss the basic properties of the functional groups and briefly present a small collection of illustrative and inspirational examples from the literature for their construction in different complex settings. Next, I will provide an overview of our work on the macrocyclic APD-CLD natural products, rakicidin A and BE-43547A1, involving the development of an efficient macrocyclization strategy and the development of methods to construct the hallmark APD group: a modified enamide. The synthesis of the meroterpenoid strongylophorine-26 is discussed next, where we developed an oxidative quinone methoxylation to build a vinylogous ester group in the final step of the synthesis and employed FeCl3-mediated cascade reactions for the rapid assembly of the overall scaffold to enable a short semisynthesis from isocupressic acid. An efficient core scaffold assembly was also in focus in our synthesis of the alkaloid streptazone A with the signature enaminone system being assembled through a rhodium-catalyzed Pauson-Khand reaction. Sequential, site-selective redox manipulations were developed to arrive at strepatzone A and additional members of the natural product family. Finally, I discuss our work to prepare analogs of complex polyether ionophores featuring functionalized tetronic acids as cation-binding groups. A method for the construction of a suitably protected chloromethylidene-modified tetronate is presented which enabled its installation in the full structure through a C-acylation reaction. This work exemplifies how components of abundant polyether ionophores can be recycled and used to access new structures which may possess enhanced biological activities.

Comparative Analyses of the Conformational Dynamics Between the Soluble and Membrane-Bound Cytokine Receptors

Cytokine receptors receive extracellular cues by binding with cytokines to transduce a signaling cascade leading to gene transcription in cells. Their soluble isoforms, functioning as decoy receptors, contain only the ectodomain. Whether the ectodomains of cytokine receptors at the membrane exhibit different conformational dynamics from their soluble forms is unknown. Using Stimulation-2 (ST2) as an example, we performed microsecond molecular dynamics (MD) simulations to study the conformational dynamics of the soluble and the membrane-bound ST2 (sST2 and ST2). Combined use of accelerated and conventional MD simulations enabled extensive sampling of the conformational space of sST2 for comparison with ST2. Using the interdomain loop conformation as the reaction coordinate, we built a Markov State Model to determine the slowest implied timescale of the conformational transition in sST2 and ST2. We found that the ectodomain of ST2 undergoes slower conformational relaxation but exhibits a faster rate of conformational transition in a more restricted conformational space than sST2. Analyses of the relaxed conformations of ST2 further suggest important contributions of interdomain salt-bridge interactions to the stabilization of different ST2 conformations. Our study elucidates differential conformational properties between sST2 and ST2 that may be exploited for devising strategies to selectively target each isoform.

Fe(II)/Fe(III) Redox Process Can Significantly Modulate the Conformational Dynamics and Electrostatics of Pirin in NF-κB Regulation.

Pirin is an iron (Fe)-dependent regulatory protein of nuclear factor κB (NF-κB) transcription factors. Binding studies have suggested that the oxidative state of iron plays a crucial role in modulating the binding of Pirin to NF-κB p65, in turn enhancing the binding of p65 to DNA. The Fe(III) form of Pirin is the active form and binds to NF-κB, whereas the Fe(II) form does not bind to NF-κB. However, the surprising consequence of a single charge perturbation in the functional modulation of NF-κB is not well understood. Here, we use quantum mechanical calculations and microsecond-long molecular dynamics simulations to explore the free-energy landscapes of the Fe(II) and Fe(III) forms of Pirin. We show that the restricted conformational space and electrostatic complementarity of the Fe(III) form of Pirin are crucial for binding and regulation of NF-κB. Our results suggest that a subtle single-electron redox trigger could significantly modulate the conformational dynamics and electrostatics of proteins in subcellular allosteric regulatory processes.

Distal substitutions drive divergent DNA specificity among paralogous transcription factors through subdivision of conformational space

Many genomes contain families of paralogs--proteins with divergent function that evolved from a common ancestral gene after a duplication event. To understand how paralogous transcription factors evolve divergent DNA specificities, we examined how the glucocorticoid receptor and its paralogs evolved to bind activating response elements [(+)GREs] and negative glucocorticoid response elements (nGREs). We show that binding to nGREs is a property of the glucocorticoid receptor (GR) DNA-binding domain (DBD) not shared by other members of the steroid receptor family. Using phylogenetic, structural, biochemical, and molecular dynamics techniques, we show that the ancestral DBD from which GR and its paralogs evolved was capable of binding both nGRE and (+)GRE sequences because of the ancestral DBD's ability to assume multiple DNA-bound conformations. Subsequent amino acid substitutions in duplicated daughter genes selectively restricted protein conformational space, causing this dual DNA-binding specificity to be selectively enhanced in the GR lineage and lost in all others. Key substitutions that determined the receptors' response element-binding specificity were far from the proteins' DNA-binding interface and interacted epistatically to change the DBD's function through DNA-induced allosteric mechanisms. These amino acid substitutions subdivided both the conformational and functional space of the ancestral DBD among the present-day receptors, allowing a paralogous family of transcription factors to control disparate transcriptional programs despite high sequence identity.

An engineered scorpion toxin analogue with improved Kv1.3 selectivity displays reduced conformational flexibility.

The voltage-gated Kv1.3 K+ channel plays a key role in the activation of T lymphocytes. Kv1.3 blockers selectively suppress immune responses mediated by effector memory T cells, which indicates the great potential of selective Kv1.3 inhibitors in the therapy of certain autoimmune diseases. Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin is a high affinity blocker of Kv1.3, but also blocks Kv1.2 with similar potency. We designed and produced three AnTx variants: ([F32T]-AnTx, [N17A]-AnTx, [N17A/F32T]-AnTx) using solid-phase synthesis with the goal of improving the selectivity of the toxin for Kv1.3 over Kv1.2 while keeping the high affinity for Kv1.3. We used the patch-clamp technique to determine the blocking potency of the synthetic toxins on hKv1.3, mKv1.1, hKv1.2 and hKCa3.1 channels. Of the three variants [N17A/F32T]-AnTx maintained the high affinity of the natural peptide for Kv1.3 but became more than 16000-fold selective over Kv1.2. NMR data and molecular dynamics simulations suggest that the more rigid structure with restricted conformational space of the double substituted toxin compared to the flexible wild-type one is an important determinant of toxin selectivity. Our results provide the foundation for the possibility of the production and future therapeutic application of additional, even more selective toxins targeting various ion channels.

Enthalpy-Entropy Compensation and Isoequilbria Implicate Solvation as the Driving Force for Amino Acid Conformational Propensity

The existence of residual structure in intrinsically disordered proteins (IDPs) raise questions regarding how disorder/order preferences are encoded in the peptide sequence. In addition to formation of local secondary structure, individual amino acids sample unique and restricted conformational space, contrary to the predictions of the random coil model. To understand the thermodynamic driving forces underlying these newly discovered amino acid conformational biases, we have examined the thermodynamics governing these intrinsic conformational propensities in a series of host-guest experiments on model “GxG” peptides in aqueous solution. Global two-state pPII/β thermodynamic analysis of UVCD and HNMR data revealed the existence of a nearly exact enthalpy-entropy compensation for the pPII-β equilibrium for all residues. The obtained ΔH/ΔS values exhibit a nearly perfect linear relationship reflecting compensation temperature of 295K ± 2 K. We identified iso-equilibria for two subsets of the investigated peptides, indicating that pPII and β propensities of most residues become similar at physiological temperature, in spite of rather large differences between respective enthalpic and entropic differences. Interestingly, alanine, aspartic acid, and threonine, which have largely biased intrinsic conformational propensities, are the only amino acid residues that do not share iso-equilibria. We assign the isoequilibria to a common enthalpy-entropy compensation which occurs in the hydration shell of side chains with considerable hydrophobic content. Aspartic acid and threonine deviate from this behavior owing to their capability to form hydrogen bonds with the backbone even in extended structures, whereas the solvation energy of alanine is dominated by backbone - solvent interactions.

High-Temperature Reactivity of the Strongly Electrophilic Pristine 9H-9-Borafluorene

Temperature treatment (120 °C, C6H6 or toluene, sealed glass ampule) of the C1-symmetric dimer of 9H-9-borafluorene (1)2 leads to the ring-opened dimer 1,2:1,2-bis(2,2′-biphenylylene)diborane(6) (2), 2,2′-bis(9-borafluoren-9-yl)biphenyl (3), and 1,2-(2,2′-biphenylylene)diborane(6) (4). 2 readily crystallizes from the thermolysis mixture in 34% yield. The ditopic borane 3 does not form in preparatively useful quantities but has been made accessible through salt metathesis between 9-bromo-9-borafluorene and donor-free 2,2′-dilithiobiphenyl in almost quantitative yields. Even at 330 K, the 1H and 13C{1H} NMR spectra of 3 are broadened almost beyond interpretability. X-ray crystallography and molecular modeling studies on 3 indicate a highly restricted conformational space. 3 binds THF and SMe2 only weakly but readily forms a stable B–N adduct upon addition of 2 equiv of pyridine (py). The resulting 3·py2 (X-ray crystal structure) shows perfectly well resolved NMR spectra.

Restricted N-glycan Conformational Space in the PDB and Its Implication in Glycan Structure Modeling

Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycan's crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures.

N-Glycan Structure Modeling and in Silico Glycosylation: Template-Based Structure Prediction of Carbohydrate Structures of Glycoconjugates

Obtaining the crystal structure of glycoconjugate is challenging due to the flexibility of the carbohydrate chains. Alternatively, computational modeling, which combines the primary sequence information of glycans determined by the mass spectrometry and known N-glycan structure, is an appealing approach. Here we present a survey of N-glycan structures of 35 different glycan sequences in the PDB, showing that N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background. This suggests that N-glycan chains can be confidently modeled to a glycoprotein if there exists a template N-glycan structure whose parent glycoprotein shares sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins have not shown significant structure similarity. However, despite that the global N-glycan structures are different, the internal substructure of those N-glycans found on the non-homologous glycoproteins, particularly, the substructure that are closer to the protein, showed significantly similar structure. Increased interaction with protein might be responsible to the restricted conformational space of N-glycan chains. Our results so far suggest that computational structure prediction of N-glycan portion of glycoconjugate using structure database would be effective, but different approaches must be needed depending on the availability of template structure. In addition, we also present a database for PDB glycan structural fragments (substructures) as well as PDB glycan-protein database, which are useful for glycan structure modeling.

Design of Cyclic Peptides Featuring Proline Predominantly in the cis Conformation under Physiological Conditions

Turns are secondary-structure elements that are omnipresent in natively folded polypeptide chains. A large variety of four-residue β-turns exist, which differ mainly in the backbone dihedral angle values of the two central residues i+1 and i+2. The βVI-type turns are of particular biological interest because the i+2 residue is always a proline in the cis conformation and might thus serve as target of peptidyl prolyl cis/trans isomerases (PPIases). We have designed cyclic hexapeptides containing two proline residues that predominantly adopt the cis conformation in aqueous solution. NMR data and MD calculations indicated that the cyclic peptide sequences c-(-DXaa-Ser-Pro-DXaa-Lys-Pro-) result in highly symmetric backbone structures when both prolines are in the cis conformation and the D-amino acids are either alanine or phenylalanine residues. Replacement of the serine residue either by phosphoserine or by tyrosine compromises this symmetry, but further increases the cis conformation content of both prolines. As a result, we obtained a cyclic hexapeptide that exists almost exclusively as the cis-Pro/cis-Pro conformer but shows no cis/trans interconversion even in the presence of the PPIase Pin1, apparently due to an energetically quite favorable but highly restricted conformational space.

Effects of CpG Methylation on the Structure and Assembly of a Nucleosome

We studied the mechanism of nucleosome assembly with a histone chaperon Nap1 and the effects of CpG methylation on the structure and the assembly of nucleosomes with fluorescence techniques. Based on single molecule and ensemble fluorescence measurements, we found that CpG methylation induces overwrapping of nucleosomal DNA accompanied by a topology change. Real-time monitoring of nucleosome assembly with the Selex 601 sequence and Nap1 revealed that nucleosome assembly takes place largely in four steps. Reduced kinetic rates for DNA wrapping during the assembly upon CpG methylation strongly suggest that DNA becomes rigid with methylated CpG dinucleotides. Free energy profiles of the nucleosome assembly process were also constructed based on the results. According to the profiles, all the intermediate states are kinetically stabilized but thermodynamically destabilized upon CpG methylation. The decreased kinetic rates upon CpG methylation may be due to the elevated transition state energy caused by the restricted conformational space of the rigidified DNA in the transition states. Based on these results, we propose a hypothesis where rigidified DNA upon CpG methylation inhibits nucleosome disassembly by destabilizing the transition states.

Structure-inhibitory activity relationships of pyrrolnitrin analogues on its biosynthesis

Pyrrolnitrin is a bacterial metabolite, served as a natural lead of agricultural fungicides. In a previous study, fenpiclonil was proven to inhibit the oxidative transformation of aminopyrrolnitrin to pyrrolnitrin, catalyzed by aminopyrrolnitrin oxidase (PrnD). This monooxygenase has an interesting catalytic activity of selective oxidation of aromatic amines, rather than aliphatic amines. However, its structural details are not well understood. In this study, various analogues of pyrrolnitrin were prepared to elucidate the structures of active site of PrnD through structure-activity relationships. In vivo pyrrolnitrin biosynthesis inhibition was determined with Burkholderia sp. O33 and Pseudomonas fluorescens Pf-5. Quantitative analysis of pyrrolnitrin and precursors indicates that 2,3-disubstituted phenyl at 3rd carbon and small substituents at 4th carbon of pyrrole are strictly required to give strong inhibitory effects. In addition, dissociable proton of pyrrole is also critical for inhibitory activity. Molecular simulation with homology-based PrnD model suggests a highly restricted conformational space in active site. The results may help more detailed understanding of this unusual enzyme. In addition, the information will be useful for the development of novel fungicide, compatible with pyrrolnitrin-producing bacterium.

Starch phosphorylation—Maltosidic restrains upon 3′‐ and 6′‐phosphorylation investigated by chemical synthesis, molecular dynamics and NMR spectroscopy

Phosphorylation is the only known in vivo substitution of starch, yet no structural evidence has been provided to explain its implications of the amylosidic backbone and its stimulating effects on starch degradation in plants. In this study, we provide evidence for a major influence on the glucosidic bond in starch specifically induced by the 3-O-phosphate. Two phosphorylated maltose model compounds were synthesized and subjected to combined molecular dynamics (MD) studies and 950 MHz NMR studies. The two phosphorylated disaccharides represent the two possible phosphorylation sites observed in natural starches, namely maltose phosphorylated at the 3'- and 6'-position (maltose-3'-O-phosphate and maltose-6'-O-phosphate). When compared with maltose, both of the maltose-phosphates exhibit a restricted conformational space of the alpha(1-->4) glycosidic linkage. When maltose is phosphorylated in the 3'-position, MD and NMR show that the glucosidic space is seriously restricted to one narrow potential energy well which is strongly offset from the global potential energy well of maltose and almost 50 degrees degrees from the Phi angle of the alpha-maltose crystal structure. The driving force is primarily steric, but the configuration of the structural waters is also significantly altered. Both the favored conformation of the maltose-3'-phosphate and the maltose-6'-phosphate align well into the 6-fold double helical structure of amylopectin when the effects on the glucosidic bond are not taken into account. However, the restrained geometry of the glucosidic linkage of maltose-3'-phosphate cannot be accommodated in the helical structure, suggesting a major local disturbing effect, if present in the starch granule semi-crystalline lattice.

Synthesis of a conformationally restricted polyoxygenated crownophane

A new polyoxygenated crownophane has been synthesized from syringaldehyde and diethyleneglycol by means of McMurry pinacol reaction, whereas ring closing metathesis with Grubbs’ catalyst failed in producing the macrocyclization to the corresponding stilbenophane. The NMR data of the crownophane show a restricted conformational space accessible to the phenyl rings.

Solution Structure of Prosurvival Mcl-1 and Characterization of Its Binding by Proapoptotic BH3-only Ligands

The B cell lymphoma-2 (Bcl-2) homologs myeloid cell leukemia-1 (Mcl-1) and A1 are prosurvival factors that selectively bind a subset of proapoptotic Bcl homology (BH) 3-only proteins. To investigate the molecular basis of the selectivity, we determined the solution structure of the C-terminal Bcl-2-like domain of Mcl-1. This domain shares features expected of a prosurvival Bcl-2 protein, having a helical fold centered on a core hydrophobic helix and a surface-exposed hydrophobic groove for binding its cognate partners. A number of residues in the binding groove differentiate Mcl-1 from its homologs, and in contrast to other Bcl-2 homologs, Mcl-1 has a binding groove in a conformation intermediate between the open structures characterized by peptide complexes and the closed state observed in unliganded structures. Mutagenesis of potential binding site residues was used to probe the contributions of groove residues to the binding properties of Mcl-1. Although mutations in Mcl-1 had little impact on binding, a single mutation in the BH3-only ligand Bad enabled it to bind both Mcl-1 and A1 while retaining its binding to Bcl-2, Bcl-xL, and Bcl-w. Elucidating the selective action of certain BH3-only ligands is required for delineating their mode of action and will aid the search for effective BH3-mimetic drugs.

Naturally-occurring Modification Restricts the Anticodon Domain Conformational Space of tRNAPhe

Post-transcriptional modifications contribute chemistry and structure to RNAs. Modifications of tRNA at nucleoside 37, 3′-adjacent to the anticodon, are particularly interesting because they facilitate codon recognition and negate translational frame-shifting. To assess if the functional contribution of a position 37-modified nucleoside defines a specific structure or restricts conformational flexibility, structures of the yeast tRNAPhe anticodon stem and loop (ASLPhe) with naturally occurring modified nucleosides differing only at position 37, ASLPhe-(Cm32,Gm34,m5C40), and ASLPhe-(Cm32,Gm34,m1G37,m5C40), were determined by NMR spectroscopy and restrained molecular dynamics. The ASL structures had similarly resolved stems (RMSD∼0.6Å) of five canonical base-pairs in standard A-form RNA. The “NOE walk” was evident on the 5′ and 3′ sides of the stems of both RNAs, and extended to the adjacent loop nucleosides. The NOESY cross-peaks involving U33 H2′ and characteristic of tRNA's anticodon domain U-turn were present but weak, whereas those involving the U33 H1′ proton were absent from the spectra of both ASLs. However, ASLPhe-(Cm32,Gm34,m1G37,m5C40) exhibited the downfield shifted 31P resonance of U33pGm34 indicative of U-turns; ASLPhe-(Cm32,Gm34,m5C40) did not. An unusual “backwards” NOE between Gm34 and A35 (Gm34/H8 to A35/H1′) was observed in both molecules. The RNAs exhibited a protonated A+38 resulting in the final structures having C32·A+38 intra-loop base-pairs, with that of ASLPhe-(Cm32,Gm34,m1G37,m5C40) being especially well defined. A single family of low-energy structures of ASLPhe-(Cm32,Gm34, m1G37,m5C40) (loop RMSD 0.98Å) exhibited a significantly restricted conformational space for the anticodon loop in comparison to that of ASLPhe-(Cm32,Gm34,m5C40) (loop RMSD 2.58Å). In addition, the ASLPhe-(Cm32,Gm34,m1G37,m5C40) average structure had a greater degree of similarity to that of the yeast tRNAPhe crystal structure. A comparison of the resulting structures indicates that modification of position 37 affects the accuracy of decoding and the maintenance of the mRNA reading frame by restricting anticodon loop conformational space.

Structure, function, and dynamics of the dimerization and DNA-binding domain of oncogenic transcription factor v-Myc

The protein product (c-Myc) of the protooncogene c-myc is a transcriptional regulator playing a key role in cellular growth, differentiation, and apoptosis. Deregulated myc genes, like the transduced retroviral v-myc allele, are oncogenic and cause cell transformation. The C-terminal bHLHZip domain of v-Myc, encompassing protein dimerization (helix-loop-helix, leucine zipper) and DNA contact (basic region) surfaces, was expressed in bacteria as a highly soluble p15v-myc recombinant protein. Dissociation constants (Kd) for the heterodimer formed with the recombinant bHLHZip domain of the Myc binding partner Max (p14max) and for the Myc-Max-DNA complex were estimated using circular dichroism (CD) spectroscopy and quantitative electrophoretic mobility shift assay (EMSA). Multi-dimensional NMR spectroscopy was used to characterize the solution structural and dynamic properties of the v-Myc bHLHZip domain. Significant secondary chemical shifts indicate the presence of two separated α-helical regions. The C-terminal leucine zipper region forms a compact α-helix, while the N-terminal basic region exhibits conformational averaging with substantial α-helical content. Both helices lack stabilizing tertiary side-chain interactions and represent exceptional examples for loosely coupled secondary structural segments in a native protein. These results and CD thermal denaturation data indicate a monomeric state of the v-Myc bHLHZip domain. The 15N relaxation data revealed backbone mobilities which corroborate the existence of a partially folded state, and suggest a “beads-on-a-string” motional behaviour of the v-Myc bHLHZip domain in solution. The preformation of α-helical regions was confirmed by CD thermal denaturation studies, and quantification of the entropy changes caused by the hydrophobic effect and the reduction of conformational entropy upon protein dimerization. The restricted conformational space of the v-Myc bHLHZip domain reduces the entropy penalty associated with heterodimerization and allows rapid and accurate recognition by the authentic Myc binding partner Max.

Ab initio protein structure prediction using physicochemical potentials and a simplified off-lattice model.

This study describes a computational method for ab inito protein structure prediction. Protein conformation has been modeled by using six optimized backbone torsion angles and fixed side chains approximating rotationally averaged real side chains. The approximations aim to keep complexity of the structure description to a minimum without seriously compromising the accuracy of the structural representation. An evolutionary Monte Carlo algorithm has been developed to search through this restricted conformational space to locate low-energy protein structures. A simple physicochemical force field has been developed to assess the energies of different conformations within this structural description. The corresponding residue interaction energies are based on hydrophobic, hydrophilic, steric, and hydrogen-bonding potentials. The search procedure has been used to locate native energy minima from primary sequence alone. The 3-D structures of polypeptides up to 38 residues with both beta and alpha secondary structural elements have been accurately predicted. The search procedure has been found to be highly efficient and follows an energetically and structurally plausible pathway to locate native populations. The simple force field described in the study has been compared with a more complex all-atom model and been found to be similarly effective in predicting the structures of proposed independent folding units. Proteins 2001;43:186-202.

Photomodulation of conformational states. I. Mono- and bicyclic peptides with (4-amino)phenylazobenzoic acid as backbone constituent.

The thioredoxin reductase active-site fragment H-Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-OH [134-141], which is known for its high tendency to assume an almost identical conformation as in the intact enzyme, was backbone cyclized with the photoresponsive (4-amino)phenylazobenzoic acid (APB) to produce a monocyclic and disulfide-bridged bicyclic APB-peptide. Light-induced reversible cis/trans isomerization occurs at identical extents in both the linear and the two cyclic forms. Nuclear magnetic resonance conformational analysis clearly revealed that in the bicyclic APB-peptide both as a trans- and cis-azo-isomer the constraints imparted by the bicyclic structure do not allow the molecule to relax into a defined low energy conformation, thus making the molecule a frustrated system that flip-flops between multiple conformational states. Conversely, the monocyclic APB peptide folds into a well-defined lowest energy structure as a trans-azo-isomer, which upon photoisomerization to the cis-azo configuration relaxes into a less restricted conformational space. First femtosecond spectroscopic analysis of the dynamics of the photoreaction confirm a fast first phase on the femtosecond time scale related to the cis/trans isomerization of the azobenzene moiety followed by a slower phase in the picosecond time scale that involves an adjustment of the peptide backbone. Due to the well- defined photoresponsive two-state transition of this monocyclic peptide molecule, it represents a model system well suited for studying the ultrafast dynamics of conformational transitions by time-resolved spectroscopy.