Stimulation of the HIV‐1 integrase enzymatic activity and cDNA integration by a peptide derived from the integrase protein

Here we describe the features of a peptide that was selected from the human immunodeficiency virus Type 1 (HIV‐1) Integrase (IN) peptide library which interacts with both, the viral Rev and IN proteins. Because of its ability to stimulate the IN enzymatic activity this peptide was designated INS (IN stimulatory). Modification of its amino acid sequence revealed that replacement of its C‐terminal lysine by glutamic acid (INS K188E) converts it from a stimulatory peptide to an inhibitory one. Both peptides promoted the dissociation of a previously described complex formed between Rev and IN whose formation results in IN inactivation. INS and INS K188E penetrated HIV‐1‐infected cells and caused stimulation and inhibition of viral genome integration, respectively. Using cultured cells infected with a ΔRev HIV revealed that INS can directly activate the viral IN. These results suggest that the stimulatory effect of INS in wild‐type virus‐infected cells is due to a dual effect: it dissociates the inactive Rev‐IN complex and directly activates the free IN. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 740–751, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Viral structural transition mechanisms revealed by multiscale molecular dynamics/order parameter extrapolation simulation

On the basis of an all‐atom multiscale analysis theory of nanosystem dynamics, a multiscale molecular dynamics/order parameter extrapolation (MD/OPX) approach has recently been developed. It accelerates MD for long‐time simulation of large bionanosystems and addresses rapid atomistic fluctuations and slowly varying coherent dynamics simultaneously. In this study, MD/OPX is optimized and implemented to simulate viral capsid structural transitions. Specifically, 200 ns MD/OPX simulation of the swollen state of cowpea chlorotic mottle virus capsid reveals that it undergoes significant energy‐driven shrinkage in vacuum, which is a symmetry‐breaking process involving local initiation and front propagation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 61–73, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Insight into the stability of cross‐β amyloid fibril from molecular dynamics simulation

Amyloid fibrils are considered to play causal roles in the pathogenesis of amyloid‐related degenerative diseases such as Alzheimer's disease, type II diabetes mellitus, the transmissible spongiform encephalopathies, and prion disease. The mechanism of fibril formation is still hotly debated and remains an important open question. In this study, we utilized molecular dynamics (MD) simulation to analyze the stability of hexamer for eight class peptides. The MD results suggest that VEALYL and MVGGVV‐1 are the most stable ones, then SNQNNY, followed by LYQLEN, MVGGVV‐2, VQIVYK, SSTSAA, and GGVVIA. The statistics result indicates that hydrophobic residues play a key role in stabilizing the zipper interface. Single point and two linkage mutants of MVGGVV‐1 confirmed that both Met1 and Val2 are key hydrophobic residues. This is consistent with the statistics analysis. The stability results of oligomer for MVGGVV‐1 suggest that the intermediate state should be trimer (3‐0) and tetramer (2‐2). These methods can be used in stabilization study of other amyloid fibril. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 578–586, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Peptidic modulators of protein‐protein interactions: Progress and challenges in computational design

With the decline in productivity of drug‐development efforts, novel approaches to rational drug design are being introduced and developed. Naturally occurring and synthetic peptides are emerging as novel promising compounds that can specifically and efficiently modulate signaling pathways in vitro and in vivo. We describe sequence‐based approaches that use peptides to mimic proteins in order to inhibit the interaction of the mimicked protein with its partners. We then discuss a structure‐based approach, in which protein‐peptide complex structures are used to rationally design and optimize peptidic inhibitors. We survey flexible peptide docking techniques and discuss current challenges and future directions in the rational design of peptidic inhibitors. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 505–513, 2009. This article was originally published online as an accepted preprint. The “Published Online”date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The role of the Val57 amino‐acid residue in the hinge loop of the human cystatin C. Conformational studies of the beta2‐L1‐beta3 segments of wild‐type human cystatin C and its mutants

Human cystatin C (HCC) is one of the amyloidogenic proteins to be shown to oligomerize via a three‐dimensional domain swapping mechanism. This process precedes the formation of a stable dimer and proceeds particularly easily in the case of the L68Q mutant. According to the proposed mechanism, dimerization of the HCC precedes conformational changes within the β2 and β3 strands. In this article, we present conformational studies, using circular dichroism and MD methods, of the β2‐L1‐β3 (His43‐Thr72) fragment of the HCC involved in HCC dimer formation. We also carried out studies of the β2‐L1‐β3 peptide, in which the Val57 residue was replaced by residues promoting β‐turn structure formation (Asp, Asn, or Pro). The present study established that point mutation could modify the structure of the L1 loop in the β‐hairpin peptide. Our results showed that the L1 loop in the peptide excised from human cystatin C is broader than that in cystatin C. In the HCC protein, broadening of the L1 loop together with the unfavorable L68Q mutation in the hydrophobic pocket could be a force sufficient to cause the partial unfolding and then the opening of HCC or its L68Q mutant structure for further dimerization. We presume further that the Asp57 and Asn57 mutations in the L1 loop of HCC could stabilize the closed form of HCC, whereas the Pro57 mutation could lead to the opening of the HCC structure and then to dimer/oligomer formation. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 373–383, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Rationally designed dehydroalanine (ΔAla)‐containing peptides inhibit amyloid‐β (Aβ) peptide aggregation

Among the pathological hallmarks of Alzheimer's disease (AD) is the deposition of amyloid‐β (Aβ) peptides, primarily Aβ (1–40) and Aβ (1–42), in the brain as senile plaques. A large body of evidence suggests that cognitive decline and dementia in AD patients arise from the formation of various aggregated forms of Aβ, including oligomers, protofibrils and fibrils. Hence, there is increasing interest in designing molecular agents that can impede the aggregation process and that can lead to the development of therapeutically viable compounds. Here, we demonstrate the ability of the specifically designed α,β‐dehydroalanine (ΔAla)‐containing peptides P1 (K‐L‐V‐F‐ΔA‐I‐ΔA) and P2 (K‐F‐ΔA‐ΔA‐ΔA‐F) to inhibit Aβ (1–42) aggregation. The mechanism of interaction of the two peptides with Aβ (1–42) seemed to be different and distinct. Overall, the data reveal a novel application of ΔAla‐containing peptides as tools to disrupt Aβ aggregation that may lead to the development of anti‐amyloid therapies not only for AD but also for many other protein misfolding diseases. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 456–465, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Exploration of human serum albumin binding sites by docking and molecular dynamics flexible ligand–protein interactions

Five‐nanosecond molecular dynamics (MD) simulations were performed on human serum albumin (HSA) to study the conformational features of its primary ligand binding sites (I and II). Additionally, 11 HSA snapshots were extracted every 0.5 ns to explore the binding affinity (K_d) of 94 known HSA binding drugs using a blind docking procedure. MD simulations indicate that there is considerable flexibility for the protein, including the known sites I and II. Movements at HSA sites I and II were evidenced by structural analyses and docking simulations. The latter enabled the study and analysis of the HSA–ligand interactions of warfarin and ketoprofen (ligands binding to sites I and II, respectively) in greater detail. Our results indicate that the free energy values by docking (K_d observed) depend upon the conformations of both HSA and the ligand. The 94 HSA–ligand binding K_d values, obtained by the docking procedure, were subjected to a quantitative structure‐activity relationship (QSAR) study by multiple regression analysis. The best correlation between the observed and QSAR theoretical (K_d predicted) data was displayed at 2.5 ns. This study provides evidence that HSA binding sites I and II interact specifically with a variety of compounds through conformational adjustments of the protein structure in conjunction with ligand conformational adaptation to these sites. These results serve to explain the high ligand‐promiscuity of HSA. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 161–170, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Solution structure of a novel T‐cell adhesion inhibitor derived from the fragment of ICAM‐1 receptor: Cyclo(1,8)‐Cys‐Pro‐Arg‐Gly‐Gly‐Ser‐Val‐Cys

This study is aimed at elucidating the structure of a novel T‐cell adhesion inhibitor, cyclo(1,8)‐CPRGGSVC using one‐ and two‐dimensional (2D) ¹H NMR and molecular dynamics (MD) simulation. The peptide is derived from the sequence of its parent peptide cIBR (cyclo(1,12)‐PenPRGGSVLVTGC), which is a fragment of intercellular adhesion molecule‐1 (ICAM‐1). Our previous results show that the cyclo(1,8)‐CPRGGSVC peptide binds to the LFA‐1 I‐domain and inhibits heterotypic T‐cell adhesion, presumably by blocking the LFA‐1/ICAM‐1 interactions. The structure of the peptide was determined using NMR and MD simulation in aqueous solution. Our results indicate that the peptide adopts type‐I β‐turn conformation at the Pro2‐Arg3‐Gly4‐Gly5 (PRGG) sequence. The β‐turn structure at the PRGG motif is well conserved in cIBR peptide and ICAM‐1 receptor, which suggests the importance of the PRGG motif for the biological activity of cyclo(1,8)‐CPRGGSVC peptide. Meanwhile, the Gly5‐Ser6‐Val7‐Cys8‐Cys1 (GSVCC) sequence forms a “turn‐like” random coil structure that does not belong to any structured motif. Therefore, cyclo(1,8)‐CPRGGSVC peptide has only one structured region at the PRGG sequence, which may play an important role in the binding of the peptide to the LFA‐1 I‐domain. The conserved β‐turn conformation of the PRGG motif in ICAM‐1, cIBR, and cyclo(1,8)‐CPRGGSVC peptides can potentially be used to design peptidomimetics. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 633–641, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structural studies of SNARE complex and its interaction with complexin by molecular dynamics simulation

Since neurotransmitter releasing into the synaptic space delivers electrical signals from presynaptic neural cell to the postsynaptic cell, neurotransmitter secretion must be much orchestrated. Crowded intracellular vesicles involving neurotransmitters present a question of the how secretory vesicles fuse onto the plasma membrane in a fast synchronized fashion. Complexin is one of the most experimentally studied proteins that regulate assembly of fusogenic four‐helix SNARE complex to synchronized neurotransmitter secretion. We used MD simulation to investigate the interaction of complexin with the neural SNARE complex in detail. Our results show that the SNARE complex interacts with the complexin central helix by forming salt bridges and hydrogen bonds. Complexin also can interact with the Q‐SNARE complex instead of synaptobrevin to decrease the Q‐SNARE flexibility. The complexin alpha‐accessory helix and the C‐terminal region of synaptobrevin can interact with the same region of syntaxin. Although the alpha‐accessory helix aids the tight binding of the central helix to the SNARE complex, its proximity with synaptobrevin causes the destabilization of syntaxin and Sn1 helices. This study suggests that the alpha‐accessory helix of complexin can be an inhibiting factor for membrane fusion by competing with synaptobrevin for binding to the Q‐SNARE complex. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 560–570, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Mg2+ ions bind at the C‐terminal region of skeletal muscle α‐tropomyosin

Tropomyosin (Tm) is a dimeric coiled‐coil protein that polymerizes through head‐to‐tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may form sites for divalent cation binding. In a previous study, we showed that the Mg²⁺‐induced increase in stability of the C‐terminal half of Tm is sensitive to mutations near the C‐terminus. In the present report, we study the interaction between Mg²⁺ and full‐length Tm and smaller fragments corresponding to the last 65 and 26 Tm residues. Although the smaller Tm peptide (Tm_{259‐284(W269)}) is flexible and to large extent unstructured, the larger Tm_{220‐284(W269)} fragment forms a coiled coil in solution whose stability increases significantly in the presence of Mg²⁺. NMR analysis shows that Mg²⁺ induces chemical shift perturbations in both Tm_{220‐284(W269)} and Tm_{259‐284(W269)} in the vicinity of His276, in which are located several negatively charged residues. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 583–590, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Specific DNA G‐quadruplexes bind to ethanolamines

A significant number of G‐quadruplex‐forming sequences have been revealed in human genome by bioinformatic searches, implying that G‐quadruplexes may be involved in important biological processes and may be new chemotherapeutic targets. Therefore, it is important to discover the potential interactions of G‐quadruplexes with other molecules or groups. Here we describe a class of G‐quadruplexes, which can bind to ethanolamine groups that widely exist in biomolecules and drug molecules. The specific interaction of these G‐quadruplexes with ethanolamine groups was identified by high performance affinity chromatography (HPAC) using immobilized ethanolamine and diethanolamine as stationary phase reagents. The circular dichroism (CD) spectra and native polyacrylamide gel electrophoresis (PAGE) show that these ethanolamine binding quadruplexes adopt an intramolecularly parallel structure. The relationship of ethanolamine binding and G‐quadruplexe structure provides new clues for the G‐quadruplex‐related studies as well as for the molecular designs of therapeutic reagents. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 874–883, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Potential allosteric modulators of the proteasome activity

Proteasome, consisting of a tube‐shaped proteolytic core particle and attached to it regulatory modules, is a multifunctional enzymatic complex essential for the ubiquitin‐proteasome metabolic pathway. Due to its immense involvement in regulation of cellular physiology, the proteasome is an acknowledged anticancer drug target and potential target to treat inflammatory or degenerative diseases. So far, competitive inhibitors of the core particle gain most consideration as drugs. We postulate that noncompetitively‐acting small‐molecule compounds would provide excellent means to precisely regulate actions of the proteasome. In this study, we evaluated five short peptides based on sequences of two proteins known to interact with the core proteasome: HIV‐1 Tat and PA28/REG activator. We performed Circular Dichroism (CD), Fourier Transformed Infrared Spectroscopy (FTIR), and Nuclear Magnetic Resonance (NMR) analysis, supplemented by MD simulations, and tested influence of the peptides on performance of the core particle active sites and functioning of regulatory modules. We found that PP2‐containing Tat peptides are noncompetitive inhibitors of the core, interfering with the actions of PA28αβ activator. In addition, at low concentrations the turn‐prone Tat2 is able to activate the latent core. The random coil‐structured PA28‐derived peptides display only weak or nondetectable direct effects on the core activities, exhibiting, however, a positive cooperation with activity‐enhancing actions of PA28αβ. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 481–495, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Modulation of SHP‐1 phosphatase activity by monovalent and bivalent SH2 phosphopeptide ligands

A sequence derived from the epithelial receptor tyrosine kinase Ros (pY2267) represents a high‐affinity binding partner for protein tyrosine phosphatase SHP‐1 and was recently used as lead structure to analyze the recognition requirements for the enzyme's N‐SH2 domain. Here, we focused on a set of peptides comprising C‐terminally extended linear and conformationally constrained side chain‐bridged cyclic N‐SH2 ligands based on the consensus sequence LxpYhxh(h/b)(h/b) (x = any amino acid, h = hydrophobic, and b = basic residue). Furthermore, the bivalent peptides described were designed to modulate the activity of SHP‐1 through binding to both, the N‐SH2 domain as well as an independent binding site on the surface of the catalytic domain (PTP domain). Consistent with previous experimental findings, surface plasmon resonance experiments revealed dissociation constants of most compounds in the low micromolar range. One peptide, EGLNpYc[KVD]MFPAPEEE? NH₂, displayed favorable binding affinity, but reduced ability to stimulate SHP‐1. Docking experiments revealed that the binding of this ligand occurs in binding mode I, recently described to lead to an inhibited activation of SHP‐1. In summary, results presented in this study suggest that inhibitory N‐SH2 ligands of SHP‐1 may be obtained by designing bivalent compounds that associate with the N‐SH2 domain and simultaneously occupy a specific binding site on the PTP domain. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 102–112, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Importance of the spatial display of charged residues in heparin–peptide interactions

Many studies have examined consensus sequences required for protein‐glycosaminoglycan interactions. Through the synthesis of helical heparin binding peptides, this study probes the relationship between spatial arrangement of positive charge and heparin binding affinity. Peptides with a linear distribution of positive charge along one face of the α‐helix had the highest affinity for heparin. Moving the basic residues away from a single face resulted in drastic changes in heparin binding affinity of up to three orders of magnitude. These findings demonstrate that amino acid sequences, different from the known heparin binding consensus sequences, will form high affinity protein‐heparin binding interactions when the charged residues are aligned linearly. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 290–298, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

ILPR repeats adopt diverse G‐quadruplex conformations that determine insulin binding

The insulin‐linked polymorphic region (ILPR) is a VNTR region located upstream of the insulin (INS) gene consisting of the repeat 5′‐ACAGGGGTGTGGGG (repeat a) and several less abundant sequence repeats (b–n). Here, we have investigated the structural polymorphism of G‐quadruplexes formed from the most common repeat sequences (a–c) and their effect on insulin protein binding. We first established that the ILPR repeats “b” and “c” can form quadruplex structures. Insulin has previously been shown to bind a G‐quadruplex formed by a dimer of the repeat “a”. Our findings show that insulin binds preferentially to the repeat “a” G‐quadruplex (K_d = 0.17 ± 0.03 μM) over G‐quadruplexes formed from other ILPR repeats that were tested (K_ds from 0.71 ± 0.15 to 1.07 ± 0.09 μM). Additionally, the Watson‐Crick complementary relationship between the loop regions of repeat “a” (ACA and TGT) seemingly play an important role in favoring a specific G‐quadruplex conformation, which based on our data is critical for insulin binding. Affinity for insulin is reduced in sequences lacking the putative WC complementarity, however upon engineered restoration of complementarity, insulin binding is recovered. A DMS footprinting assay on the repeat “a” G‐quadruplex in the presence of insulin, combined with binding affinities for ILPR mutants led to identification of a loop nucleotide critical for binding. Uniquely, insulin shows clear preference for binding to the G‐quadruplexes with the more antiparallel feature. Collectively, our results illustrate the specific nature of insulin binding to the ILPR G‐quadruplexes and begin to provide molecular details on such interactions. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 21–31, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Conformational stability of the full‐atom hexameric model of the ClpB chaperone from Escherichia coli

The Escherichia coli heat shock protein ClpB, a member of the Hsp100 family, plays a crucial role in cellular thermotolerance. In co‐operation with the Hsp70 chaperone system, it is able to solubilize proteins aggregated by heat shock conditions and refold them into the native state in an ATP‐dependent way. It was established that the mechanism of ClpB action depends on the formation of a ring‐shaped hexameric structure and the translocation of a protein substrate through an axial channel. The structural aspects of this process are not fully known. By means of homology modeling and protein–protein docking, we obtained a model of the hexameric arrangement of the full‐length ClpB protein complexed with ATP. A molecular dynamics simulation of this model was performed to assess its flexibility and conformational stability. The high mobility of the “linker” M‐domain, essential for the renaturing activity of ClpB, was demonstrated, and the size and shape of central channel were analyzed. In this model, we propose the coordinates for a loop between b4 and B6 structural elements, not defined in previous structural research, which faces the inside of the channel and may therefore play a role in substrate translocation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 47–60, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Exploring the conformational and binding properties of unphosphorylated/phosphorylated monomeric and trimeric Bcl‐2 through docking and molecular dynamics simulations

B‐cell lymphoma (Bcl‐2) is commonly associated with the progression and preservation of cancer and certain lymphomas; therefore, it is considered as a biological target against cancer. Nevertheless, evidence of all its structural binding sites has been hidden because of the lack of a complete Bcl‐2 model, given the presence of a flexible loop domain (FLD), which is responsible for its complex behavior. FLD region has been implicated in phosphorylation, homotrimerization, and heterodimerization associated with Bcl‐2 antiapoptotic function. In this contribution, homology modeling, molecular dynamics (MD) simulations in the microsecond (µs) time‐scale and docking calculations were combined to explore the conformational complexity of unphosphorylated/phosphorylated monomeric and trimeric Bcl‐2 systems. Conformational ensembles generated through MD simulations allowed for identifying the most populated unphosphorylated/phosphorylated monomeric conformations, which were used as starting models to obtain trimeric complexes through protein–protein docking calculations, also submitted to µs MD simulations. Principal component analysis showed that FLD represents the main contributor to total Bcl‐2 mobility, and is affected by phosphorylation and oligomerization. Subsequently, based on the most representative unphosphorylated/phosphorylated monomeric and trimeric Bcl‐2 conformations, docking studies were initiated to identify the ligand binding site of several known Bcl‐2 inhibitors to explain their influence in homo‐complex formation and phosphorylation. Docking studies showed that the different conformational states experienced by FLD, such as phosphorylation and oligomerization, play an essential role in the ability to make homo and hetero‐complexes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 393–413, 2016.     

Substrate directs enzyme dynamics by bridging distal sites: UDP-galactopyranose mutase

UDP-Galactopyranose mutase (UGM) is a flavoenzyme that catalyzes interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf); its activity depends on FAD redox state. The enzyme is vital to many pathogens, not native to mammals, and is an important drug target. We have probed binding of substrate, UDP-Galp, and UDP to wild type and W160A UGM from K. pneumoniae, and propose that substrate directs recognition loop dynamics by bridging distal FAD and W160 sites; W160 interacts with uracil of the substrate and is functionally essential. Enhanced Trp fluorescence upon substrate binding to UGM indicates conformational changes remote from the binding site because the fluorescence is unchanged upon binding to W70F/W290F UGM where W160 is the sole Trp. MD simulations map these changes to recognition loop closure to coordinate substrate. This requires galactose-FAD interactions as Trp fluorescence is unchanged upon substrate binding to oxidized UGM, or binding of UDP to either form of the enzyme, and MD show heightened recognition loop mobility in complexes with UDP. Consistent with substrate-directed loop closure, UDP binds 10-fold more tightly to oxidized UGM, yet substrate binds tighter to reduced UGM. This requires the W160-U interaction because redox-switched binding affinity of substrate reverses in the W160A mutant where it only binds when oxidized. Without the anchoring W160-U interaction, an alternative binding mode for UDP is detected, and STD-NMR experiments show simultaneous binding of UDP-Galp and UDP to different subsites in oxidized W160A UGM: Substrate no longer directs recognition loop dynamics to coordinate tight binding to the reduced enzyme.     

Study of the interactions between a proline‐rich protein and a flavan‐3‐ol by NMR: Residual structures in the natively unfolded protein provides anchorage points for the ligands

Astringency is one of the major organoleptic properties of food and beverages that are made from plants, such as tea, chocolate, beer, or red wine. This sensation is thought to be due to interactions between tannins and salivary proline‐rich proteins, which are natively unfolded proteins. A human salivary proline‐rich protein, namely IB‐5, was produced by the recombinant method. Its interactions with a model tannin, epigallocatechin gallate (EGCG), the major flavan‐3‐ol in green tea, were studied here. Circular dichroism experiments showed that IB‐5 presents residual structures (PPII helices) when the ionic strength is close to that in saliva. In the presence of these residual structures, IB‐5 undergoes an increase in structural content upon binding to EGCG. NMR data corroborated the presence of preformed structural elements within the protein prior to binding and a partial assignment was proposed, showing partial structuration. TOCSY experiments showed that amino acids that are involved in PPII helices are more likely to interact with EGCG than those in random coil regions, as if they were anchorage points for the ligand. The signal from IB‐5 in the DOSY NMR spectrum revealed an increase in polydispersity upon addition of EGCG while the mean hydrodynamic radius remained unchanged. This strongly suggests the formation of IB‐5/EGCG aggregates. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 745–756, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Model peptide‐based system used for the investigation of metal ions binding to histidine‐containing polypeptides

The reaction of histidine‐containing polypeptides with toxic and essential metals and the molecular mechanism of complexation has yet to be determined, particularly with respect to the conformational changes of the interacting macromolecules. Therefore, a system of oligopeptides containing histidine residues in various positions of Ala or Gly sequences has been designed and used in heavy metal comparatively binding experiments. The role of spacing residues (Gly and Ala repeats) in selecting the various conformations was investigated. The newly synthesized peptides and metal ion adducts have been characterized by Fourier transform infrared spectroscopy (FTIR) as well as electrospray ion trap mass spectrometry (ESI–MS) and circular dichroism (CD). The analysis of CD‐spectra of the four peptides in water revealed that the secondary structure depends much on the position of each amino acid in the peptide backbone. Our peptides system reveals various binding mechanisms of metal ions to peptides depending on the position of histidine residue and the corresponding conformations of Ala or Gly sequences. Biological and medical consequences of conformational changes of metal‐bound peptides are further discussed. Thus, the binding of heavy metals to four peptides may serve as a model system with respect to the conformational consequences of the metal addition on the amino acid repeats situated in prion protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93:497–508, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com