Mapping the Dynamics Landscape of Conformational Transitions in Enzyme: The Adenylate Kinase Case.

Conformational transition describes the essential dynamics and mechanism of enzymes in pursuing their various functions. The fundamental and practical challenge to researchers is to quantitatively describe the roles of large-scale dynamic transitions for regulating the catalytic processes. In this study, we tackled this challenge by exploring the pathways and free energy landscape of conformational changes in adenylate kinase (AdK), a key ubiquitous enzyme for cellular energy homeostasis. Using explicit long-timescale (up to microseconds) molecular dynamics and bias-exchange metadynamics simulations, we determined at the atomistic level the intermediate conformational states and mapped the transition pathways of AdK in the presence and absence of ligands. There is clearly chronological operation of the functional domains of AdK. Specifically in the ligand-free AdK, there is no significant energy barrier in the free energy landscape separating the open and closed states. Instead there are multiple intermediate conformational states, which facilitate the rapid transitions of AdK. In the ligand-bound AdK, the closed conformation is energetically most favored with a large energy barrier to open it up, and the conformational population prefers to shift to the closed form coupled with transitions. The results suggest a perspective for a hybrid of conformational selection and induced fit operations of ligand binding to AdK. These observations, depicted in the most comprehensive and quantitative way to date, to our knowledge, emphasize the underlying intrinsic dynamics of AdK and reveal the sophisticated conformational transitions of AdK in fulfilling its enzymatic functions. The developed methodology can also apply to other proteins and biomolecular systems.

Illuminating Flash Point: Comprehensive Prediction Models.

Flash point is an important property of chemical compounds that is widely used to evaluate flammability hazard. However, there is often a significant gap between the demand for experimental flash point data and their availability. Furthermore, the determination of flash point is difficult and costly, particularly for some toxic, explosive, or radioactive compounds. The development of a reliable and widely applicable method to predict flash point is therefore essential. In this paper, the construction of a quantitative structure - property relationship model with excellent performance and domain of applicability is reported. It uses the largest data set to date of 9399 chemically diverse compounds, with flash point spanning from less than -130 °C to over 900 °C. The model employs only computed parameters, eliminating the need for experimental data that some earlier computational models required. The model allows accurate prediction of flash point for a broad range of compounds that are unavailable or not yet synthesized. This single model with a very broad range of chemical and flash point applicability will allow accurate predictions of this important property to be made for a broad range of new materials.

Molecular dynamics reveal a novel kinase-substrate interface that regulates protein translation.

A key control point in gene expression is the initiation of protein translation, with a universal stress response being constituted by inhibitory phosphorylation of the eukaryotic initiation factor 2α (eIF2α). In humans, four kinases sense diverse physiological stresses to regulate eIF2α to control cell differentiation, adaptation, and survival. Here we develop a computational molecular model of eIF2α and one of its kinases, the protein kinase R, to simulate the dynamics of their interaction. Predictions generated by coarse-grained dynamics simulations suggest a novel mode of action. Experimentation substantiates these predictions, identifying a previously unrecognized interface in the protein complex, which is constituted by dynamic residues in both eIF2α and its kinases that are crucial to regulate protein translation. These findings call for a reinterpretation of the current mechanism of action of the eIF2α kinases and demonstrate the value of conducting computational analysis to evaluate protein function.

Operating mechanism and molecular dynamics of pheromone-binding protein ASP1 as influenced by pH.

Odorant binding protein (OBP) is a vital component of the olfactory sensation system. It performs the specific role of ferrying odorant molecules to odorant receptors. OBP helps insects and types of animal to sense and transport stimuli molecules. However, the molecular details about how OBPs bind or release its odorant ligands are unclear. For some OBPs, the systems' pH level is reported to impact on the ligands' binding or unbinding capability. In this work we investigated the operating mechanism and molecular dynamics in bee antennal pheromone-binding protein ASP1 under varying pH conditions. We found that conformational flexibility is the key factor for regulating the interaction of ASP1 and its ligands, and the odorant binds to ASP1 at low pH conditions. Dynamics, once triggered by pH changes, play the key roles in coupling the global conformational changes with the odorant release. In ASP1, the C-terminus, the N-terminus, helix α2 and the region ranging from helices α4 to α5 form a cavity with a novel 'entrance' of binding. These are the major regions that respond to pH change and regulate the ligand release. Clearly there are processes of dynamics and hydrogen bond network propagation in ASP1 in response to pH stimuli. These findings lead to an understanding of the mechanism and dynamics of odorant-OBP interaction in OBP, and will benefit chemsensory-related biotech and agriculture research and development.

Mg²âº coordinating dynamics in Mg:ATP fueled motor proteins.

The coordination of Mg(2+) with the triphosphate group of adenosine triphosphate (ATP) in motor proteins is investigated using data mining and molecular dynamics. The possible coordination structures available from crystal data for actin, myosin, RNA polymerase, DNA polymerase, DNA helicase, and F1-ATPase are verified and investigated further by molecular dynamics. Coordination states are evaluated using structural analysis and quantified by radial distribution functions, coordination numbers, and pair interaction energy calculations. The results reveal a diverse range of both transitory and stable coordination arrangements between Mg(2+) and ATP. The two most stable coordinating states occur when Mg(2+) coordinates two or three oxygens from the triphosphate group of ATP. Evidence for five-site coordination is also reported involving water in addition to the triphosphate group. The stable states correspond to a pair interaction energy of either ∼-2750 kJ/mol or -3500 kJ/mol. The role of water molecules in the hydration shell surrounding Mg(2+) is also reported.

Conformational dynamics of ATP/Mg:ATP in motor proteins via data mining and molecular simulation.

The conformational diversity of ATP/Mg:ATP in motor proteins was investigated using molecular dynamics and data mining. Adenosine triphosphate (ATP) conformations were found to be constrained mostly by inter cavity motifs in the motor proteins. It is demonstrated that ATP favors extended conformations in the tight pockets of motor proteins such as F(1)-ATPase and actin whereas compact structures are favored in motor proteins such as RNA polymerase and DNA helicase. The incorporation of Mg(2+) leads to increased flexibility of ATP molecules. The differences in the conformational dynamics of ATP/Mg:ATP in various motor proteins was quantified by the radius of gyration. The relationship between the simulation results and those obtained by data mining of motor proteins available in the protein data bank is analyzed. The data mining analysis of motor proteins supports the conformational diversity of the phosphate group of ATP obtained computationally.

Identification of catalytic residues using a novel feature that integrates the microenvironment and geometrical location properties of residues.

Enzymes play a fundamental role in almost all biological processes and identification of catalytic residues is a crucial step for deciphering the biological functions and understanding the underlying catalytic mechanisms. In this work, we developed a novel structural feature called MEDscore to identify catalytic residues, which integrated the microenvironment (ME) and geometrical properties of amino acid residues. Firstly, we converted a residue's ME into a series of spatially neighboring residue pairs, whose likelihood of being located in a catalytic ME was deduced from a benchmark enzyme dataset. We then calculated an ME-based score, termed as MEscore, by summing up the likelihood of all residue pairs. Secondly, we defined a parameter called Dscore to measure the relative distance of a residue to the center of the protein, provided that catalytic residues are typically located in the center of the protein structure. Finally, we defined the MEDscore feature based on an effective nonlinear integration of MEscore and Dscore. When evaluated on a well-prepared benchmark dataset using five-fold cross-validation tests, MEDscore achieved a robust performance in identifying catalytic residues with an AUC1.0 of 0.889. At a ≤ 10% false positive rate control, MEDscore correctly identified approximately 70% of the catalytic residues. Remarkably, MEDscore achieved a competitive performance compared with the residue conservation score (e.g. CONscore), the most informative singular feature predominantly employed to identify catalytic residues. To the best of our knowledge, MEDscore is the first singular structural feature exhibiting such an advantage. More importantly, we found that MEDscore is complementary with CONscore and a significantly improved performance can be achieved by combining CONscore with MEDscore in a linear manner. As an implementation of this work, MEDscore has been made freely accessible at http://protein.cau.edu.cn/mepi/.

Identifying the molecular mechanics and binding dynamics characteristics of potent inhibitors to HIV-1 protease.

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the primary inhibition targets for chemotherapy of AIDS because of its critical role in the replication cycle of the HIV. In this work, a combinatory coarse-grained and atomistic simulation method was developed for dissecting molecular mechanisms and binding process of inhibitors to the active site of HIV-1 PR, in which 35 typical inhibitors were trialed. We found that the molecular size and stiffness of the inhibitors and the binding energy between the inhibitors and PR play important roles in regulating the binding process. Comparatively, the smaller and more flexible inhibitors have larger binding energy and higher binding rates; they even bind into PR without opening the flaps. In contrast, the larger and stiffer inhibitors have lower binding energy and lower binding rate, and their binding is subjected to the opening and gating of the PR flaps. Furthermore, the components of binding free energy were quantified and analyzed by their dependence on the molecular size, structures, and hydrogen bond networks of inhibitors. Our results also deduce significant dynamics descriptors for determining the quantitative structure and property relationship in potent drug ligands for HIV-1 PR inhibition.

Coarse-grained molecular dynamics of ligands binding into protein: The case of HIV-1 protease inhibitors.

Binding dynamics and pathways of ligands or inhibitors to target proteins are challenging both experimental and theoretical biologists. A dynamics understanding of inhibitors interacting with protein is essential for the design of novel potent drugs. In this work we applied a coarse-grained molecular dynamics method for simulating inhibitors entering the binding cavity of human immunodeficiency virus type 1 protease (PR). It shows that the coarse-grained dynamics, consistent with the experimental results, can capture the essential molecular dynamics of various inhibitors binding into PR. The primary driving force for the binding processes is the nonbond interaction between inhibitors and PR. The size and topology of inhibitors and the interacting strength between inhibitors and PR have great influence on the binding mode and processes. The interaction strength between the PR and various inhibitors is also analyzed by atomistic molecular mechanics and Poisson-Boltzmann solvation area method.

Coarse-grained dynamics of the receiver domain of NtrC: fluctuations, correlations and implications for allosteric cooperativity.

Receiver domains are key molecular switches in bacterial signaling. Structural studies have shown that the receiver domain of the nitrogen regulatory protein C (NtrC) exists in a conformational equilibrium encompassing both inactive and active states, with phosphorylation of Asp54 allosterically shifting the equilibrium towards the active state. To analyze dynamical fluctuations and correlations in NtrC as it undergoes activation, we have applied a coarse-grained dynamics algorithm using elastic network models. Normal mode analysis reveals possible dynamical pathways for the transition of NtrC from the inactive state to the active state. The diagonalized correlation between the inactive and the active (phosphorylated) state shows that most correlated motions occur around the active site of Asp54 and in the region Thr82 to Tyr101. This indicates a coupled correlation of dynamics in the "Thr82-Tyr101" motion. With phosphorylation inducing significant flexibility changes around the active site and alpha3 and alpha4 helices, we find that this activation makes the active-site region and the loops of alpha3/beta4 and alpha4/beta5 more stable. This means that phosphorylation entropically favors the receiver domain in its active state, and the induced conformational changes occur in an allosteric manner. Analyses of the local flexibility and long-range correlated motion also suggest a dynamics criterion for determining the allosteric cooperativity of NtrC, and may be applicable to other proteins.

Individual detection of genetically modified maize varieties in non-identity-preserved maize samples.

In many countries, the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved GM varieties. The GMO content in a maize sample containing the combined-trait (stacked) GM maize as determined by the currently available methodology is likely to be overestimated. However, there has been little information in the literature on the mixing level and varieties of stacked GM maize in real sample grains. For the first time, the GMO content of non-identity-preserved (non-IP) maize samples imported from the United States has been successfully determined by using a previously developed individual kernel detection system coupled to a multiplex qualitative PCR method followed by multichannel capillary gel electrophoresis system analysis. To clarify the GMO content in the maize samples imported from the United States, determine how many stacked GM traits are contained therein, and which GM trait varieties frequently appeared in 2005, the GMO content (percent) on a kernel basis and the varieties of the GM kernels in the non-IP maize samples imported from the United States were investigated using the individual kernel analysis system. The average (+/-standard deviation) of the GMO contents on a kernel basis in five non-IP sample lots was determined to be 51.0+/-21.6%, the percentage of a single GM trait grains was 39%, and the percentage of the stacked GM trait grains was 12%. The MON810 grains and NK603 grains were the most frequent varieties in the single GM traits. The most frequent stacked GM traits were the MON810xNK603 grains. In addition, the present study would provide the answer and impact for the quantification of GM maize content in the GM maize kernels on labeling regulation.

Use of multiplex PCR and CE for gene dosage quantification and its biomedical applications for SMN, PMP22, and alpha-globin genes.

Many genetic diseases are caused by the presence of point mutations, small insertions, and deletions in respective genes, and the number of diseases known to be caused by deletions and duplications involving large DNA genomes is increasing. These changes lead to underexpression or overexpression of the gene, according to changes in gene dosage. The methods for the detection of point mutations, small insertions, and deletions are well established, but the detection of larger genomic deletions or duplications is more difficult. Due to the lack of efficient and technically feasible protocols for gene dosage quantification, we describe a diagnostic protocol employing a combination of available methods. The efficient and accurate gene dosage quantification platform is combined with multiplex PCR and CE, and applied to detect dosages of several genes, including SMN, PMP22, and alpha-globin genes. The reliability of this novel methodology shows that it is a relatively speedy and low-cost procedure and a significant tool for genetic diagnosis. Its sensitivity and specificity for identifying deletion and duplication genotypes approach 100%. Moreover, once we establish this powerful system, we will further apply this technique to the rapid detection of trisomy syndromes and microdeletion syndromes, including trisomy 13, Down syndrome, DiGeorge syndrome, and others.

Dynamics of the SPRY domain-containing SOCS box protein 2: flexibility of key functional loops.

The SPRY domain was identified originally as a sequence repeat in the dual-specificity kinase splA and ryanodine receptors and subsequently found in many other distinct proteins, including more than 70 encoded in the human genome. It is a subdomain of the B30.2/SPRY domain and is believed to function as a protein-protein interaction module. Three-dimensional structures of several B30.2/SPRY domain-containing proteins have been reported recently: murine SSB-2 in solution by NMR spectroscopy, a Drosophila SSB (GUSTAVUS), and human PRYSPRY protein by X-ray crystallography. The three structures share a core of two antiparallel beta-sheets for the B30.2/SPRY domain but show differences located mainly at one end of the beta-sandwich. Analysis of SSB-2 residues required for interactions with its intracellular ligands has provided insights into B30.2/SPRY binding specificity and identified loop residues critical for the function of this domain. We have investigated the backbone dynamics of SSB-2 by means of Modelfree analysis of its backbone (15)N relaxation parameters and carried out coarse-grained dynamics simulation of B30.2/SPRY domain-containing proteins using normal mode analysis. Translational self-diffusion coefficients of SSB-2 measured using pulsed field gradient NMR were used to confirm the monomeric state of SSB-2 in solution. These results, together with previously reported amide exchange data, highlight the underlying flexibility of the loop regions of B30.2/SPRY domain-containing proteins that have been shown to be important for protein-protein interactions. The underlying flexibility of certain regions of the B30.2/SPRY domain-containing proteins may also contribute to some apparent structural differences observed between GUSTAVUS or PRYSPRY and SSB-2.

Dynamic and coordinating domain motions in the active subunits of the F1-ATPase molecular motor.

F1-ATPase is a rotary molecular motor crucial for various cellular functions. In F1-ATPase, the rotation of the gammadeltaepsilon subunits against the hexameric alpha(3)beta(3) subunits is highly coordinative, driven by ATP hydrolysis and structural changes at three beta subunits. However, the dynamical and coordinating structural transitions in the beta subunits are not fully understood at the molecular level. Here we examine structural transitions and domain motions in the active subunits of F1-ATPase via dynamical domain analysis of the alpha(3)beta(3)gammadeltaepsilon complex. The domain movement and hinge axes and bending residues have been identified and determined for various conformational changes of the beta-subunits. P-loop and the ATP-binding pocket are for the first time found to play essential mechanical functions additional to the catalytic roles. The cooperative conformational changes pertaining to the rotary mechanism of F1-ATPase appears to be more complex than Boyer's 'bi-site' activity. These findings provide unique molecular insights into dynamic and cooperative domain motions in F1-ATPase.

Quantitative assay of deletion or duplication genotype by capillary electrophoresis system: Application in Prader-Willi syndrome and Duchenne muscular dystrophy.

BACKGROUND: Deletions and duplications involving large DNA segments result in underexpression or overexpression, depending on the changes in allele dose, and are known to cause many common disorders. Detection of allele dose variations in the human genome is increasingly important in medical genetic diagnosis. METHODS: We used multiplex quantitative PCR coupled with capillary electrophoresis for accurate allele dose determination. In cases of Prader-Willi syndrome (PWS), a total of 24 patients with PWS, as well as 205 control individuals from the general population, were analyzed by use of multiplex quantitative PCR to amplify the FGFR2 gene, the KRIT1 gene, and the SNRPN gene simultaneously. In cases of Duchenne muscular dystrophy (DMD), we optimized the multiplex quantitative PCR to amplify 38 exons to analyze the DMD gene for rapid diagnosis of 12 DMD-affected males, 12 obligate carriers from families, and 50 unaffected female controls. RESULTS: We were able to unambiguously diagnose the deletion genotype in PWS patients and identify all deletion or duplication genotypes and carrier status in DMD-affected cases with 100% sensitivity and specificity. CONCLUSIONS: This report describes a novel single assay that can rapidly quantify allele dose to provide accurate clinical genetic diagnosis. This technique offers a valuable alternative for the rapid detection of genomic deletions or duplications and decreases costs because it does not require expensive fluorescent reagents.

Determination of SMN1/SMN2 gene dosage by a quantitative genotyping platform combining capillary electrophoresis and MALDI-TOF mass spectrometry.

BACKGROUND: Spinal muscular atrophy (SMA) is a common inherited and fatal neuromuscular disease caused by deletions and/or mutations that lead to altered concentrations of proteins encoded by the survival motor neuron genes SMN1 and SMN2. Because of the high incidence (at least 1 in 10,000 live births and a carrier frequency of 1 in 35 to 1 in 50) and severity of the disease, precise quantification of SMN1 and SMN2 gene copy numbers is essential for diagnosis and genetic counseling. METHODS: We developed a genotyping platform combining capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to quantify absolute gene dosage. The absolute gene dosage can be determined by a multiplexed competitive PCR protocol followed by capillary electrophoresis analysis. The relative SMN1/SMN2 ratio can be analyzed by PinPoint assay followed by MALDI-TOF MS analysis. RESULTS: The complementary assays were evaluated in confirmed cases including 9 affected patients, 33 carriers, and 478 healthy individuals from the general population. We were able to determine all genotypes with different SMN1/SMN2 gene copy number ratios, which unambiguously diagnosed carrier status and the severity of SMA with 100% specificity. CONCLUSIONS: This quantitative genotyping platform is suitable for detection of SMA. The described approach may serve as a general quantitative genotyping method for molecular diagnosis of other inheritable diseases.

Cooperativity in the motor activities of the ATP-fueled molecular motors.

Kinesin, myosin and F1-ATPase are multi-domain molecular motors with multiple catalytic subunits. The motor mechanochemics are achieved via the conversion of ATP hydrolysis energy into forces and motions. We find that the catalysis of these molecular motors do not follow the simple Michaelis-Menten mechanism. The motor activities, such as the hydrolysis or processive rates, of kinesin, myosin and F1-ATPase have a complex ATP-dependent cooperativity. To understand this complexity in kinetics and mechanochemics, we develop a conformation correlation theory of cooperativity for the ATP-fueled motor proteins. The quantitative analysis and simulations indicate that cooperativity is induced by the conformational coupling of binding states of different subunits and prevails in the motor activities.

Polymerase chain reaction method to identify Down syndrome model segmentally trisomic mice.

The Ts65Dn segmentally trisomic mouse possesses an extra copy of a segment of chromosome 16 translocated to chromosome 17. This segment includes the mouse homolog of the Down syndrome critical region of human chromosome 21. The Ts65Dn mouse serves as a useful model to study the developmental regulation of the Down syndrome phenotype. To identify mice bearing the extra chromosome 16 segment, we developed a polymerase chain reaction (PCR) method as an alternative to karyotyping. Conditions under which segments of genes on chromosome 16 (App and Dyrk1a) could be coamplified with a control gene on chromosome 8 (Acta1) so that the yield of each PCR product was proportional to the amount of its template were determined. The amplification products were resolved and quantified by two methods. In the first method, the DNA segments were separated by agarose gel electrophoresis and stained with ethidium bromide. The fluorescence yields were quantified by photodensitometry. In the second method, the fragments were resolved and quantified by the high-performance DNA analysis system, a high-throughput, multichannel, microcapillary electrophoresis instrument. The results of both methods were within 10% of the expected ratio of 1.5. Application of these methods has allowed the maintenance of a Ts65Dn breeding colony through six generations and should permit the precise and efficient identification of trisomic and disomic animals at any developmental stage with minimally invasive procedures.

Complex cooperativity of ATP hydrolysis in the F(1)-ATPase molecular motor.

F(1)-ATPase catalyses ATP hydrolysis and converts the cellular chemical energy into mechanical rotation. The hydrolysis reaction in F(1)-ATPase does not follow the widely believed Michaelis-Menten mechanism. Instead, the hydrolysis mechanism behaves in an ATP-dependent manner. We develop a model for enzyme kinetics and hydrolysis cooperativity of F(1)-ATPase which involves the binding-state changes to the coupling catalytic reactions. The quantitative analysis and modeling suggest the existence of complex cooperative hydrolysis between three different catalysis sites of F(1)-ATPase. This complexity may be taken into account to resolve the arguments on the binding change mechanism in F(1)-ATPase.

DNA fragment analysis by an affordable multiple-channel capillary electrophoresis system.

We are demonstrating a cost-effective multichannel capillary electrophoresis system for a high-efficiency double-stranded DNA (dsDNA) fragments analysis. This bench-type high-performance DNA analysis (HDA) system uses fluorescence-type detection with inexpensive solid-state light sources and nonmoving integrated emission collection micro-optics. DNA samples are analyzed simultaneously by using a multiple usage and disposable multicapillary cartridge, which contains integrated capillary channels, optical fibers and an integrated sieving gel reservoir. Using commercially available dsDNA size markers as indicators, the HDA system provides high resolving power in 7 min separations. The system can hold a total of 192 samples in two 96-well polymerase chain reaction (PCR) plates, which can be automatically analyzed within 2.5 h. This affordable system can be used in laboratories to replace slab gel electrophoresis for routine and high-throughput dsDNA analysis.