SDOCK: a global protein-protein docking program using stepwise force-field potentials

Fast Fourier transform (FFT) method limits the forms of scoring functions in global protein-protein docking. On the other hand, force field potentials can effectively describe the energy hyper surface of biological macromolecules. In this study, we developed a new protein-protein docking program, SDOCK, that incorporates van der Waals attractive potential, geometric collision, screened electrostatic potential, and Lazaridis-Karplus desolvation energy into the scoring function in the global searching process. Stepwise potentials were generated from the corresponding continuous forms to treat the structure flexibility. After optimization of the atom solvation parameters and the weights of different potential terms based on a new docking test set that contains 142 cases with small or moderate conformational changes upon binding, SDOCK slightly outperformed the well-known FFT based global docking program ZDOCK3.0. Among the 142 cases tested, 52.8% gave at least one near-native solutions in the top 100 solutions. SDOCK was also tested on six blind testing cases in Critical Assessment of Predicted Interactions rounds 13 to 18. In all six cases, the near-native solutions could be found within the top 350 solutions. Because the SDOCK approach performs global docking based on force-field potentials, one of its advantages is that it provides global binding free energy surface profiles for further analysis. The efficiency of the program is also comparable with that of other FFT based protein-protein docking programs. SDOCK is available for noncommercial applications at http://mdl.ipc.pku.edu.cn/cgi-bin/down.cgi.     

Diphenylacetylene‐Linked Peptide Strands Induce Bidirectional β‐Sheet Formation

In the search for synthetic mimics of protein secondary structures relevant to the mediation of protein–protein interactions, we have synthesized a series of tetrasubstituted diphenylacetylenes that display β‐sheet structures in two directions. Extensive X‐ray crystallographic and NMR solution phase studies are consistent with these proteomimetics adopting sheet structures, displaying both hydrophobic and hydrophilic amino acid side chains.     

Synthesis of Substituted Pyridyl‐Pyrimidines as Potential Protein–Protein Interaction Inhibitors

An efficient synthetic method for the preparation of pyridyl‐pyrimidines as potential inhibitors of protein–protein interactions is described. The key transformation is the reaction of a pyrimidine enaminone with phenyl ethyl acetate and NH⁴OAc to yield the desired pyridyl‐pyrimidine.     

Enrichment of low-abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions

We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.     

Electrophoretic behavior of DNA‐methyl‐CpG‐binding domain protein complexes revealed by capillary electrophoreses laser‐induced fluorescence

The free solution electrophoretic behavior of DNA‐protein complexes depends on their charge and mass in a certain experimental condition, which are two fundamental properties of DNA‐protein complexes in free solution. Here, we used CE LIF to study the free solution behavior of DNA‐methyl‐CpG‐binding domain protein (MBD2b) complexes through exploring the relationship between the mobilities, charge, and mass of DNA‐protein complexes. This method is based on the effective separation of free DNA and DNA‐protein complexes because of their different electrophoretic mobility in a certain electric field. In order to avoid protein adsorption, a polyacrylamide‐coated capillary was used. Based on the evaluation of the electrophoretic behavior of formed DNA‐MBD2b complexes, we found that the values of (μ₀/μ)‐1 were directly proportional to the charge‐to‐mass ratios of formed complexes, where the μ₀ and μ are the mobility of free DNA probe and DNA‐protein complex, respectively. The models were further validated by the complex mobilities of protein with various lengths of DNA probes. The deviation of experimental and calculated charge‐to‐mass ratios of formed complexes from the theoretical data was less than 10%, suggesting that our models are useful to analyze the DNA‐binding properties of the purified MBD2b protein and help to analyze other DNA‐protein complexes. Additionally, this study enhances the understanding of the influence of the charge‐to‐mass ratios of formed DNA‐protein complexes on their separation and electrophoretic behaviors.     

Clean STD-NMR spectrum for improved detection of ligand-protein interactions at low concentration of protein

Saturation transfer difference (STD)-NMR has been widely used to screen ligand compound libraries for their binding activities to proteins and to determine the binding epitopes of the ligands. We report herein, a Clean STD-NMR method developed to overcome false positives (artifacts) observed in the STD-NMR spectrum due to the power spillover of RF irradiation. The method achieved higher degree of resonance saturation through digital editing of two STD-NMR spectra to generate a concatenated difference spectrum and three times of sensitivity enhancement for a loose binding complex involving DNA oligonucleotide and an RNA-binding protein, CUGBP-1ab (25.2 kDa). The interesting binding characteristics of the complex dCTGTCT-CUGBP1ab were obtained. The method was applied to a mixture of small ligand and bovine serum albumin protein (BSA, 66.3 kDa), and detected the intermolecular contacts at a BSA concentration as low as 0.1 μM, a working concentration useful for the detection of proteins of low solubility at biologically relevant conditions.     

Characterizing amyloid‐beta protein misfolding from molecular dynamics simulations with explicit water

Extracellular deposition of amyloid‐beta (Aβ) protein, a fragment of membrane glycoprotein called β‐amyloid precursor transmembrane protein (βAPP), is the major characteristic for the Alzheimer's disease (AD). However, the structural and mechanistic information of forming Aβ protein aggregates in a lag phase in cell exterior has been still limited. Here, we have performed multiple all‐atom molecular dynamics simulations for physiological 42‐residue amyloid‐beta protein (Aβ42) in explicit water to characterize most plausible aggregation‐prone structure (APS) for the monomer and the very early conformational transitions for Aβ42 protein misfolding process in a lag phase. Monitoring the early sequential conformational transitions of Aβ42 misfolding in water, the APS for Aβ42 monomer is characterized by the observed correlation between the nonlocal backbone H‐bond formation and the hydrophobic side‐chain exposure. Characteristics on the nature of the APS of Aβ42 allow us to provide new insight into the higher aggregation propensity of Aβ42 over Aβ40, which is in agreement with the experiments. On the basis of the structural features of APS, we propose a plausible aggregation mechanism from APS of Aβ42 to form fibril. The structural and mechanistic observations based on these simulations agree with the recent NMR experiments and provide the driving force and structural origin for the Aβ42 aggregation process to cause AD. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

Module Assembly for Designing Multivalent Mid‐Sized Inhibitors of Protein‐Protein Interactions

Developing clinically relevant synthetic agents that are capable of disrupting protein‐protein interactions (PPIs) is now a major goal of scientific research. In an effort to explore new methodologies that are applicable to the design of synthetic PPI inhibitors, we examined a strategy based on the assembly of small module compounds to create multivalent mid‐sized agents. This personal account describes three particular approaches based on module assembly: metal‐chelating‐based ligand assembly, covalent chemical ligation templated by a targeted protein, and bivalent inhibitor design for simultaneous targeting of the active pocket and protein surface. These strategies were shown to be useful for synthesizing minimally sized synthetic agents for targeting PPIs and may enable development of agents that are applicable to inhibition of intracellular PPIs.     

Self‐Assembled Cage‐Like Protein Structures

Proteins and protein‐based assemblies represent the most structurally and functionally diverse molecules found in nature. Protein cages, viruses and bacterial microcompartments are highly organized structures that are composed primarily of protein building blocks and play important roles in molecular ion storage, nucleic acid packaging and catalysis. The outer and inner surface of protein cages can be modified, either chemically or genetically, and the internal cavity can be used to template, store and arrange molecular cargo within a defined space. Owing to their structural, morphological, chemical and thermal diversity, protein cages have been investigated extensively for applications in nanotechnology, nanomedicine and materials science. Here we provide a concise overview of the most common icosahedral viral and nonviral assemblies, their role in nature, and why they are highly attractive scaffolds for the encapsulation of functional materials.     

Dead-end elimination for multistate protein design

Multistate protein design is the task of predicting the amino acid sequence that is best suited to selectively and stably fold to one state out of a set of competing structures. Computationally, it entails solving a challenging optimization problem. Therefore, notwithstanding the increased interest in multistate design, the only implementations reported are based on either genetic algorithms or Monte Carlo methods. The dead-end elimination (DEE) theorem cannot be readily transfered to multistate design problems despite its successful application to single-state protein design. In this article we propose a variant of the standard DEE, called type-dependent DEE. Our method reduces the size of the conformational space of the multistate design problem, while provably preserving the minimal energy conformational assignment for any choice of amino acid sequence. Type-dependent DEE can therefore be used as a preprocessing step in any computational multistate design scheme. We demonstrate the applicability of type-dependent DEE on a set of multistate design problems and discuss its strength and limitations.     

Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein‐folding liquid‐chromatography columns

Protein‐folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high‐performance size‐exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low‐urea gradient‐elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS–18% PAGE, Matrix‐Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI‐TOF‐MS) and the biological activity assay by HP‐RPLC. Copyright © 2014 John Wiley & Sons, Ltd.     

Mass spectrometric studies of potent inhibitors of farnesyl protein transferase--detection of pentameric noncovalent complexes

Farnesyl protein transferase (FPT) inhibition is an interesting and promising approach to noncytotoxic anticancer therapy. Research in this area has resulted in several orally active compounds that are in clinical trials. Electrospray ionization (ESI) time-of-flight mass spectrometry (TOF-MS) was used for the direct detection of a 95 182 Da pentameric noncovalent complex of alpha/beta subunits of FPT containing Zn, farnesyl pyrophosphate (FPP) and SCH 66336, a compound currently undergoing phase III clinical trials as an anticancer agent. It was noted that the desalting of protein samples was an important factor in the detection of the complex. This study demonstrated that the presence of FPP in the system was necessary for the detection of the FPT-inhibitor complex. No pentameric complex was detected in the spectrum when the experiment was carried out in the absence of the FPP. An indirect approach was also applied to confirm the noncovalent binding of SCH 66336 to FPT by the use of an off-line size exclusion chromatography followed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) for the detection of the inhibitor.     

Protein Interactions with Polymer Coatings and Biomaterials

Protein adsorption is considered to be the most important factor of the interaction between polymeric biomaterials and body fluids or tissues. Water‐mediated hydrophobic and hydration forces as well as electrostatic interactions are believed to be the major factors of protein adsorption. A systematic analysis of various monolayer systems has resulted in general guidelines, the so‐called “Whitesides rules”. These concepts have been successfully applied for designing various protein‐resistant surfaces and are being studied to expand the understanding of protein–material interactions beyond existing limitations. Theories on the mechanisms of protein adsorption are constantly being improved due to the fast‐developing analytical technologies. This Review is aimed at improving these empirical guidelines with regard to present theoretical and analytical advances. Current analytical methods to test mechanistic hypotheses and theories of protein–surface interactions will be discussed. Special focus will be given to state‐of‐the‐art bioinert and biospecific coatings and their applications in biomedicine.