Azobenzene-modified poly(l-glutamic acid) (AZOPLGA): its conformational and photodynamic properties

Azobenzene-modified poly(l-glutamic acid) (AZOPLGA) polymers with 22 and 35 mol % of azo chromophores in the side chains have been synthesized by condensing 4-methoxy-4'-aminoazobenzene and poly(l-glutamic acid). These polymers have been characterized by NMR, FT-IR, and UV-visible spectroscopic techniques. The conformational features of the polymer backbone chains in the films that were cast from the polymer solutions prepared in different solvents have been investigated by circular dichroism spectroscopy. Experimental data suggested that the thermal cis-trans relaxation and photoinduced birefringence, which are related to the azo chromophores in the side chains of polymer, are not affected by the conformations of polymer backbones. However, the modulations of the surface relief gratings, the result of photoinduced mass transport process, recorded on these polymers are sensitive to polymer main chain conformation, as well as the degree of functionalization.     

Poly(β-amino ester)-nanoparticle mediated transfection of retinal pigment epithelial cells in vitro and in vivo

A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, might be excellent targets for gene delivery as treatment. A major challenge in non-viral gene delivery remains finding a safe and effective delivery system. Poly(beta-amino ester)s (PBAEs) have shown great potential as gene delivery reagents because they are easily synthesized and they transfect a wide variety of cell types with high efficacy in vitro. We synthesized a combinatorial library of PBAEs and evaluated them for transfection efficacy and toxicity in retinal pigment epithelial (ARPE-19) cells to identify lead polymer structures and transfection formulations. Our optimal polymer (B5-S5-E7 at 60 w/w polymer:DNA ratio) transfected ARPE-19 cells with 44±5% transfection efficacy, significantly higher than with optimized formulations of leading commercially available reagents Lipofectamine 2000 (26±7%) and X-tremeGENE HP DNA (22±6%); (p<0.001 for both). Ten formulations exceeded 30% transfection efficacy. This high non-viral efficacy was achieved with comparable cytotoxicity (23±6%) to controls; optimized formulations of Lipofectamine 2000 and X-tremeGENE HP DNA showed 15±3% and 32±9% toxicity respectively (p>0.05 for both). Our optimal polymer was also significantly better than a gold standard polymeric transfection reagent, branched 25 kDa polyethyleneimine (PEI), which achieved only 8±1% transfection efficacy with 25±6% cytotoxicity. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×10(3)-fold and 1.5±0.7×10(3)-fold increased GFP expression in the retinal pigment epithelium (RPE)/choroid and neural retina respectively, compared to injection of DNA alone (p?=?0.003 for RPE/choroid, p<0.001 for neural retina). The successful transfection of the RPE in vivo suggests that these nanoparticles could be used to study a number of genetic diseases in the laboratory with the potential to treat debilitating eye diseases.     

Solving and analyzing side-chain positioning problems using linear and integer programming

MOTIVATION: Side-chain positioning is a central component of homology modeling and protein design. In a common formulation of the problem, the backbone is fixed, side-chain conformations come from a rotamer library, and a pairwise energy function is optimized. It is NP-complete to find even a reasonable approximate solution to this problem. We seek to put this hardness result into practical context. RESULTS: We present an integer linear programming (ILP) formulation of side-chain positioning that allows us to tackle large problem sizes. We relax the integrality constraint to give a polynomial-time linear programming (LP) heuristic. We apply LP to position side chains on native and homologous backbones and to choose side chains for protein design. Surprisingly, when positioning side chains on native and homologous backbones, optimal solutions using a simple, biologically relevant energy function can usually be found using LP. On the other hand, the design problem often cannot be solved using LP directly; however, optimal solutions for large instances can still be found using the computationally more expensive ILP procedure. While different energy functions also affect the difficulty of the problem, the LP/ILP approach is able to find optimal solutions. Our analysis is the first large-scale demonstration that LP-based approaches are highly effective in finding optimal (and successive near-optimal) solutions for the side-chain positioning problem.     

Backbone dependency further improves side chain prediction efficiency in the Energy‐based Conformer Library (bEBL)

Side chain optimization is an integral component of many protein modeling applications. In these applications, the conformational freedom of the side chains is often explored using libraries of discrete, frequently occurring conformations. Because side chain optimization can pose a computationally intensive combinatorial problem, the nature of these conformer libraries is important for ensuring efficiency and accuracy in side chain prediction. We have previously developed an innovative method to create a conformer library with enhanced performance. The Energy‐based Library (EBL) was obtained by analyzing the energetic interactions between conformers and a large number of natural protein environments from crystal structures. This process guided the selection of conformers with the highest propensity to fit into spaces that should accommodate a side chain. Because the method requires a large crystallographic data‐set, the EBL was created in a backbone‐independent fashion. However, it is well established that side chain conformation is strongly dependent on the local backbone geometry, and that backbone‐dependent libraries are more efficient in side chain optimization. Here we present the backbone‐dependent EBL (bEBL), whose conformers are independently sorted for each populated region of Ramachandran space. The resulting library closely mirrors the local backbone‐dependent distribution of side chain conformation. Compared to the EBL, we demonstrate that the bEBL uses fewer conformers to produce similar side chain prediction outcomes, thus further improving performance with respect to the already efficient backbone‐independent version of the library. Proteins 2014; 82:3177–3187. © 2014 Wiley Periodicals, Inc.     

Ternary complexes comprising polyphosphoramidate gene carriers with different types of charge groups improve transfection efficiency

To understand the influence of charge groups on transfection mediated by polymer complexes, we have synthesized a series of biodegradable and cationic polyphosphoramidates (PPAs) with an identical backbone but different side chains. Our previous study showed that PPA with a spermidine side chain (PPA-SP) showed high transfection efficiency in culture, whereas PPAs with secondary, tertiary, and quaternary amino groups were significantly less efficient. To investigate whether the coexistence of 1 degrees amino charge groups with 3 degrees and 2 degrees amino charge groups in the DNA/polymer complexes would enhance their transfection efficiency, we evaluated a ternary complex system containing DNA and PPAs with 1 degrees amino groups (PPA-SP) and 3 degrees amino groups (PPA-DMA) and a quaternary complex system containing DNA and PPAs with 1 degrees and 2 degrees and 3 degrees amino groups (PPA-EA/PPA-MEA/PPA-DMA), respectively. Ternary complexes mediated 20 and 160 times higher transfection efficiency in COS-7 cells than complexes of DNA with PPA-SP or PPA-DMA alone, respectively. Similarly, quaternary complexes exhibited 8-fold higher transfection efficiency than PPA-EA/DNA complexes. The mechanism of enhancement in transfection efficiency by the mixture carriers appears to be unrelated to the particle size, zeta potential, or DNA uptake. The titration characterization and the transfection experiments using a proton pump inhibitor suggest that the enhancement effect is unlikely due to the slightly improved buffering capacity of the mixture over PPA-SP. This approach represents a simple strategy of developing polymeric gene carriers and understanding the mechanisms of polymer-mediated gene transfer.     

Facilitation of gene transfection with well-defined degradable comb-shaped poly(glycidyl methacrylate) derivative vectors

Recently, we reported that ethanolamine (EA)-functionalized poly(glycidyl methacrylate) (PGMA) vectors (PGEAs) can produce good transfection efficiency, while exhibiting very low toxicity. Further improvement in degradability and transfection efficiency of the PGEA vectors will facilitate their application in gene therapy. Comb-shaped cationic copolymers have been of interest and importance as nonviral gene carriers. Herein, the degradable high-molecular-weight comb-shaped PGEA vectors (c-PGEAs) composed of the low-molecular-weight PGEA backbone and side chains were prepared by a combination of atom transfer radical polymerization (ATRP) and ring-opening reactions. The PGEA side chains were linked with the PGEA backbones via hydrolyzable ester bonds. Such comb-shaped c-PGEA vectors possessed the degradability, good pDNA condensation ability, low cytotoxicity, and good buffering capacity. More importantly, the comb-shaped c-PGEA vectors could enhance the gene expression levels. Moreover, the PGEA side chains of c-PGEA could also be copolymerized with some poly(poly(ethylene glycol)ethyl ether methacrylate) species to further improve the gene delivery system.     

Effects of side chain configuration and backbone spacing on the gene delivery properties of lysine-derived cationic polymers

A series of lysine-based oligomers (18 residues) that differ in side chain configuration or side chain spacing along the backbone was tested for DNA transfection activity. Although materials constructed from lysine are not the most effective polymeric transfection agents, we have chosen L-lysine-based molecules as a starting point because this system allows us to examine the functional effects of incremental changes in polycation structure. The oligomer constructed from beta(3)-homolysine (beta(3)-hLys) and that from alpha-D-lysine were superior to an alpha-L-lysine 18-mer in gene delivery assays. This improved activity is attributed to the fact that the alpha-L-peptide is a protease substrate while the other 18-mers are not. This conclusion is supported by the effects of chloroquine on transfection activity, based on the protease inhibition activity of chloroquine. To our knowledge, these results represent the first direct comparison of a D-lysine oligomer with an L-lysine oligomer in the context of gene delivery. Poly(beta(3)-hLys) was synthesized from the ring opening polymerization of the corresponding lactam. The DNA transfection ability of this polymer was compared with that of commercially available poly(L-lysine) (PLL). In each case the polymer was more active than the corresponding oligomer.     

Criteria that discriminate between native proteins and incorrectly folded models

Various theoretical concepts, such as free energy potentials, electrostatic interaction potentials, atomic packing, solvent-exposed surface, and surface charge distribution, were tested for their ability to discriminate between native proteins and misfolded protein models. Misfolded models were constructed by introducing incorrect side chains onto polypeptide backbones: side chains of the alpha-helical hemerythrin were modeled on the beta-sheeted backbone of immunoglobulin VL domain, whereas those of the VL domain were similarly modeled on the hemerythrin backbone. CONGEN, a conformational space sampling program, was used to construct the side chains, in contrast to the previous work, where incorrect side chains were modeled in all trans conformations. Capability of the conformational search procedure to reproduce native conformations was gauged first by rebuilding (the correct) side chains in hemerythrin and the VL domain: constructs with r.m.s. differences from the x-ray side chains 2.2-2.4 A were produced, and many calculated conformations matched the native ones quite well. Incorrectly folded models were then constructed by the same conformational protocol applied to incorrect amino acid sequences. All CONGEN constructs, both correctly and incorrectly folded, were characterized by exceptionally small molecular surfaces and low potential energies. Surface charge density, atomic packing, and Coulomb formula-based electrostatic interactions of the misfolded structures and the correctly folded proteins were similar, and therefore of little interest for diagnosing incorrect folds. The following criteria clearly favored the native structures over the misfolded ones: 1) solvent-exposed side-chain nonpolar surface, 2) number of buried ionizable groups, and 3) empirical free energy functions that incorporate solvent effects.     

Structure of branched poly-alpha-amino acids in dimethylformamide. II. Viscosity, sedimentation, and diffusion

The intrinsic viscosity, sedimentation and diffusion of a series of branched, multichain poly-α-amino acids having a poly(L -lysine) backbone and poly(γ-benzyl L -glutamate) and poly (β-benzyl L -aspartate) side chains was studied at room temperature in dimethylformamide. The molecules were found to be extremely compact structures in which the molecular backbone is either lying along the major axis in a slightly twisted configuration (the longer the side chain the smaller the twist) or is coiled up in the form of a disk with backbone and side chains coplanar. Heat treatment (to 70°C.) introduces only small changes in the hydrodynamic parameters showing that the heat-labile aggregates detected by light scattering are reversibly broken up during the hydrodynamic measurements. The above structural information concerns the initial metastable conformation of the molecules which is irreversibly destroyed by heat treatment.     

A combinatorial approach to protein docking with flexible side chains.

Rigid-body docking approaches are not sufficient to predict the structure of a protein complex from the unbound (native) structures of the two proteins. Accounting for side chain flexibility is an important step towards fully flexible protein docking. This work describes an approach that allows conformational flexibility for the side chains while keeping the protein backbone rigid. Starting from candidates created by a rigid-docking algorithm, we demangle the side chains of the docking site, thus creating reasonable approximations of the true complex structure. These structures are ranked with respect to the binding free energy. We present two new techniques for side chain demangling. Both approaches are based on a discrete representation of the side chain conformational space by the use of a rotamer library. This leads to a combinatorial optimization problem. For the solution of this problem, we propose a fast heuristic approach and an exact, albeit slower, method that uses branch-and-cut techniques. As a test set, we use the unbound structures of three proteases and the corresponding protein inhibitors. For each of the examples, the highest-ranking conformation produced was a good approximation of the true complex structure.     

An immunomodulating pectic polymer from Glinus oppositifolius

An immunomodulating pectic polymer, GOA1, obtained from the aerial parts of the Malian medicinal plant Glinus oppositifolius (L.) Aug. DC. (Aizoaceae) has previously been reported to consist of arabinogalactans type I and II, probably linked to a rhamnogalacturonan backbone. To further elucidate the structure of the polymer GOA1, enzymatic degradation studies and weak acid hydrolysis were performed. Five different glycosidases were used, endo-alpha-D-(1-->4)-polygalacturonase, exo-alpha-L-arabinofuranosidase, endo-alpha-L-(1-->5)-arabinanase, endo-beta-D-(1-->4)-galactanase and exo-beta-D-galactosidase. It appears that GOA1 may contain a structural moiety consisting of a 1,3-linked galactopyranosyl (Galp) main chain with 1,6-linked Galp side chains attached to position 6 of the main chain. The 1,6-linked Galp side chain may be branched in position 3 with arabinofuranosyl (Araf) side chains. A 1,4-linked Galp backbone which might carry side chains or glycosyl units attached to position 3 is also a structural element in the polymer. We further show that GOA1 induce proliferation of B cells and the secretion of IL-1beta by macrophages, in addition to a marked increase of mRNA for IFN-gamma in NK-cells. To elucidate structure-activity relations the native polymer and the digested fractions were tested for complement fixing activity and intestinal immune stimulating activity. The partial removal of Araf residues after enzymatic degradations did not affect the bioactivities, while the acid hydrolysed fraction showed reduced complement fixing activity. A decrease in Araf units, 1,3,6-linked Galp units and a partial hydrolysed rhamnogalacturonan backbone, in addition to a reduction in molecular weight are factors that might have contributed to reduced bioactivity.     

Cationic spacer arm design strategy for control of antimicrobial activity and conformation of amphiphilic methacrylate random copolymers

Antimicrobial and hemolytic activities of amphiphilic random copolymers were modulated by the structure of the cationic side chain spacer arms, including 2-aminoethylene, 4-aminobutylene, and 6-aminohexylene groups. Cationic amphiphilic random copolymers with ethyl methacrylate (EMA) comonomer were prepared with a range of comonomer fractions, and the library of copolymers was screened for antimicrobial and hemolytic activities. Copolymers with 4-aminobutylene cationic side chains showed an order of magnitude enhancement in their antimicrobial activity relative to those with 2-aminoethylene spacer arms, without causing adverse hemolysis. When the spacer arms were further elongated to hexylene, the copolymers displayed potent antimicrobial and hemolytic activities. The 4-aminobutylene side chain appears to be the optimal spacer arm length for maximal antimicrobial potency and minimal hemolysis, when combined with hydrophobic ethylmethacrylate in a roughly 70/30 ratio. The copolymers displayed relatively rapid bactericidal kinetics and broad-spectrum activity against a panel of Gram-positive and Gram-negative bacteria. The effect of the spacer arms on the polymer conformation in the membrane-bound state was investigated by molecular dynamics simulations. The polymer backbones adopt an extended chain conformation, parallel to the membrane surface. A facially amphiphilic conformation at the membrane surface was observed, with the primary ammonium groups localized at the lipid phoshophate region and the nonpolar side chains of EMA comonomers buried in the hydrophobic membrane environment. This study demonstrates that the antimicrobial activity and molecular conformation of amphiphilic methacrylate random copolymers can be modulated by adjustment of cationic side chain spacer arms.     

Addition of "charge-shifting" side chains to linear poly(ethyleneimine) enhances cell transfection efficiency

We reported recently that the addition of ester-functionalized, "charge-shifting" side chains to linear poly(ethyleneimine) (LPEI) can be used to design polyamines that promote both self-assembly and self-disassembly with DNA in aqueous environments. This investigation sought to characterize the influence of charge-shifting side chains on the ability of LPEI to mediate cell transfection and understand the extent to which increases (or decreases) in levels of transfection could be understood in terms of time-dependent changes in the net charges of these polymers. We report that the addition of "charge-shifting" side chains to LPEI leads to significant increases in levels of LPEI-mediated transfection. In particular, polymer 1e, functionalized with 20 mol % ester-functionalized side chains, mediates levels of transgene expression in vitro up to 8-fold higher than LPEI. Experiments using an amide-functionalized analog of polymer 1e demonstrated that the esters in polymer 1e play an important role in promoting increased levels of transfection. These results, in combination with the results of additional gel electrophoresis experiments, provide support for the view that increases in transfection result from time-dependent changes in the net charge of polymer 1e and the disruption of ionic interactions in polyplexes. Additional support for this view is provided by the results of confocal microscopy experiments and measurements of fluorescence resonance energy transfer, which suggest that polymer 1e promotes the disruption of polyplexes in intracellular environments effectively. The approach reported here provides a means of addressing one important "late-stage" obstacle to polyplex-mediated transfection (polyplex unpackaging). If integrated successfully with methods that have been developed to address other important barriers to transfection, this general approach could lead to the development of multifunctional polyplexes that mimic more effectively the range of functions of viruses as agents for the delivery of DNA.     

The interplay between transient α-helix formation and side chain rotamer distributions in disordered proteins probed by methyl chemical shifts

The peptide backbones of disordered proteins are routinely characterized by NMR with respect to transient structure and dynamics. Little experimental information is, however, available about the side chain conformations and how structure in the backbone affects the side chains. Methyl chemical shifts can in principle report the conformations of aliphatic side chains in disordered proteins and in order to examine this two model systems were chosen: the acid denatured state of acyl-CoA binding protein (ACBP) and the intrinsically disordered activation domain of the activator for thyroid hormone and retinoid receptors (ACTR). We find that small differences in the methyl carbon chemical shifts due to the γ-gauche effect may provide information about the side chain rotamer distributions. However, the effects of neighboring residues on the methyl group chemical shifts obscure the direct observation of γ-gauche effect. To overcome this, we reference the chemical shifts to those in a more disordered state resulting in residue specific random coil chemical shifts. The (13)C secondary chemical shifts of the methyl groups of valine, leucine, and isoleucine show sequence specific effects, which allow a quantitative analysis of the ensemble of χ(2)-angles of especially leucine residues in disordered proteins. The changes in the rotamer distributions upon denaturation correlate to the changes upon helix induction by the co-solvent trifluoroethanol, suggesting that the side chain conformers are directly or indirectly related to formation of transient α-helices.     

Structure of Plant Cell Walls : XII. Identification of Seven Differently Linked Glycosyl Residues Attached to O-4 of the 2,4-Linked l-Rhamnosyl Residues of Rhamnogalacturonan I

Seven differently linked glycosyl residues have been found to be glycosidically linked to O-4 of the branched 2,4-linked l-rhamnosyl residues contained in the rhamnosyl and galacturonosyl backbone of the cell wall pectic polysaccharide rhamnogalacturonan I. These seven glycosyl residues are, therefore, the first residues of at least seven different side chains attached to the rhamnogalacturonan backbone. These first side chain glycosyl residues are 5-linked l-arabinofuranosyl and terminal 3-, 4-, 6-, 2,6-, and 3,6-linked d-galactopyranosyl residues. The existence of at least seven different side chains in rhamnogalacturonan I indicates that rhamnogalacturonan I is either an exceedingly complex polysaccharide or that rhamnogalacturonan I is a family of polysaccharides with similar or identical rhamnogalacturonan backbones substituted with different side chains.     

New characteristic signatures from time-dependent static light scattering during polymer depolymerization,with application to proteoglycan subunit degradation

Models were developed for the time-dependent light scattering intensity for simply branched (“comb”) polymers undergoing one or more of three distinguishable degradation mechanisms: (a) stripping off the side chains, (b) randomly degrading off the side chains, and (c) randomly degrading the backbone. The model equations were applied to the analysis of different types of degradation of simply branched biopolymers—bovine nasal cartilage proteoglycan subunits (or “monomers”); NaOH stripped off the glycosaminoglycan (GAG) chains from the protein backbone [mechanism (a)], whereas hyaluronidase seemed to randomly cleave the GAG side chains [mechanism (b)], and HCl both stripped the GAG side chains and randomly cleaved the protein backbone [combined mechanisms (a) and (c)]. The reactions were followed with time-dependent multiangle, static light scattering. The time-resolved total scattering technique allowed degradation rate constants and percentage of material in the branched polymer backbone and side chains to be determined, in addition to the mechanisms involved. These new time-dependent light scattering profiles are added to the growing library of functions from which deductions can be made concerning polymer structure and associated degradation mechanisms and kinetics. These conclusions, drawn from time-dependent “batch” light scattering, are substantiated by preliminary size exclusion chromatography results and chemical binding assays for sulfated GAGs. © 1995 John Wiley & Sons, Inc.     

Dynamic simulations of the molecular conformations of wild type and mutant xanthan polymers suggest that conformational differences may contribute to observed differences in viscosity

Xanthan gum is an exopolysaccharide secreted by the bacterium Xanthamonas campestris whose ability to make solutions viscous at low concentrations and over a pH and temperature range have generated much interest in both academic and industrial environments. Mutant Xanthamonas strains have been derived that produce xanthan gums with an altered or variant subunit chemical structure and different measured viscosities when compared with the wild type (wt) form of the polymer. Two variant gums were targeted as potentially interesting in this study, these being the nonacetylated tetramer (natct; and the acetylated tetramer (atet), which both lack a side-chain terminal mannose residue and in one case (natet) lacks an acetate group on an internal mannose residue. Solutions of these tetrameric gums possess viscosities higher (natet) and lower (atet) than the wt gum, and therefore we have attempted to determine whether these molecules possess unique conformational preferences when compared with the wt and with each other. In this manner we can initiate an understanding of how a polysaccharide's conformation contributes to its solution properties. The GEGOP software permits a sampling of the static and dynamic equilibrium states of carbohydrate molecules, and this software was employed to calculate equilibrium states of representative oligosaccharides with chemical structures representative of xanthan-like molecules. Energy minimization techniques revealed similar local minima for all three molecules. Some of these minima are comprised of elongate backbone conformations (A type) in which side chains fold onto backbone surfaces. Other minima with A backbones possessed side chains in less intimate backbone contact especially when calculations were performed with a low dielectric constant. This phenomenon was particularly pronounced in the wt molecule where an increased number of negatively charged side-chain residues experience charge repulsion resulting in reduced side-chain-backbone contact. Metropolis Monte Carlo (MMC) dynamic simulations performed with an elevated temperature factor (1000 K) allowed a better qualitative representation of conformational space than 300 K simulations. Employing a nonhierarchical cluster analysis method (population density profile: PDP) coupled with a classification scheme, it was possible to partition resulting MMC data sets into conformational families. This analysis revealed that in simulations performed with different dielectric constant values (10, 25, and ∞) all molecules possessed primarily A-type backbones. Less elongate, more open helical backbone forms (B, C, D, J, and Flat-a) did occur during the simulations but were populated to a lesser extent. In the natet molecule significantly open helical backbones existed (E, F, G, H, and I) that did not occur in the lower viscosity wt and atet molecules. PDP clustering methods and subsequent conformational classification applied to the first residue (mannose) of the side chain permitted a determination of side-chain orientation. Comparison of all three molecules indicated a larger population of side-chain conformational families in less direct backbone contact for the wt molecule than either of the variant molecules (natet/atet) suggesting that the side chains in the wt are more flexible. Thus, a major conformational difference between the high viscosity natet. In addition, the significant difference between the higher viscosity wt and the lower viscosity atet is the increase side-chain flexibility in the wt. We hypothesize that conformational differences of this kind could form a partial explanation of the observed differences in viscosity between these xanthan-like polymers. © 1996 John Wiley & Sons, Inc.     

NMR characterization of the electrostatic interaction of the basic residues in HDGF and FGF2 during heparin binding

Electrostatic interaction is a major driving force in the binding of proteins to highly acidic glycosaminoglycan, such as heparin. Although NMR backbone chemical shifts have generally been used to identify the heparin-binding site on a protein, however, there is no correlation between the binding free energies and the perturbed backbone chemical shifts for individual residues. The binding event occurs at the end of a side chain of basic residue, and does not require causing significant alterations in the backbone environment at a distance of multiple bonds. We used the H₂CN NMR pulse sequence to detect heparin binding through the side-chain resonances Hε–Cε–Nζ of Lys and Hδ–Cδ–Nε of Arg in the two proteins of hepatoma-derived growth factor (HDGF) and basic fibroblast growth factor (FGF2). H₂CN titration experiments revealed chemical shift perturbations in the side chains, which were correlated with the free energy changes in various mutants. The residues K19 in HDGF and K125 in FGF2 demonstrated the most significant perturbations, consistent with our previous observation that the two residues are crucial for binding. The result suggests that H₂CN NMR provides a precise evaluation for the electrostatic interactions. The discrepancy observed between backbone and side chain chemical shifts is correlated to the solvent accessibility of residues that the K19 and K125 backbones are highly buried with the restricted backbone conformation and are not strongly affected by the events at the end of the side chains.     

Evaluation of short-range interactions as secondary structure energies for protein fold and sequence recognition.

Short-range interactions for secondary structures of proteins are evaluated as potentials of mean force from the observed frequencies of secondary structures in known protein structures which are assumed to have an equilibrium distribution with the Boltzmann factor of secondary structure energies. A secondary conformation at each residue position in a protein is described by a tripeptide, including one nearest neighbor on each side. The secondary structure potentials are approximated as additive contributions from neighboring residues along the sequence. These are part of an empirical potential to provide a crude estimate of protein conformational energy at a residue level. Unlike previous works, interactions are decoupled into intrinsic potentials of residues, potentials of backbone-backbone interactions, and of side chain-backbone interactions. Also interactions are decoupled into one-body, two-body, and higher order interactions between peptide backbone and side chain and between backbones. These decouplings are essential to correctly evaluate the total secondary structure energy of a protein structure without overcounting interactions. Each interaction potential is evaluated separately by taking account of the correlation in the amino acid order of protein sequences. Interactions among side chains are neglected, because of the relatively limited number of protein structures. Proteins 1999;36:347-356. Published 1999 Wiley-Liss, Inc.