Preference For Protein Research Articles

Structural differences between hypoxanthine phosphoribosyltransferase family members highlight opportunities for antiparasitic drug design in neglected diseases

Approaches to identify novel druggable targets for treating neglected diseases include computational studies that predict possible interactions of drugs and their molecular targets. Hypoxanthine phosphoribosyltransferase (HPRT) plays a central role in the purine salvage pathway. This enzyme is essential for the survival of the protozoan parasite T. cruzi, the causal agent of Chagas disease, and other parasites related to neglected diseases. Here we found dissimilar functional behaviours between TcHPRT and the human homologue, HsHPRT, in the presence of substrate analogues that can lie in differences in their oligomeric assemblies and structural features. To shed light on this issue, we carried out a comparative structural analysis between both enzymes. Our results show that HsHPRT is considerably more resistant to controlled proteolysis than TcHPRT. Moreover, we observed a variation in the length of two key loops depending on the structural arrangement of each protein (groups D1T1 and D1T1′). Such variations might be involved in inter-subunit communication or influencing the oligomeric state. Besides, to understand the molecular basis that govern D1T1 and D1T1′ folding groups, we explored the distribution of charges on the interaction surfaces of TcHPRT and HsHPRT, respectively. To know whether the rigidity degree bears effect on the active site, we studied the flexibility of both proteins. The analysis performed here illuminates the underlying reasons and significance behind each protein's preference for one or the other quaternary arrangement that can be exploited for therapeutic approaches.

Accurate positioning of functional residues with robotics-inspired computational protein design

SignificanceComputational protein design promises to advance applications in medicine and biotechnology by creating proteins with many new and useful functions. However, new functions require the design of specific and often irregular atom-level geometries, which remains a major challenge. Here, we develop computational methods that design and predict local protein geometries with greater accuracy than existing methods. Then, as a proof of concept, we leverage these methods to design new protein conformations in the enzyme ketosteroid isomerase that change the protein's preference for a key functional residue. Our computational methods are openly accessible and can be applied to the design of other intricate geometries customized for new user-defined protein functions.

X-ray crystallographic analysis of time-dependent binding of guanidine hydrochloride to HEWL: First steps during protein unfolding

Time-dependent binding of guanidine hydrochloride (GuHCl) to hen egg-white lysozyme (HEWL), and effects of this binding on the protein structure have been investigated by solving X-ray structures of crystalline complexes. The complexes have been prepared by soaking, for different periods of time, native lysozyme crystals in solutions containing 2.5M GuHCl. In the refined structures, the number of water molecules in the protein's first solvent shell has progressively decreased from 152 to 115, showing protein's preference for guanidinium over water. Guanidinium ions preferentially hydrogen bond with the backbone carbonyl oxygen atoms. In their van der Waals interactions, they do not show any preference for apolar residues. Guanidinium ions have replaced water molecules that form cages around exposed hydrophobic residues. Guanidinium binding has decreased the average length of water-water hydrogen bond by 0.1Å. The hydrogen bonds between main chain atoms have been weakened by GuHCl, and this may be the reason for increased potency of GuHCl compared to urea. Guanidinium binding destabilizes the β-domain by causing loss of hydrogen bonds involving Asn 59 side chain. Interestingly, this loss is almost identical to that observed in structures of amyloidogenic variants of human lysozyme. Compounds preventing this loss could be anti-amyloidogenic.

Assembly and Kinetic Folding Pathways of a Tetrameric β-Sheet Complex: Molecular Dynamics Simulations on Simplified Off-Lattice Protein Models

We have performed discontinuous molecular dynamic simulations of the assembly and folding kinetics of a tetrameric β-sheet complex that contains four identical four-stranded antiparallel β-sheet peptides. The potential used in the simulation is a hybrid Go-type potential characterized by the bias gap parameter g, an artificial measure of a model protein's preference for its native state, and the intermolecular contact parameter η, which measures the ratio of intermolecular to intramolecular native attractions. The formation of the β-sheet complex and its equilibrium properties strongly depend on the size of the intermolecular contact parameter η. The ordered β-sheet complex in the folded state and nonaligned β-sheets or tangled chains in the misfolded state are distinguished by measuring the squared radius of gyration R g 2 and the fraction of native contacts Q. The folding yield for the folded state is high at intermediate values of η, but is low at both small and large values of η. The folded state at small η is liquid-like, but is solid-like at both intermediate and large η. The misfolded state at small η contains nonaligned β-sheets and tangled chains with poor secondary structure at large η. Various folding pathways via dimeric and trimeric intermediates are observed, depending on η. Comparison with experimental results on protein aggregation indicates that intermediate η values are most appropriate for modeling fibril formation and small η values are most appropriate for modeling the formation of amorphous aggregates.

Protein Folding Pathways and Kinetics: Molecular Dynamics Simulations of β-Strand Motifs

The folding pathways and the kinetic properties for three different types of off-lattice four-strand antiparallel β-strand protein models interacting via a hybrid Go-type potential have been investigated using discontinuous molecular dynamics simulations. The kinetic study of protein folding was conducted by temperature quenching from a denatured or random coil state to a native state. The progress parameters used in the kinetic study include the squared radius of gyration R g 2, the fraction of native contacts within the protein as a whole Q, and between specific strands Q ab. In the time series of folding, the denatured proteins undergo a conformational change toward the native state. The model proteins exhibit a variety of kinetic folding pathways that include a fast-track folding pathway without passing through an intermediate and multiple pathways with trapping into more than one intermediate. The kinetic folding behavior of the β-strand proteins strongly depends on the native-state geometry of the model proteins and the size of the bias gap g, an artificial measure of a model protein's preference for its native state.

Heme distortions in sperm-whale carbonmonoxy myoglobin: correlations between rotational strengths and heme distortions in MD-generated structures.

We have investigated the effects of heme rotational isomerism in sperm-whale carbonmonoxymyoglobin using computational techniques. Several molecular dynamics simulations have been performed for the two rotational isomers A and B, which are related by a 180 degrees rotation around the alpha-gamma axis of the heme, of sperm-whale carbonmonoxy myoglobin in water. Both neutron diffraction and NMR structures were used as starting structures. In the absence of an experimental structure, the structure of isomer B was generated by rotating the heme in the structure of isomer A. Distortions of the heme from planarity were characterized by normal coordinate structural decomposition and by the angle of twist of the pyrrole rings from the heme plane. The heme distortions of the neutron diffraction structure were conserved in the MD trajectories, but in the NMR-based trajectories, where the heme distortions are less well defined, they differ from the original heme deformations. The protein matrix induced similar distortions on the hemes in orientations A and B. Our results suggest that the binding site prefers a particular macrocycle conformation, and a 180 degrees rotation of the heme does not significantly alter the protein's preference for this conformation. The intrinsic rotational strengths of the two Soret transitions, separated according to their polarization in the heme plane, show strong correlations with the ruffling deformation and the average twist angle of the pyrrole rings. The total rotational strength, which includes contributions from the chromophores in the protein, shows a weaker correlation with heme distortions.

The interactions between cellular retinol‐binding protein (CRBP‐I) and retinal: A vibrational spectroscopic study

Preresonance Raman difference spectra have been obtained for all-trans retinal in dilute CCl4 solution, complexed with cellular retinol-binding protein (CRBP-I) and retinol-binding protein (RBP). These spectra indicate that retinal is of a slightly more planar conformation within the binding pocket of CRBP-I than in solution or hydrophobically complexed with RBP. Compared to retinal in solution or bound to RBP, the conformation of the polyene tail of the retinal chromophore is perturbed from C8 through C11. This perturbation is probably due to the close proximity of the Lys40 in the CRBP-I binding pocket to the above-mentioned carbons. The C(DOUBLE BOND)O stretching vibration of bound retinal carbonyl has been found to shift from 1664 cm−1 solubilized in CCl4 to 1650 and 1645 cm−1 in RBP and CRBP-I, respectively, and significantly broadened in both cases. The frequency shift and broadening have been attributed to hydrogen bonding. These have been compared to calibrations of frequency shift (ΔνC(DOUBLE BOND)O) vs. ΔH and ΔG of all-trans retinal complexed with a series of phenol derivatives of incremental proton-donating ability as obtained by the relationship of van't Hoff. By this relationship, the binding enthalpy of the all-trans retinal carbonyl moiety bound to CRBP-I and RBP is −28.1 kJ/mol (−6.7 kcal/mol) and −23.5 kJ/mol (−5.6 kcal/mol), respectively. The free energy of binding of the retinal carbonyl bound to CRBP-I and RBP has been determined to be −10.5 kJ/mol (−2.5 kcal/mol) and −7.2 kJ/mol (−1.7 kcal/mol), respectively. The hydrogen-bonded C(DOUBLE BOND)O moiety of retinal complexed with CRBP-I accounts for a substantial (25%) but not overriding amount of the binding energy of CRBP-I for retinal, and it also accounts for the protein's preference for binding retinol. © 1997 John Wiley & Sons, Inc. Biospect 3: 131–142, 1997

The interactions between cellular retinol-binding protein (CRBP-I) and retinal: A vibrational spectroscopic study

Preresonance Raman difference spectra have been obtained for all-trans retinal in dilute CCl4 solution, complexed with cellular retinol-binding protein (CRBP-I) and retinol-binding protein (RBP). These spectra indicate that retinal is of a slightly more planar conformation within the binding pocket of CRBP-I than in solution or hydrophobically complexed with RBP. Compared to retinal in solution or bound to RBP, the conformation of the polyene tail of the retinal chromophore is perturbed from C8 through C11. This perturbation is probably due to the close proximity of the Lys40 in the CRBP-I binding pocket to the above-mentioned carbons. The C(DOUBLE BOND)O stretching vibration of bound retinal carbonyl has been found to shift from 1664 cm−1 solubilized in CCl4 to 1650 and 1645 cm−1 in RBP and CRBP-I, respectively, and significantly broadened in both cases. The frequency shift and broadening have been attributed to hydrogen bonding. These have been compared to calibrations of frequency shift (ΔνC(DOUBLE BOND)O) vs. ΔH and ΔG of all-trans retinal complexed with a series of phenol derivatives of incremental proton-donating ability as obtained by the relationship of van't Hoff. By this relationship, the binding enthalpy of the all-trans retinal carbonyl moiety bound to CRBP-I and RBP is −28.1 kJ/mol (−6.7 kcal/mol) and −23.5 kJ/mol (−5.6 kcal/mol), respectively. The free energy of binding of the retinal carbonyl bound to CRBP-I and RBP has been determined to be −10.5 kJ/mol (−2.5 kcal/mol) and −7.2 kJ/mol (−1.7 kcal/mol), respectively. The hydrogen-bonded C(DOUBLE BOND)O moiety of retinal complexed with CRBP-I accounts for a substantial (25%) but not overriding amount of the binding energy of CRBP-I for retinal, and it also accounts for the protein's preference for binding retinol. © 1997 John Wiley & Sons, Inc. Biospect 3: 131–142, 1997

Crystal structure of the dipeptide binding protein from Escherichia coli involved in active transport and chemotaxis

The Escherichia coli periplasmic dipeptide binding protein functions in both peptide transport and taxis toward peptides. The structure of the dipeptide binding protein in complex with Gly-Leu (glycyl-L-leucine) has been determined at 3.2 A resolution. The binding site for dipeptides is designed to recognize the ligand's backbone while providing space to accommodate a variety of side chains. Some repositioning of protein side chains lining the binding site must occur when the dipeptide's second residue is larger than leucine. The protein's fold is very similar to that of the Salmonella typhimurium oligopeptide binding protein, and a comparison of the structures reveals the structural basis for the dipeptide binding protein's preference for shorter peptides.

Site-specific Mutants of Oncomodulin: 1H NMR and optical stopped-flow studies of the effect on the metal binding properties of an Asp59 → Glu59 substitution in the calcium-specific site

Abstract High resolution 1H nuclear magnetic resonance spectroscopy and optical stopped-flow techniques have been used to study the metal binding properties of a site-specific mutant of bacterial recombinant oncomodulin in which glutamate has replaced a liganding aspartate at position 59 in the CD calcium-binding site. In particular we have followed the replacement of calcium by lutetium in bacterial recombinant oncomodulin and D59E oncomodulin to provide a measure of the protein's preferences for metal ions of different ionic radii. The result of the Asp----Glu substitution is to make the mutant oncomodulin more similar to rat parvalbumin in terms of its relative CD- and EF-domain affinities for lutetium(III), that is to increase its affinity for metal ions with smaller ionic radii. This finding supports the original hypothesis that the presence of Asp at sequence position 59 is an important factor in the reduced preference of the CD site of oncomodulin for smaller metals such as magnesium (Williams, T. C., Corson, D. C., Sykes, B. D., and MacManus, J. P. (1987) J. Biol. Chem. 262, 6248-6256). However, our studies show that both the CD and the EF sites are affected by this single residue substitution suggesting that many factors play a role in the metal binding affinity and interaction between the two sites.