Sairam S. Mallajosyula

Influence of Solvent and Intramolecular Hydrogen Bonding on the Conformational Properties of O-Linked Glycopeptides

A detailed investigation of the conformational properties of all the biologically relevant O-glycosidic linkages using the Hamiltonian replica exchange (HREX) simulation methodology and the recently developed CHARMM carbohydrate force field parameters is presented. Fourteen biologically relevant O-linkages between the five sugars N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), D-glucose (Glc), D-mannose (Man), and L-fucose (Fuc) and the amino acids serine and threonine were studied. The force field was tested by comparing the simulation results of the model glycopeptides to various NMR (3)J coupling constants, NOE distances, and data from molecular dynamics with time-averaged restraints (tar-MD). The results show the force field to be in overall agreement with experimental and previous tar-MD simulations, although some small limitations are identified. An in-depth hydrogen bond and bridging water analysis revealed an interplay of hydrogen bonding and bridge water interactions influencing the geometry of the underlying peptide backbone, with the O-linkages favoring extended β-sheet and polyproline type II (PPII) conformations over the compact α(R)-helical conformation. The newly developed parameters were also able to identify hydrogen bonding and water mediated interactions between O-linked sugars and proteins. These results indicate that the newly developed parameters in tandem with HREX conformational sampling provide the means to study glycoproteins in the absence of targeted NMR restraint data.

Organometallic vanadium-borazine systems: efficient one-dimensional half-metallic spin filters

Using density functional theory, we have investigated the electronic and magnetic properties of finite-size as well as infinitely periodic organometallic vanadium-borazine systems (Vn(B3N3H6)n + 1 for their possible applications in spintronics devices. From our calculations, we find the Vn(B3N3H6)n + 1 systems to be structurally more stable in comparison to their isoelectronic benzene counterparts (Vn(C6H6)n + 1). All the Vn(B3N3H6)n + 1 systems are found to be ferromagnetically stabilized, with the infinite one-dimensional [V(B3N3H6)]∞ wire exhibiting robust half-metallic behaviour. The Vn(B3N3H6)n + 1clusters are also found to exhibit efficient spin filter properties when coupled to graphene electrodes.

Conformational Properties of α- or β-(1→6)-Linked Oligosaccharides: Hamiltonian Replica Exchange MD Simulations and NMR Experiments

Conformational sampling for a set of 10 α- or β-(1→6)-linked oligosaccharides has been studied using explicit solvent Hamiltonian replica exchange (HREX) simulations and NMR spectroscopy techniques. Validation of the force field and simulation methodology is done by comparing calculated transglycosidic J coupling constants and proton–proton distances with the corresponding NMR data. Initial calculations showed poor agreement, for example, with >3 Hz deviation of the calculated 3J(H5,H6R) values from the experimental data, prompting optimization of the ω torsion angle parameters associated with (1→6)-linkages. The resulting force field is in overall good agreement (i.e., within ∼0.5 Hz deviation) from experimental 3J(H5,H6R) values, although some small limitations are evident. Detailed hydrogen bonding analysis indicates that most of the compounds lack direct intramolecular H-bonds between the two monosaccharides; however, minor sampling of the O6···HO2′ hydrogen bond is present in three compounds. The results verify the role of the gauche effect between O5 and O6 atoms in gluco- and manno-configured pyranosides causing the ω torsion angle to sample an equilibrium between the gt and gg rotamers. Conversely, galacto-configured pyranosides sample a population distribution in equilibrium between gt and tg rotamers, while the gg rotamer populations are minor. Water radial distribution functions suggest decreased accessibility to the O6 atom in the (1→6)-linkage as compared to the O6′ atom in the nonreducing sugar. The role of bridging water molecules between two sugar moieties on the distributions of ω torsion angles in oligosaccharides is also explored.

Fluctuations at the base pair level effecting charge transfer in DNA.

We investigate the energetics of the basepair degrees of freedom and their effects on the overall charger transfer processes in DNA. We find that the rotational and translational basepair degrees of freedom can be broadly classified into soft and hard vibrational modes, with the stiffness of the modes depending on the nature of the basepair. We also find that the intrabasepair charge transfer, in the A:T and G:C basepairs, is strongly influenced by open (sigma) and stretch (Sy) vibrational modes. Our calculations for the AT-GC and GC-AT dinucleotide steps suggest that the fluctuations in the G:C basepair strongly influence the site energies when compared to fluctuations in the A:T basepair. However, for both the dinucleotide steps, we find that the charge transfer integrals are strongly influenced by the fluctuations at the basepair level. Overall, our studies suggest that for a better understanding of the overall charge transfer processes, it is important to account for the basepair fluctuations.

Perturbation of Long-Range Water Dynamics as the Mechanism for the Antifreeze Activity of Antifreeze Glycoprotein

Very little is known about the mechanism of antifreeze action of antifreeze glycoproteins (AFGPs) present in Antarctic teleost fish. Recent NMR and CD studies assisted with total synthesis of synthetic AFGP variants have provided insight into the structure of short AFGP glycopeptides, though the observations did not yield information on the antifreeze mechanism of action. In this study, we use Hamiltonian replica exchange (HREX) molecular dynamics simulations to probe the structure and surrounding aqueous environments of both the natural (AFGP8) and synthetic (s-AFGP4) AFGPs. AFGPs can adopt both amphiphilic and pseudoamphiphilic conformations, the preference of which is related to the proline content of the peptide. The arrangement of carbohydrates allows the hydroxyl groups on terminal galactose units to form stable water bridges which in turn influence the hydrogen-bond network, structure, and dynamics of the surrounding solvent. Interestingly, these local effects lead to the perturbation of the tetrahedral environment for water molecules in hydration layers far (10.0–12.0 Å) from the AFGPs. This structure-induced alteration of long-range hydration dynamics is proposed to be the major contributor to antifreeze activity, a conclusion that is in line with terahertz spectroscopy experiments. The detailed structure–mechanism correlation provided in this study could lead to the design of better synthetic AFGP variants.

Conformational determinants of the activity of Antiproliferative factor glycopeptide

The antiproliferative factor (APF) involved in interstitial cystitis is a glycosylated nonapeptide (TVPAAVVVA) containing a sialylated core 1 α-O-disaccharide linked to the N-terminal threonine. The chemical structure of APF was deduced using spectroscopic techniques and confirmed using total synthesis. The synthetic APF provided a platform to study amino acid modifications and their effect on APF activity, based on which a structure-activity relationship (SAR) for APF activity was previously proposed. However, this SAR model could not explain the change in activity associated with minor alterations in the peptide sequence. Presented is computational analysis of 14 APF derivatives to identify structural trends from which a more detailed SAR is obtained. The APF activity is found to be dictated by the close interplay between carbohydrate-peptide and peptide-peptide interactions. The former involves hydrogen bond and hydrophobic interactions, and the latter is dominated by hydrophobic interactions. The highly flexible hydrophobic peptide adopts collapsed conformations separated by low energy barriers. APF activity correlates with hydrophobic clustering associated with amino acids 4A, 6V, and 8V. Peptide conformations are highly sensitive to single point mutations, which explain the experimental trends. The presented SAR will act as a guide for lead optimization of more potent APF analogues of potential therapeutic utility.

Insight into Early-Stage Unfolding of GPI-Anchored Human Prion Protein

Prion diseases are fatal neurodegenerative disorders, which are characterized by the accumulation of misfolded prion protein (PrPSc) converted from a normal host cellular prion protein (PrPC). Experimental studies suggest that PrPC is enriched with α-helical structure, whereas PrPSc contains a high proportion of β-sheet. In this study, we report the impact of N-glycosylation and the membrane on the secondary structure stability utilizing extensive microsecond molecular dynamics simulations. Our results reveal that the HB (residues 173 to 194) C-terminal fragment undergoes conformational changes and helix unfolding in the absence of membrane environments because of the competition between protein backbone intramolecular and protein-water intermolecular hydrogen bonds as well as its intrinsic instability originated from the amino acid sequence. This initiation of the unfolding process of PrPC leads to a subsequent increase in the length of the HB-HC loop (residues 195 to 199) that may trigger larger rigid body motions or further unfolding around this region. Continuous interactions between prion protein and the membrane not only constrain the protein conformation but also decrease the solvent accessibility of the backbone atoms, thereby stabilizing the secondary structure, which is enhanced by N-glycosylation via additional interactions between the N-glycans and the membrane surface.

Molecular Dynamics Simulations of Glycoproteins Using CHARMM

Molecular dynamics simulations are an effective tool to study the structure, dynamics, and thermodynamics of carbohydrates and proteins. However, the simulations of heterogeneous glycoprotein systems have been limited due to the lack of appropriate molecular force field parameters describing the linkage between the carbohydrate and the protein regions as well as the tools to prepare these systems for modeling studies. In this work we outline the recent developments in the CHARMM carbohydrate force field to treat glycoproteins and describe in detail the step-by-step procedures involved in building glycoprotein geometries using CHARMM-GUI Glycan Reader.

Phosphorylation versus O-GlcNAcylation: Computational Insights into the Differential Influences of the Two Competitive Post-Translational Modifications

Phosphorylation and O-GlcNAcylation are rapidly cycling intracellular protein post-translational modifications (PTMs) that can compete for the same serine (S) and threonine (T) sites. Limited crystal structure information is available on the direct influence of these PTMs on the underlying protein structure, especially for O-GlcNAcylation. NMR and CD studies show that these competitive-PTMs can have the same or differential influence on the overall secondary structure. In Tau derived peptide fragments, it was found that phosphorylation stabilized PPII conformations while O-GlcNAcylation destabilized the same. In the absence of substantial structural information, we have performed a systematic computational study utilizing PDB analysis, QM calculations, and MD simulations to identify key structural trends upon PTM. Our analysis of the limited PDB data set revealed conformational shifts from PPII to α-helical geometry upon serine phosphorylation and in the opposite direction, from α-helical to PPII geometry upon threonine phosphorylation. Gas phase QM calculations covering the complete Ramachandran ϕ/ψ space using model native, phosphorylated, and O-GlcNAcylated dipeptide systems revealed preferences toward α-helical conformations. However, the major structural transitions were observed in the MD simulations upon the inclusion of solvation. The model dipeptide simulations revealed a preference for PPII and α-helical conformations for phosphorylated serine and threonine, while O-GlcNAcylated dipeptides exhibited a complete shift toward extended conformations, β-sheet and PPII, disfavoring the α-helical conformation. For the Baldwin α-helix simulations, it was found that both phosphorylation and O-GlcNAcylation destabilized the helix; however, the destabilization was governed by H-bonding and electrostatic interactions in the former, while the latter was controlled by hydrophobic collapse and steric interactions. The presence of lysine in close proximity of phosphate leads to potentially stable salt bridge interactions, which can influence the structure on the basis of the relative placement of the lysine with respect to the PTM site. Similar strong lysine-phosphate contacts were observed in the model Tau peptides, which steers the conformations toward PPII geometries, highlighting the direct influence of the PTM on function.

Influence of Polarization on Carbohydrate Hydration: A Comparative Study Using Additive and Polarizable Force Fields

Carbohydrates are known to closely modulate their surrounding solvent structures and influence solvation dynamics. Spectroscopic investigations studying far-IR regions (below 1000 cm(-1)) have observed spectral shifts in the libration band (around 600 cm(-1)) of water in the presence of monosaccharides and polysaccharides. In this paper, we use molecular dynamics simulations to gain atomistic insight into carbohydrate-water interactions and to specifically highlight the differences between additive (nonpolarizable) and polarizable simulations. A total of six monosaccharide systems, α and β anomers of glucose, galactose, and mannose, were studied using additive and polarizable Chemistry at HARvard Macromolecular Mechanics (CHARMM) carbohydrate force fields. Solvents were modeled using three additive water models TIP3P, TIP4P, and TIP5P in additive simulations and polarizable water model SWM4 in polarizable simulations. The presence of carbohydrate has a significant effect on the microscopic water structure, with the effects being pronounced for proximal water molecules. Notably, disruption of the tetrahedral arrangement of proximal water molecules was observed due to the formation of strong carbohydrate-water hydrogen bonds in both additive and polarizable simulations. However, the inclusion of polarization resulted in significant water-bridge occupancies, improved ordered water structures (tetrahedral order parameter), and longer carbohydrate-water H-bond correlations as compared to those for additive simulations. Additionally, polarizable simulations also allowed the calculation of power spectra from the dipole-dipole autocorrelation function, which corresponds to the IR spectra. From the power spectra, we could identify spectral signatures differentiating the proximal and bulk water structures, which could not be captured from additive simulations.