Synthetic flavinyl peptides related to the active site of mitochondrial monoamine oxidase II. Fluorescence properties.

The fluorescence properties of various 8alpha-sulfur-linked flavinyl peptides and related flavin analogues were investigated as the pH solvent, temperature, and flavin concentration were varied. Substitution in the 8alpha position by a thioether-linked peptide brings about a marked quenching of fluorescence (up to 98% in water), a slight bathochromic shift and broadening of the fluorescence emission spectra, and a slight decrease in the fluorescence lifetimes. Oxidation of the thioether function to a sulfone partially releases this fluorescence quenching without further changes in the fluorescence emission spectra. The primary effect on the fluorescence intensity is due to an interaction between the nonbonding electrons of the thioether, the hydrogen-bonding, polar solvent, and the isoalloxazine ring. Dissolving these flavinyl peptides in nonaqueous solvents increases the fluorescence intensity as much as 20-fold. A secondary effect on flavinyl fluorescence can be attributed to a collisional quenching by the vicinal tyrosyl residue within tyrosine-containing flavinyl peptides. The fluorescence properties provide further confirmation of the identity of the synthetic and naturally obtained flavinyl peptides and of the interaction between the free-hydroxyl functions of the ribityl side chain and the thioether.     

A study of the preferred environment of amino acid residues in globular proteins

In the native folded conformation of a globular protein, amino acid residues distant along the polypeptide chain come together to form the compact structure. This spatial structure is such that most of the polar residues are on the surface and have contact with the solvent medium and the nonpolar residues buried in the interior which have contact with similar nonpolar side chains. This cooperativity and mutual interaction among the randomly aligned amino acid residues suggest that each type of residue may prefer to have a specific environment. To gain more insight into this aspect of residue-residue cooperativity, a detailed analysis of the preferred environment associated with each of the 20 different amino acid residues in a number of protein crystals has been carried out. The variation of nonpolar nature computed for different sizes of spheres shows that the spatial region between radii of 6 and 8 Å is more favored for hydrophobic interactions and indicates that the influence of each residue over the surrounding medium extends predominantly up to a distance of 8 Å. The analysis of the surrounding amino acid residues associated with each type of residue shows that there is a definite tendency for each type of residue to have association with specific residues. The variation in environment is found even within the polar group as well as in the nonpolar group of residues. The surrounding residues associated with isoleucine, leucine, and valine are purely nonpolar. Proline, a nonpolar residue, is often surrounded by polar residues. The surrounding nonpolar nature of the tryptophan and tyrosine residues implies that even a single polar atom in a nonpolar side chain is sufficient to reduce their hydrophobic environment. There exists a high degree of mutual residue-residue cooperativity between the pairs glutamic acid-lysine, methionine-arginine, asparagine-tryptophan, and glutamine-proline, and the mutual residue-residue noncooperativity is high for the pairs methionine-aspartic acid, cysteine-glutamic acid, histidine-glutamine, and leucine-asparagine. The formation of secondary and tertiary structures is discussed in terms of the preferred environment and mutual cooperativity among various types of amino acid residues.     

Structural basis for the unusual specificity of Escherichia coli aminopeptidase N

Aminopeptidase N from Escherichia coli is a M1 class aminopeptidase with the active-site region related to that of thermolysin. The enzyme has unusual specificity, cleaving adjacent to the large, nonpolar amino acids Phe and Tyr but also cleaving next to the polar residues Lys and Arg. To try to understand the structural basis for this pattern of hydrolysis, the structure of the enzyme was determined in complex with the amino acids L-arginine, L-lysine, L-phenylalanine, L-tryptophan, and L-tyrosine. These amino acids all bind with their backbone atoms close to the active-site zinc ion and their side chain occupying the S1 subsite. This subsite is in the form of a cylinder, about 10 A in cross-section and 12 A in length. The bottom of the cylinder includes the zinc ion and a number of polar side chains that make multiple hydrogen-bonding and other interactions with the alpha-amino group and the alpha-carboxylate of the bound amino acid. The walls of the S1 cylinder are hydrophobic and accommodate the nonpolar or largely nonpolar side chains of Phe and Tyr. The top of the cylinder is polar in character and includes bound water molecules. The epsilon-amino group of the bound lysine side chain and the guanidinium group of arginine both make multiple hydrogen bonds to this part of the S1 site. At the same time, the hydrocarbon part of the lysine and arginine side chains is accommodated within the nonpolar walls of the S1 cylinder. This combination of hydrophobic and hydrophilic binding surfaces explains the ability of ePepN to cleave Lys, Arg, Phe, and Tyr. Another favored substrate has Ala at the P1 position. The short, nonpolar side chain of this residue can clearly be bound within the hydrophobic part of the S1 cylinder, but the reason for its facile hydrolysis remains uncertain.     

Stabilizing nonpolar/polar side-chain interactions in the alpha-helix.

A simplistic, yet often used, view of protein stability is that amino acids attract other amino acids with similar polarity, whereas nonpolar and polar side chains repel. Here we show that nonpolar/polar interactions, namely Val or Ile bonding to Lys or Arg in alpha-helices, can in fact be stabilizing. Residues spaced i, i + 4 in alpha-helices are on the same face of the helix, with potential to favorably interact and stabilize the structure. We observe that the nonpolar/polar pairs Ile-Lys, Ile-Arg, and Val-Lys occur in protein helices more often than expected when spaced i, i + 4. Partially helical peptides containing pairs of nonpolar/polar residues were synthesized. Controls with i, i + 5 spacing have the residues on opposite faces of the helix and are less helical than the test peptides with the i, i + 4 interactions. Experimental circular dichroism results were analyzed with helix-coil theory to calculate the free energy for the interactions. All three stabilize the helix with DeltaG between -0.14 and -0.32 kcal x mol(-1). The interactions are hydrophobic with contacts between Val or Ile and the alkyl groups in Arg or Lys. Side chains such as Lys and Arg can thus interact favorably with both polar and nonpolar residues.     

Structural evidence for a dominant role of nonpolar interactions in the binding of a transport/chemosensory receptor to its highly polar ligands.

The receptor, a maltose/maltooligosaccharide-binding protein, has been found to be an excellent system for the study of molecular recognition because its polar and nonpolar binding functions are segregated into two globular domains. The X-ray structures of the "closed" and "open" forms of the protein complexed with maltose and maltotetraitol have been determined. These sugars have approximately 3 times more accessible polar surface (from OH groups) than nonpolar surface (from small clusters of sugar ring CH bonds). In the closed structures, the oligosaccharides are buried in the groove between the two domains of the protein and bound by extensive hydrogen bonding interactions of the OH groups with the polar residues confined mostly in one domain and by nonpolar interactions of the CH clusters with four aromatic residues lodged in the other domain. Substantial contacts between the sugar hydroxyls and aromatic residues are also formed. In the open structures, the oligosaccharides are bound almost exclusively in the domain rich in aromatic residues. This finding, along with the analysis of buried surface area due to complex formations in the open and closed structures, supports a major role for nonpolar interactions in initial ligand binding even when the ligands have significantly greater potential for highly specific polar interactions.     

Functional interactions in bacteriorhodopsin: a theoretical analysis of retinal hydrogen bonding with water.

The light-driven proton pump, bacteriorhodopsin (bR) contains a retinal molecule with a Schiff base moiety that can participate in hydrogen-bonding interactions in an internal, water-containing channel. Here we combine quantum chemistry and molecular mechanics techniques to determine the geometries and energetics of retinal Schiff base-water interactions. Ab initio molecular orbital calculations are used to determine potential surfaces for water-Schiff base hydrogen-bonding and to characterize the energetics of rotation of the C-C single bond distal and adjacent to the Schiff base NH group. The ab initio results are combined with semiempirical quantum chemistry calculations to produce a data set used for the parameterization of a molecular mechanics energy function for retinal. Using the molecular mechanics force field the hydrated retinal and associated bR protein environment are energy-minimized and the resulting geometries examined. Two distinct sites are found in which water molecules can have hydrogen-bonding interactions with the Schiff base: one near the NH group of the Schiff base in a polar region directed towards the extracellular side, and the other near a retinal CH group in a relatively nonpolar region, directed towards the cytoplasmic side.     

Use of a potential of mean force to analyze free energy contributions in protein folding.

A method for calculation of the free energy of residues as a function of residue burial is proposed. The method is based on the potential of mean force, with a reaction coordinate expressed by residue burial. Residue burials are calculated from high-resolution protein structures. The largest individual contributions to the free energy of a residue are found to be due to the hydrophobic interactions of the nonpolar atoms, interactions of the main chain polar atoms, and interactions of the charged groups of residues Arg and Lys. The contribution to the free energy of folding due to the uncharged side chain polar atoms is small. The contribution to the free energy of folding due to the main chain polar atoms is favorable for partially buried residues and less favorable or unfavorable for fully buried residues. Comparison of the accessible surface areas of proteins and model spheres shows that proteins deviate considerably from a spherical shape and that the deviations increase with the size of a protein. The implications of these results for protein folding are also discussed.     

Discrimination in resolving systems. VI. Comparison of the diastereomers of deoxyephedrinium and ephedrinium 4'-fluoromandelates

(-)-(R)-Deoxyephedrine forms poorly discriminating diastereomeric salts with 4'-fluoromandelic acid from 95% ethanol. Both less-soluble (L) (S)-4'-fluoromandelate and more-soluble (M) (R)-4'-fluoromandelate phases are monoclinic and unsolvated. Their solubility ratio (M/L) in 95% ethanol is only 1.2, which correlates with the similarity and small differences in their respective heats of fusion and fusion temperatures. The (R)-deoxyephedrinium and the related (1R;2S)-ephedrinium 4'-fluoromandelate systems show L-salts with higher ion-pair volumes and lower densities than their M-salts. (R)-Deoxyephedrinium salts have higher volumes than the comparable (1R;2S)-ephedrinium salts even though the resolving base (R)-deoxyephedrine lacks the benzylic hydroxy. In the solids, bilayered structures segregate polar and nonpolar molecular regions. The principle interionic interactions are hydrogen bonds between protonated secondary ammonium ions and carboxylates forming infinite chains with a six-atom repeating unit H-N(+)-H...O-C(-)-O [C(2)(2)(6)]. These are buttressed by mandelate hydroxy to carboxylate hydrogen bonds. Differing interactions between phenyl and 4'-fluorophenyl rings in the nonpolar layers of the salts correlate with the density and stability inversion.     

Transmembrane segments of nascent polytopic membrane proteins control cytosol/ER targeting during membrane integration

During cotranslational integration of a eukaryotic multispanning polytopic membrane protein (PMP), its hydrophilic loops are alternately directed to opposite sides of the ER membrane. Exposure of fluorescently labeled nascent PMP to the cytosol or ER lumen was detected by collisional quenching of its fluorescence by iodide ions localized in the cytosol or lumen. PMP loop exposure to the cytosol or lumen was controlled by structural rearrangements in the ribosome, translocon, and associated proteins that occurred soon after a nascent chain transmembrane segment (TMS) entered the ribosomal tunnel. Each successive TMS, although varying in length, sequence, hydrophobicity, and orientation, reversed the structural changes elicited by its predecessor, irrespective of loop size. Fluorescence lifetime data revealed that TMSs occupied a more nonpolar environment than secretory proteins inside the aqueous ribosome tunnel, which suggests that TMS recognition by the ribosome involves hydrophobic interactions. Importantly, the TMS-triggered structural rearrangements that cycle nascent chain exposure between cytosolic and lumenal occur without compromising the permeability barrier of the ER membrane.     

Structural details of urea binding to barnase: a molecular dynamics analysis.

BACKGROUND: The molecular mechanism of urea-induced protein unfolding has not been established. It is generally thought that denaturation results from the stabilizing interactions of urea with portions of the protein that are buried in the native state and become exposed upon unfolding of the protein. RESULTS: We have performed molecular dynamics simulations of barnase (a 110 amino acid RNase from Bacillus amyloliquefaciens) with explicit water and urea molecules at 300 K and 360 K. The native conformation was unaffected in the 300 K simulations at neutral and low pH. Two of the three runs at 360 K and low pH showed some denaturation, with partial unfolding of the hydrophobic core 2. The first solvation shell has a much higher density of urea molecules (water/urea ratio ranging from 2.07 to 2.73) than the bulk (water/urea ratio of 4.56). About one half of the first-shell urea molecules are involved in hydrogen bonds with polar or charged groups on the barnase surface, and between 15% and 18% of the first-shell urea molecules participate in multiple hydrogen bonds with barnase. The more stably bound urea molecules tend to be in crevices or pockets on the barnase surface. CONCLUSIONS: The simulation results indicate that an aqueous urea solution solvates the surface of a polypeptide chain more favorably than pure water. Urea molecules interact more favorably with nonpolar groups of the protein than water does, and the presence of urea improves the interactions of water molecules with the hydrophilic groups of the protein. The results suggest that urea denaturation involves effects on both nonpolar and polar groups of proteins.     

Noncontact dipole effects on channel permeation. V. Computed potentials for fluorinated gramicidin

Experimental and theoretical calculations indicate that the dipole moment of the four Trp side chains in gramicidin A (gA) channels modify channel conductance through long-range electrostatic interactions. Electrostatic ion/side-chain interaction energies along the channel were computed with CHARMM using ab initio atom charges for native and 4-, 5-, or 6-fluorinated Trp side chains. The bulk water reaction to the polar side chains was included using the method of images as implemented by, and channel waters in idealized structures were included. Ion/Trp interaction energies were approximately -0.6 kcal/mol throughout the channel for all four of the native Trp pairs. Channel waters produced a modest reduction in the magnitude of interactions, essentially offsetting images representing the bulk water outside the channel. The effects of side-chain fluorination depended on ring position and, to a lesser extent, residue number. Compared with native Trp, 5-fluorination reduces the translocation barrier with minor effects on the exit barrier. In contrast, 6-fluorination primarily reduces exit barrier. 4-Fluorination produces a more complex double-well energy profile. Effects of measured side-chain movements resulting from fluorination or change in lipid bilayer were negligible whereas thermal side chain librations cause large effects, especially in the region of the ion-binding sites.     

Non-canonical interactions of porphyrins in porphyrin-containing proteins

In this study we have described the non-canonical interactions between the porphyrin ring and the protein part of porphyrin-containing proteins to better understand their stabilizing role. The analysis reported in this study shows that the predominant type of non-canonical interactions at porphyrins are CH···O and CH···N interactions, with a small percentage of CH···π and non-canonical interactions involving sulfur atoms. The majority of non-canonical interactions are formed from side-chains of charged and polar amino acids, whereas backbone groups are not frequently involved. The main-chain non-canonical interactions might be slightly more linear than the side-chain interactions, and they have somewhat shorter median distances. The analysis, performed in this study, shows that about 44% of the total interactions in the dataset are involved in the formation of multiple (furcated) non-canonical interactions. The high number of porphyrin–water interactions show importance of the inclusion of solvent in protein–ligand interaction studies. Furthermore, in the present study we have observed that stabilization centers are composed predominantly from nonpolar amino acid residues. Amino acids deployed in the environment of porphyrin rings are deposited in helices and coils. The results from this study might be used for structure-based porphyrin protein prediction and as scaffolds for future porphyrin-containing protein design.     

Structure investigation,enrichment analysis and structure-based repurposing of FDA-approved drugs as inhibitors of BET-BRD4

We report herein detailed structural insights into the ligand recognition modes guiding bromodomain selectivity, enrichment analysis and docking-based database screening for the identification of the FDA-approved drugs that have potential to be the human BRD4 inhibitors. Analysis of multiple X-ray structures prevailed that the lysine-recognition sites are highly conserved, and apparently, the dynamic ZA loop guides the specific ligand-recognition. The protein–ligand interaction profiling revealed that both BRD2 and BRD4 shared hydrophobic interaction of bound ligands with PRO-98/PRO-82, PHE-99/PHE-83, LEU-108/LEU-92 and direct H-bonding with ASN-156/ASN-140 (BRD2/BRD4), while on the other hand the water-mediated H-bonding of bound ligands with PRO-82, GLN-85, PRO-86, VAL-87, ASP-88, LEU-92, TYR-97 and MET-132, and aromatic π–π stacking with TRP-81 prevailed as unique interaction in BRD4, and were not observed in BRD2. Subsequently, through ROC curve analysis, the best enrichment was found with PDB-ID 4QZS of BRD4 structures. Finally, through docking-based database screening study, we found that several drugs have better binding affinity than the control candidate lead (+)-JQ1 (Binding affinity?=?-7.9?kcal/mol), a well-known BRD4 inhibitor. Among the top-ranked drugs, azelastine, a selective histamine H1 receptor antagonist, showed the best binding affinity of –9.3?kcal/mol and showed interactions with several key residues of the acetyl lysine binding pocket. Azelastine may serve as a promising template for further medicinal chemistry. These insights may serve as basis for structure-based drug design, drug repurposing and the discovery of novel BRD4 inhibitors.

Communicated by Ramaswamy H. Sarma     

Secondary and tertiary structure of the A-state of cytochrome c from resonance Raman spectroscopy.

Ferricytochrome c can be converted to the partially folded A-state at pH 2.2 in the presence of 1.5 M NaCl. The structure of the A-state has been studied in comparison with the native and unfolded states, using resonance Raman spectroscopy with visible and ultraviolet excitation wavelengths. Spectra obtained with 200 nm excitation show a decrease in amide II intensity consistent with loss of structure for the 50s and 70s helices. The 230-nm spectra contain information on vibrational modes of the single Trp 59 side chain and the four tyrosine side chains (Tyr 48, 67, 74, and 97). The Trp 59 modes indicate that the side chain remains in a hydrophobic environment but loses its tertiary hydrogen bond and is rotationally disordered. The tyrosine modes Y8b and Y9a show disruption of tertiary hydrogen bonding for the Tyr 48, 67, and 74 side chains. The high-wavenumber region of the 406.7-nm resonance Raman spectrum reveals a mixed spin heme iron atom, which arises from axial coordination to His 18 and a water molecule. The low-frequency spectral region reports on heme distortions and indicates a reduced degree of interaction between the heme and the polypeptide chain. A structural model for the A-state is proposed in which a folded protein subdomain, consisting of the heme and the N-terminal, C-terminal, and 60s helices, is stabilized through nonbonding interactions between helices and with the heme.     

Statistical analysis of protein structures suggests that buried ionizable residues in proteins are hydrogen bonded or form salt bridges

It is well known that nonpolar residues are largely buried in the interior of proteins, whereas polar and ionizable residues tend to be more localized on the protein surface where they are solvent exposed. Such a distribution of residues between surface and interior is well understood from a thermodynamic point: nonpolar side chains are excluded from the contact with the solvent water, whereas polar and ionizable groups have favorable interactions with the water and thus are preferred at the protein surface. However, there is an increasing amount of information suggesting that polar and ionizable residues do occur in the protein core, including at positions that have no known functional importance. This is inconsistent with the observations that dehydration of polar and in particular ionizable groups is very energetically unfavorable. To resolve this, we performed a detailed analysis of the distribution of fractional burial of polar and ionizable residues using a large set of ?2600 nonhomologous protein structures. We show that when ionizable residues are fully buried, the vast majority of them form hydrogen bonds and/or salt bridges with other polar/ionizable groups. This observation resolves an apparent contradiction: the energetic penalty of dehydration of polar/ionizable groups is paid off by favorable energy of hydrogen bonding and/or salt bridge formation in the protein interior. Our conclusion agrees well with the previous findings based on the continuum models for electrostatic interactions in proteins. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Single-channel studies on linear gramicidins with altered amino acid side chains. Effects of altering the polarity of the side chain at position 1 in gramicidin A.

The modulation of gramicidin A single-channel characteristics by the amino acid side chains was investigated using gramicidin A analogues in which the NH2 terminal valine was chemically replaced by other amino acids. The replacements were chosen such that pairs of analogues would have essentially isosteric side chains of different polarities at position 1 (valine vs. trifluorovaline or hexafluorovaline; norvaline vs. S-methyl-cysteine; and norleucine vs. methionine). Even though the side chains are not in direct contact with the permeating ions, the single-channel conductances for Na+ and Cs+ are markedly affected by the changes in the physico-chemical characteristics of the side chains. The maximum single-channel conductance for Na+ is decreased by as much as 10-fold in channels formed by analogues with polar side chains at position 1 compared with their counterparts with nonpolar side chains, while the Na+ affinity is fairly insensitive to these changes. The relative conductance changes seen with Cs+ were less than those seen with Na+; the ion selectivity of the channels with polar side chains at position 1 was increased. Hybrid channels could form between compounds with a polar side chain at position 1 and either valine gramicidin A or their counterparts with a nonpolar side chain at position 1. The structure of channels formed by the modified gramicidins is thus essentially identical to the structure of channels formed by valine gramicidin A. The polarity of the side chain at position 1 is an important determinant of the permeability characteristics of the gramicidin A channel. We discuss the importance of having structural information when interpreting the functional consequences of site-directed amino acid modifications.     

Characterization of Protomer Interfaces in HslV Protease; the Bacterial Homologue of 20S Proteasome

HslVU, a two-component proteasome-related prokaryotic system is composed of HslV protease and HslU ATPase. HslV protomers assemble in a dodecamer of two-stacked hexameric rings that form a complex with HslU hexamers. The intra- and inter-ring protomer interfaces in the HslV dodecamer underpin the integrity and functionality of HslVU. Structural characterization of HslV from different bacteria illustrated considerable differences in interacting residues, accessible surface and gap volumes at the intra-ring interface that is primarily stabilized by polar interactions. Amino acid residues Lys28, Arg83 and Asp111 have envisaged as hot spots at this HslU-interacting interface. The inter-ring interfaces that are made up of side chain packing of hydrophobic residues are structurally conserved. Hyperthermostable bacterium T. maritima HslV has extensively networked polar/nonpolar interactions and highly packed environment at all interfaces. Present data demonstrates that HslV protomer interfaces perform distinct functions; whereas intra-ring interface participates in HslV:HslU interaction resulting in allosteric activation of HslV protease by HslU, the inter-ring interfaces uphold the oligomeric form of HslV.     

The interaction of flavins with egg white riboflavin-binding protein

The properties of the riboflavin-binding site in the riboflavin-binding protein from egg white have been elucidated by determining constants for binding of flavin analogs to the protein and by changes in absorption spectra of free and bound flavins. The spectral changes and unfavorable interaction of the protein with charged species indicate that the overall flavin environment in the holoprotein is hydrophobic. Modification of either ring or side-chain portions of flavin usually results in a decrease of binding energy. Although no one portion of the structure is absolutely essential, both 7- and 8-methyl groups and 2′-hydroxyl group contribute most significantly to binding. The binding site at the region of C-2 and N-3 of the isoalloxazine is rather insensitive to the relative site of a substituent and thus relatively open, whereas considerable steric limitation is imposed at C-8, N-10, especially C-1′, and 4carbonyl positions. The hydroxyl groups of the N-10 side chain contribute in a stereoselective manner by formation of hydrogen bonds. Studies with model compounds that represent only a part of flavin suggest that the dimethylbenzenoid portion of the ring is involved in primary interactions of binding, and relatively buried in the protein. The quenching of protein fluorescence upon binding is mainly due to ground-state stacking interaction between a trytophanyl residue at the binding site and the quinoxaline portion, and not to Förster energy transfer.