Contributions of tryptophan side chains to the far-ultraviolet circular dichroism of proteins

It has often been assumed that the role of aromatic side chains in the far-ultraviolet region of protein circular dichroism (CD) is negligible. However, some proteins have positive CD bands in the 220–230 nm region which are almost certainly due to aromatic side chains. The contributions to the CD of interactions between tryptophan side chains and the nearest neighbor peptide groups have been studied, focusing on the indole B_b transition which occurs near 220 nm. Calculations on idealized peptide conformations show that the CD depends strongly on both backbone and side-chain conformation. Because of the low symmetry of indole, rotation about the CC bond (dihedral angle ₂) by 180° generally leads to large changes in the CD, often causing the B_b band to reverse sign. When side-chain conformational preferences are taken into account, there is no strong bias for either positive or negative B_b rotational strengths. The observation that simple tryptophan derivatives such as N-acetyl-L-tryptophan methylamide have positive CD near 220 nm implies either that these derivatives prefer the _R region over the region, or that there is little preference for ₂ < 180° over ₂ > 180°. Nearest-neighbor-only calculations on individual tryptophans in 15 globular proteins also reveal a small bias toward positive B_b bands. Rotational strengths of the B_b transition for some conformations can be as large as 1.0 Debye-Bohr magnetons in magnitude, corresponding to maximum molar ellipticities greater than 10⁵ degcm²/dmol. Although a substantial amount of cancellation occurs in most of the examples considered here, such CD contributions could be significant, especially in proteins of low helix content.     

Infrared absorption of tyrosine side chains in proteins

Poly-L -tyrosine absorbs strongly at 1515 cm.^?1, and a band at this frequency has been found in a number of proteins and has been assigned to the tyrosine residue. The assignment was confirmed by examination of spectra of deuterated proteins, which usually exhibit a residual band at 1513 cm.^?1. In proteins, this band correlates linearly with known tyrosine content, but the point corresponding to poly-L -tyrosine itself does not fall on the correlation line. The possibility and limitations of using the infrared method for tyrosine determinations is discussed.     

Picosecond dynamics of tyrosine side chains in proteins.

To probe the details of small amplitude motions in proteins, a dynamical analysis of the orientation fluctuations of two tyrosine side chains in the bovine pancreatic trypsin inhibitor is presented. Detailed results are given for the time history and correlation functions obtained for the ring motion from a molecular dynamics simulation of the entire protein. It is shown that even on a picosecond time scale orientational fluctuations of +/-30 degrees from the average position occur for the tyrosine rings in the interior of the protein. It is found that the Langevin equation is applicable to the ring torsional motion, which corresponds to that of an angular harmonic oscillator with near-critical damping. Two possible microscopic models for the observed damping effects are outlined. One of these, analogous to liquid behavior, is based on kinetic theory and takes account of the collisions which occur between atoms of the protein; the other, more analogous to solid behavior, involves the coupling among a large number of harmonic oscillators. The collisional model with parameters obtained from theoretical estimates leads to good agreement with the correlation functions from the dynamic simulation. However, the dephasing of harmonic oscillations can yield similar short-time results so that a distinction between the two models is difficult. The importance of damping effects on the motions involved in conformational transitions and enzymatic reactions is discussed.     

The role of tryptophan side chains in membrane protein anchoring and hydrophobic mismatch

Tryptophan (Trp) is abundant in membrane proteins, preferentially residing near the lipid–water interface where it is thought to play a significant anchoring role. Using a total of 3 μs of molecular dynamics simulations for a library of hydrophobic WALP-like peptides, a long poly-Leu α-helix, and the methyl-indole analog, we explore the thermodynamics of the Trp movement in membranes that governs the stability and orientation of transmembrane protein segments. We examine the dominant hydrogen-bonding interactions between the Trp and lipid carbonyl and phosphate moieties, cation–π interactions to lipid choline moieties, and elucidate the contributions to the thermodynamics that serve to localize the Trp, by ~ 4 kcal/mol, near the membrane glycerol backbone region. We show a striking similarity between the free energy to move an isolated Trp side chain to that found from a wide range of WALP peptides, suggesting that the location of this side chain is nearly independent of the host transmembrane segment. Our calculations provide quantitative measures that explain Trp's role as a modulator of responses to hydrophobic mismatch, providing a deeper understanding of how lipid composition may control a range of membrane active peptides and proteins.     

Protein-RNA interactions and virus stability as probed by the dynamics of tryptophan side chains

The correlation between dynamics and stability of icosahedral viruses was studied by steady-state and time-resolved fluorescence approaches. We compared the environment and dynamics of tryptophan side chains of empty capsids and ribonucleoprotein particles of two icosahedral viruses from the comovirus group: cowpea mosaic virus (CPMV) and bean pod mottle virus (BPMV). We found a great difference between tryptophan fluorescence emission spectra of the ribonucleoprotein particles and the empty capsids of BPMV. For CPMV, time-resolved fluorescence revealed differences in the tryptophan environments of the capsid protein. The excited-state lifetimes of tryptophan residues were significantly modified by the presence of RNA in the capsid. More than half of the emission of the tryptophans in the ribonucleoprotein particles of CPMV originates from a single exponential decay that can be explained by a similar, nonpolar environment in the local structure of most of the tryptophans, even though they are physically located in different regions of the x-ray structure. CPMV particles without RNA lost this discrete component of emission. Anisotropy decay measurements demonstrated that tryptophans rotate faster in empty particles when compared with the ribonucleoprotein particles. The increased structural breathing facilitates the denaturation of the empty particles. Our studies bring new insights into the intricate interactions between protein and RNA where part of the missing structural information on the nucleic acid molecule is compensated for by the dynamics.     

Beyond rotamers: a generative,probabilistic model of side chains in proteins

Background  

Accurately covering the conformational space of amino acid side chains is essential for important applications such as protein design, docking and high resolution structure prediction. Today, the most common way to capture this conformational space is through rotamer libraries - discrete collections of side chain conformations derived from experimentally determined protein structures. The discretization can be exploited to efficiently search the conformational space. However, discretizing this naturally continuous space comes at the cost of losing detailed information that is crucial for certain applications. For example, rigorously combining rotamers with physical force fields is associated with numerous problems.     

Systematic analysis of buried polar side chains and their interactions in alpha-helical proteins

Examination of 80 alpha-helical proteins and domains demonstrates that they contain from 1 to more than 20 completely buried (water-inaccessible) polar side chains. As a rule the latter have partners for H-bonding but the resulting H-bond system is often not exhaustive. Basing on statistical analysis, we determined the optimal number of H-bonds for every type of polar side chain, and discuss the structural role of vacant donors and acceptors. About half of the H-bonds formed by buried side chains pertain to interhelix contacts of the (side chain)-(side chain) and (side chain)-(main chain) types. Such interactions appear to be a most important factor determining the mutual arrangement of alpha-helices in proteins. Analysis of the frequency of occurrence of various interacting pairs reveals that these interactions are selective.     

Docking of calcium ions in proteins with flexible side chains and deformable backbones

A method of docking Ca²⁺ ions in proteins with flexible side chains and deformable backbones is proposed. The energy was calculated with the AMBER force field, implicit solvent, and solvent exposure-dependent and distance-dependent dielectric function. Starting structures were generated with Ca²⁺ coordinates and side-chain torsions sampled in 1000 Å³ cubes centered at the experimental Ca²⁺ positions. The energy was Monte Carlo-minimized. The method was tested on fourteen Ca²⁺-binding sites. For twelve Ca²⁺-binding sites the root mean square (RMS) deviation of the apparent global minimum from the experimental structure was below 1.3 and 1.7 Å for Ca²⁺ ions and side-chain heavy atoms, respectively. Energies of multiple local minima correlate with the RMS deviations from the X-ray structures. Two Ca²⁺-binding sites at the surface of proteinase K were not predicted, because of underestimation of Ca²⁺ hydration energy by the implicit-solvent method.     

CAP: conformation angles package-displaying the conformation angles of side chains in proteins

SUMMARY: A graphics package has been developed to display all side chain conformation angles of the user selected residue in a given protein structure. The proposed package is incorporated with all the protein structures (solved using X-ray diffraction and NMR spectroscopy) available in the Protein Data Bank. The package displays the multiple conformations adopted by a single amino acid residue whose structure is solved and refined at very high resolution. In addition, it shows the percentage distribution of the side chain conformation angles in different rotameric states. AVAILABILITY: http://144.16.71.146/cap/     

Environments and conformations of tryptophan side chains of gramicidin A in phospholipid bilayers studied by Raman spectroscopy.

Raman spectroscopy has been used to investigate the hydrophobic interaction of the indole ring with the environments, the water accessibility to the N1H site, and the conformation about the C beta-C3 bond for the four tryptophan side chains of gramicidin A incorporated into phospholipid bilayers. Most of the tryptophan side chains of the head-to-head helical dimer transmembrane channel are strongly interacting with the lipid hydrocarbon chains, and the hydrophobic interactions for the rest increase with increasing hydrocarbon chain length of the lipid. One tryptophan side chain (probably Trp-15) is accessible to water molecules, another (Trp-9) is deeply buried in the bilayer and inaccessible, and the accessibilities of the remaining two (Trp-11 and Trp-13) depend on the bilayer thickness. The torsional angle about the C beta-C3 bond is found to be +/- 90 degrees for all the tryptophans irrespective of the membrane thickness. Binding of the sodium cation to the channel does not change the torsional angles but decreases the water accessibilities of two tryptophans (Trp-11 and Trp-13) considerably. In conjunction with a slight spectral change in the amide III region, it is suggested that the sodium binding causes a partial change in the main-chain conformation around Trp-11 and Trp-13, which results in the movements of these side chains toward the bilayer center. Two models consistent with the present Raman data are proposed for the tryptophan orientation in the dominant channel structure.     

Roles of the histidine and tryptophan side chains in the M2 proton channel from influenza A virus

The M2 protein form influenza A virus forms a tetrameric ion channel, which enables proton passage across biological membranes when the N-terminal side is acidified. Among the amino acid residues in the transmembrane domain of the M2 protein, His37 and Trp41 are essential for the pH-regulated proton conductance. Current knowledge about the structures and interactions of His37 and Trp41 suggests a model for the M2 ion channel, in which the channel is closed by a network of His37 hydrogen bonds at neutral pH and is opened by a His37-Trp41 cation-pi interaction at acidic pH.     

Charge-transfer studies of the availability of aromatic side chains of proteins in guanidine hydrochloride.

The ability of aromatic tryptophyl and tyrosyl side-chain donors to form charge-transfer (CT) complexes with the acceptor 1-methyl-3-carbamidopyridinium chloride has been used to investigate the degree of exposure of these aromatic residues in denaturated proteins. The coplanar geometry of the CT complexes requires that virtually a full ring face of the donor be available for interaction with the acceptor, and the aromatic donor residues of lysozyme, trypsin, chymotrypsin, and the zymogens of the latter two enzymes do not appear to be wholly "exposed" in 6 M guanidine hydrochloride. Comparison of the CT proerties of the proteins with the corresponding properties of model complexes suggests that the incomplete exposure is due at least in part to statistical fluctuations in the continuously mobile, randomly coiled polypeptide chain which result in residues being alternately fully exposed and partly covered. Reduction and alkylation of the disulfide cross-links increase the apparent availability of the aromatic residues but the exposure is still less than that expected from a comparable mixture of tryptophan and tyrosine residues. Previous studies on the exposure of the aromatic residues of lysozyme and trypsin in aqueous salt solutions, when taken together with the present results, further suggest that there are two distinct kinds of surface environment possible on native proteins in solution. Some residues appear to be located in areas of the protein surface which are characterized by relatively fixed or stable local conformations, and have apparent CT association constants closely resembling these of comparable model complexes. Other residues may be located in a region where the protein conformation is flexible or continuously mobile, as evidenced by their smaller apparent association constants. It is probably significant that Trp-62 of lysozyme and Trp-215 of trypsin, both specificity site residues, appear to belong to the class of residues which can be considered as being in a flexible environment on the protein surface.     

An evaluvation of discrete and continuum search techniques for conformational analysis of side chains in proteins

Methodology for calculation of side-chain conformations in proteins is evaluated. The role and impact of corrections to idealized rotameric structures are considered, by incorporating methods for torsional optimization into rotamer-packing algorithms. Off-rotamer corrections given by continuum torsional optimization improve, over simpler rotamer-packing procedures, the accuracy with which the conformations of side chains of buried amino acids can be predicted. The analogy between protein side-chain calculations and spin systems is explored by adapting spin simulation methods to side-chain packing algorithms. Implementations of mean-field and heat-bath algorithms for side-chain packing are described and their performance tested. The procedures introduced here address the combinatorial problem in an efficient and reasonably effective manner, as evidenced by analysis of their convergence properties. Application of refined protocols yields overall prediction accuracies of 80% for χ¹ and 68percnt; for χ^1,2 pairs for a test set of 60 proteins, using a 40° cutoff to define correct placement. For buried amino acids (defined as having less than 30% relative solvent accessibility) the prediction accuracies increase to 88percnt; for χ¹ and 79percnt; for χ^1,2 pairs. The influence of the form of the potential energy function is studied by comparing results obtained with 12-6 and 9-6 potentials. The 9-6 form leads to more accurate results. Detailed comparison with previous work is presented, and the effect of combinatorial packing steps is shown to be important for all but the smallest proteins. © 1995 John Wiley & Sons, Inc.     

Simultaneous in vivo truncation of pectic side chains

Despite the wide occurrence of pectin in nature only a few source materials have been used to produce commercial pectins. One of the reasons for this is that many plant species contain pectins with high levels of neutral sugar side chains or that are highly substituted with acetyl or other groups. These modifications often prevent gelation, which has been a major functional requirement of commercial pectins until recently. We have previously shown that modification of pectin is possible through heterologous expression of pectin degrading enzymes in planta. To test the effect of simultaneous modification of the two main neutral pectic side chains in pectic rhamnogalacturonan I (RGI), we constitutively expressed two different enzymes in Arabidopsis thaliana that would either modify the galactan or the arabinan side chains, or both side chains simultaneously. Our analysis showed that the simultaneous truncation of arabinan and galactan side chains is achievable and does not severely affect the growth of Arabidopsis thaliana.     

Sterols with modified side chains.

Radio immunoassays for aldosterone and deoxycorticosterone (DOC)are described in which a simple separation procedure using ammonium sulfate stabilization of bound steroid and extraction of free steroid into toluene scintillant allows an “in vial” assay without mechanical separation of the two phase system. Extraction and thin layer Chromatographic methods for purification of aldosterone and DOC are free of solvent and plate blank effects. Normal values are given for unconjugated aldosterone and DOC in urine, for aldosterone and DOC in plasma and for aldosterone 18-glucuronide in urine.     

Evaluating the roles of the heme a side chains in cytochrome c oxidase using designed heme proteins

Heme a is a redox cofactor unique to cytochrome c oxidases and vital to aerobic respiration. Heme a differs from the more common heme b by two chemical modifications, the C-8 formyl group and the C-2 hydroxyethylfarnesyl group. The effects of these porphyrin substituents on ferric and ferrous heme binding and electrochemistry were evaluated in a designed heme protein maquette. The maquette scaffold chosen, [Delta7-H3m](2), is a four-alpha-helix bundle that contains two bis(3-methyl-l-histidine) heme binding sites with known absolute ferric and ferrous heme b affinities. Hemes b, o, o+16, and heme a, those involved in the biosynthesis of heme a, were incorporated into the bis(3-methyl-l-histidine) heme binding sites in [Delta7-H3m](2). Spectroscopic analyses indicate that 2 equiv of each heme binds to [Delta7-H3m](2), as designed. Equilibrium binding studies of the hemes with the maquette demonstrate the tight affinity for hemes containing the C-2 hydroxyethylfarnesyl group in both the ferric and ferrous forms. Coupled with the measured equilibrium midpoint potentials, the data indicate that the hydroxyethylfarnesyl group stabilizes the binding of both ferrous and ferric heme by at least 6.3 kcal/mol via hydrophobic interactions. The data also demonstrate that the incorporation of the C-8 formyl substituent in heme a results in a 179 mV, or 4.1 kcal/mol, positive shift in the heme reduction potential relative to heme o due to the destabilization of ferric heme binding relative to ferrous heme binding. The two substituents appear to counterbalance each other to provide for tighter heme a affinity relative to heme b in both the ferrous and ferric forms by at least 6.3 and 2.1 kcal/mol, respectively. These results also provide a rationale for the reaction sequence observed in the biosynthesis of heme a.     

Photo-CIDNP 1H-NMR of proteins: Exposure,mobility and orientation of the tyrosyl side chains

The photo-CIDNP technique is capable of giving information, not only on the degree of exposure of aromatic residues in proteins and on the correlation time of aromatic side chains, but also on the fine structure of -CH₂ multiplets, whose J-pattern directly reflects the side chain orientation about X ¹. Moreove, the resonance filtering achieved in the difference spectra allows selective measurement of line broadenings in binding processes, which are directly related to the association constants (K _a ). In this communication, data are reported concerning the exposure, mobility and orientation of the tyrosyl side chains in bovine PSTI at different pH values. The immobilization of tyrosyl residues in Met-enkephalin, human, and camel -endorphin in the presence of lipid micelles is also investigated; the extent of interaction of the three peptides with C₁₂PN is discussed. The binding efficiency is considerably lower for the shorter peptide; of the two endorphins, the camel hormone exhibits a higher affinity for the micelles than the human analog.Abbreviations PSTI pancreatic secretory trypsin inhibitor - CIDNP chemically induced dynamic nuclear polarization - C₁₂PN n-dodecylphosphorylcholine - NOE nuclear Overhauser enhancement