Mechanisms of Protein Circular Dichroism Insights From Computational Modeling

MECHANISMS OF PROTEIN CIRCULAR DICHROISM: INSIGHTS FROM COMPUTATIONAL MODELING
By TATYANA KARABENCHEVA AND CHRISTO CHRISTOV

Department of Biomedical Sciences, School of Life Sciences, Northumbria University, Newcastle Upon Tyne, United Kingdom
I. II.
III. IV. V. VI. VII.
VIII.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Theoretical Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Important Denitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Computational Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Insights from Model Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tyrosine Interactions and Contributions in RNase A and RNase S . . . . . . . . . . . . . Toward Improved Accuracy Using Ab Initio Parameter Sets . . . . . . . . . . . . . . . . . . . . . Far-UV Contributions of Tryptophans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of CD Spectra in Class A b-Lactamase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Strategy of the Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Analysis the MGRS of TEM-1 b-Lactamase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Electrostatic Effects on the MGRS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Conformational Effects on the MGRS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Electrostatic Inuence of the Conformational Sensitivity of MGRS . . . . . . . F. Individual Contributions of Each Aromatic and Disulde Chromophore Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
Chirality is a fundamental property of molecular systems, and stereoselectivity underlines many fundamental biomolecular processes like biological recognition and catalysis. Circular dichroism (CD) which is a consequence of molecular chirality is an important method for the investigation of protein structure and structural changes during interactions with ligands, mutations, and folding. The development of computational methods allows powerful insight to be provided into the mechanisms of generation of CD spectra in complex systems as proteins and to explain experimental data, to validate predicted structures, and to explain ne details of biomolecular interactions. In this chapter, we provide a survey on several aspects of the current investigation on the CD phenomena: the emphasis is on its mechanisms and how they can be analyzed using
ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY, Vol. 80 DOI: 10.1016/S1876-1623(10)80003-3
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Copyright 2010, Elsevier Inc. All rights reserved.
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KARABENCHEVA AND CHRISTOV
computational methods in strong reference to the experimental data. We analyzed the mechanisms of interactions of the aromatic and disulde chromophores mainly in the near-UV CD as the number is smaller and allows detailed analysis. We describe some of the investigations on model systems and improve the parameter sets for the approximated CD modeling methods and key calculations on several proteins. We also provide a comprehensive survey on the investigations done in our group on the chiropticity of class A b-lactamases.
I. Introduction
Chirality is a fundamental molecular characteristic, and stereoselectivity underlines the most important biomolecular processes as receptorligand interactions and enzyme catalysis (Ranjbar and Gill, 2009). Electronic circular dichroism (CD) is an extremely powerful method for exploration of chirality and stereoselectivity of both large and small biomolecules (Miles and Wallace, 2006; Berova et al., 2007). The method is powerful and fast source for structural information of proteins (Kelly et al., 2005). The far-ultraviolet (UV) CD is largely applied for elucidation of the secondary structure of proteins (Cantor and Schimmel, 1980; Lees and Wallace, 2002), while in the near-UV, the method is utilized for understanding ne and subtle alterations in protein tertiary structure and interactions which might underline interactions with ligands (substrates, agonists, inhibitors, allosteric modulators), mutations effects, and environmental changes (Kahn, 1979; Woody and Dunker, 1996). Modern technological developments made possible the utilization of synchrotron radiation for CD experiments, and the construction of massive synchrotron facilities made this available to the research community (Miles and Wallace, 2007). CD experiment is not high resolution method; therefore in order to relate the spectral phenomena to alternations in protein structure we could successfully apply computational methods. A combined implementation of CD experiments and theory is often the only possibility for determination of the absolute conguration of small molecules (Berova, 2007 #2360; Woody, 1996). Theoretical investigation of CD of proteins and their complexes, however, could be quite complicated due to the large size of the protein molecules, the importance of the conformational changes, electrostatic, and solvent effects. Even with the modern supercomputers and accelerated quantum chemical codes, it is
MECHANISMS OF PROTEIN CIRCULAR DICHROISM
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not feasible to calculate chiropticity of large protein complexes directly. Instead, approximated methods need to be applied and experimentally derived parameters can be included. Nevertheless, understanding of the mechanisms of protein CD generation is a crucial missing link, which can relate the protein structure and dynamics to the observed experimental spectra and can provide deeper insight into protein biophysics and engineering. This review is selectively directed toward representing several lines of the investigation of the mechanisms of generation of protein circular dichroism using computational methods. The emphasis is to provide information about the recent advances in revealing the CD phenomena of proteins, peptides, and model systems using different levels of theory, advances in application direct methods (as time-dependent density functional theory [TD-DFT]), the applications of the approximated methods (as the matrix method and dipole interacting method), and the incorporation of accurate parameters for the last group of methods. We present calculations on model systems as they can be used as benchmarks for the performance of the approaches used later for larger systems and for preparations of accurate data for isolated chromophores. Consequently, we describe the analysis of the CD spectra of ribonucleases A and S (which does not contain tryptophans), the tryptophan contributions to far-UV spectra of several proteins. The rest of the review is directed to present the investigations done in our group to reveal comprehensively the mechanisms of generation of CD in class A b-lactamases. The review is also selectively directed toward the investigation of the aromatic and disulde contributions to the CD. The contributions of the backbone were the object of other reviews (e.g., Sreerama and Woody, 2004; Bulheller et al., 2007).
II.
Theoretical Background A. Important Denitions
CD is dened as the difference between the absorption of the left and right polarized light. Most fundamental molecular characteristics of chiropticity are a Rotational (rotatory) Strength (R). It is formulated as an imaginary part of the scalar product between the vectors of the electric
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transition dipole moment and the magnetic transition dipole moment (Rosenfeld, 1928): R fm0i mi0 g 1 where m0i and mi0 are electric and magnetic transition moments of the 0 ! i electronic transitions, respectively. Most protein chromophores exhibited (with exception of the disulde and nonplanar peptide group) are not intrinsically chiral. Consequently, they have vanishing rotational strengths over all electronic transitions and they became secondary chiral under the effects of protein environment (Woody, 1996). There are three mechanisms of generation of rotational strengths in proteins (MGRS; Fig. 1): (i) one-electron mechanism, or static eld effect (Condon, 1937), where electrically and magnetically allowed transitions in the same chromophore are mixed and give nonvanishing rotational strength; (ii) coupling between electrically allowed transitions in different groups called mm mechanism (Kuhn, 1930; Kirkwood, 1937) (the exciton effect is the degenerate case of this mechanism; Moftt et al., 1957); (iii) coupling between a magnetically allowed transition in one group and an electrically allowed transition in second group, which is known as mm mechanism (Schellman, 1968). Our perception of the physical principles and nature of the above mechanisms have not changed for the past 70 years; however, the theoretical level of their description and interpretation was dramatically improved
Coupled oscillators mm mechanism mm mechanism
Static field effect (one electron)
FIG. 1. Mechanisms of generation of rotation strengths in proteins (MGRS): oneelectron effect (left panel) and coupled oscillators (right panel).
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due to the development of the theoretical and experimental methods and the computer and experimental techniques.
B. Computational Methods
The calculation of the CD spectra can be done using direct and approximate methods. From the direct methods, the most applicable is the TD-DFT (Dreuw and Head-Gordon, 2005). From the approximated methods, the most utilized in calculations of protein CD spectra is the matrix method of Baylay, Nilsen, and Schellman (Bayley et al., 1969); however, Tinocos rst-order perturbation theory (Tinoco, 1962) and dipole interaction model are also applied (Woody, 1996). TD-DFT is an extension of the KohnSham theorem for the ground state to the excited state properties utilizing the linear-response formalism. The quality of the calculations depends strongly on the choice of the specic exchange-correlation functional with the preference of the hybrid functionals (e.g., B3LYP and BP86). The method works satisfactory for a large majority of organic, bioorganic, and bioinorganic systems; however, it is demonstrated to lead to several discrepancies for charge-transfer (CT) transitions. The method can be applied for the calculations of the excited states of molecules containing tens of heavy atoms but could not be applied for large systems as proteins. Nevertheless, comparison between the results done on model systems at TDDFT level and more approximate methods (which will be discussed below) can be used as justication of the last for application for larger systems. The matrix method is an approximate method developed by Baylay, Nielsen, and Schellman which can be implemented for exploration of very large multichromophoric systems as proteins (Bayley et al., 1969). It is developed from the rst-order perturbation theory of Tinoco (1962), but for accounting of the interactions between the chromophores, it utilizes matrix diagonalization which makes it easy to implement in computer programs. The variant, which is most frequently implemented for calculation of proteins CD, includes the modication proposed by Goux and Hooker for incorporation of the matrix elements of the momentum or gradient operator, thus avoiding origin dependence of the magnetic transition moment (Goux and Hooker, 1980a). There are several programs which implement the method as MATMAC, which was developed by the group of Prof. Joerg Fleischhauer (RWTH-Aachen) (Fleischhauer et al., 2000), PROTEIN developed by Woody
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and his coworkers (Colorado State University, USA) (Sreerama and Woody, 2004), DICHROCALC server developed by Hirst and his coworkers (Nottingham University, UK) (Bulheller and Hirst, 2009). The matrix method (Bayley et al., 1969) considers the protein as a system of M chromophoric groups which initially do not interact with each other. The total excited state wave function of the macromolecule is given as a linear combination of the basis functions, including ni excitations within each monomer (chromophoric) group: Ck
M X ni X i a
cia Fia
where every basis function Fia is a product from all M monomer wave functions. The electronic excitations are possible to arise within the groups, but not between them, and in the basis function, only one group is in its excited state. In the Hamiltonian matrix, H
M X i 1
Hi
M 1 X i
M X j i 1
Vij
the diagonal elements represent the excited state energies of the isolated chromophores, while the off-diagonal elements consist of two types: (i) those which accounts for the interactions between different chromophoric groups (coupling oscillators interactions) and (ii) those which represent the mixing of the electronic transition inside one chromophore under the inuence of the rest of the molecule (static eld one-electron effect). The interactions between chromophores are considered as electrostatic and therefore they can be written as: ri 0a rirj 0b rj Vi 0a ; j 0b d ti d tj ; 4 4pe0ri ; j ri rj where ri0a and ri0b are the permanent and transition electron densities of the transition 0 ! a in group i and 0 ! b in group j, respectively. In order to save computational time, the above densities can be presented as point charges (monopoles) in Eq. (4). The Hamiltonian matrix is diagonalized by a standard unitary transformation and the diagonal elements in the new matrix represent the excited states of the interacting protein molecules. The electric and magnetic
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dipole moments of the isolated transitions in the monomer chromophores are similarly diagonalized by unitary transformation in order to represent the interactions and MGRS in the protein. Consequently, the imaginary part of their scalar product is the rotational strength of the whole protein for a particular wavelength. There are different strategies for calculations of the positions and charges of monopoles for all relevant chromophores, based on semiempirical calculations and including experimental corrections (Kurapkat et al., 1997; Sreerama and Woody, 2004) and using high-accurate multireference methods (Besley and Hirst, 1999; Rogers and Hirst, 2004a,b). Electrostatic interactions (EIs) contribute for formation of stable molecular conformation and folding and underline recognition, association processes, and enzyme catalytic mechanisms (Honig and Nicholls, 1995; Norberg and Nilsson, 2003). The protein chromophores can be sensitive to changes in electrostatic environment (Woody and Dunker, 1996; Kurapkat et al., 1997). For example, the energy and the dipole moment of La transition of tryptophans are very sensitive to environmental changes. Therefore, it is important to understand the relationship between the EIs and the CD mechanisms. The electrostatic environment around chromophores is included in the diagonal elements of the Hamiltonian matrix by not only the excitation energies of the isolated groups but also their shifts under the local EIs: X Hi DEi 0a Viaa ; j 00 Vi 00; j 00; 5
i 6j
where Viaa;j00 is the Coulomb interaction between group i in excited state and group j in ground state and Vi00;j00 is the interaction energy between both groups in ground state. The effective dielectric constant is 1. The CT between groups is not accounted for, and only single excitations are included. To generate CD spectrum from the calculated rotational strengths, the last were combined with Gaussian band functions. 1. Applicability of the Matrix Method and Comparisons to Experimental CD Spectra The matrix method has been successfully applied to predict CD spectra of transmission electron microscopy (TEM)-1 b-lactamase in near and farUV (Christov, 2002; Christov and Karabencheva, 2004; Rogers and Hirst,
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2004a,b; Christov et al., 2006), ribonuclease A, ribonuclease S (Kurapkat et al., 1997), and peptides (Fleischhauer et al., 1994; Daura et al., 2003) in good qualitative agreement with the experimental data, as well as with other free b-lactamases from class A (Christov, 2002; Karabencheva and Christov, 2004). The matrix method has also been applied to predict the qualitative CD spectra of lysozyme (Goux and Hooker, 1980b), myoglobin, hemoglobin, lactate dehydrogenase, and other proteins (Besley and Hirst, 1999; Sreerama et al., 1999; Bhattacharjee et al., 2003; Woody and Woody, 2003; Rogers and Hirst, 2004a,b; Sreerama and Woody, 2004; Oakley and Hirst, 2006; Oakley et al., 2006).
III.
Insights from Model Systems
Aromatic contributions are weaker than those generated by the peptide groups. Aromatic amino acid residues could have signicant impact on protein structure, stability, and functions as they participate in ligand binding and orientation, substrate activation, and contribute to the enzyme catalysis (Fersht, 1999; Berg et al., 2002). Their contribution is mainly in the near-UV (Woody and Dunker, 1996); however, they might inuence also in the far-UV alternating the predictions about protein secondary structures which are made in the far-UV region and this contribution can be stronger for proteins with low a-helix content (e.g., immunoglobulins and snake toxins). Hirst et al. applied TD-DFT for investigation of the p ! p* transitions of the tryptophan chromophore in the protein environment using barnase and human serum albumin as objects (Rogers et al., 2005) applying the TammDancoff approximation (TDA). The Trp chromophore was represented as indole and the method explicitly includes the local neighboring residues and presents the rest of the protein as a set of point charges. This concluded that in the gas phase the calculated vertical excitation energies to the 1L transitions are likely to be reordered with respect to the measured spectra. The authors discuss that the 1La state exhibit more the features of the 1Lb state and that is likely to be a result from the approximated nature of TD-DFT. They also indicated that the excited state characteristic might depend on the torsion angle of the side chain (in the case of albumin). The ability of TD-DFT to reproduce the near-UV excited state properties of aromatic model systems would be very important to justify its applications
93
for calculations of aromatic dimmers and clusters in proteins. Pollet and Brenner (2008) evaluated the performance of TD-DFT on tryptophanphenylalanine (Trp-Phe) dipeptide taking into account different minimum energy conformations of the molecule. It is well known that TD-DFT has good performance for the low-lying valence excited states with accuracy better than a few tenths of electron volts; however, it makes poor predictions of Rydberg and long-range CT states. In the case of (Trp-Phe) dipeptide, the emergence of the artifact long-range CT excitations is found to be enhanced when both aromatic side chains go closer. This leads to mixing of the interchromophore CT transitions with the intrachromophore Lb and La transitions. This depends on the choice of the density functional and is partially overcome by a corrected exchange-correlation potential; however, nonlocal HartreeFock-like exchange needs to be used for removing the CT transitions below the valence transitions. The intriguing feature of the aromatic CD in proteins is that they tend to form interacting pairs or clusters and to realize coupled oscillator type of interactions, and this could explain the frequently observed lack of correlation between the number of aromatic chromophores and their CD intensity. These interactions are the function of the distance, and the mutual orientation of the aromatic rings could also be inuenced by the local environment. Also, the aromatic chromophores could largely utilize the one-electron effect (mixing of electrically and magnetically allowed transitions within the same chromophore under the inuence of the protein structure; Kurapkat et al., 1997). The role of aromatic residues has been extensively studied experimentally and computationally. The experimental approach includes the chromophores to be mutated to weaker (e.g., Phe) or nonaromatic chromophore and consequent analysis of the difference between the CD spectra of the wild-type and the mutated protein. The possible drawback could be that the mutation induces small structural changes which might affect the orientations and interactions between the chromophores. Theoretical methods could overcome this utilizing in silico mutants and/or analyzing the individual contributions of the wild-type protein with mutations. The computational methods could, however, not include important environmental dynamical and electronic effects which might lead to qualitative level of predictions. The combination of both the approaches is likely to complement the drawback of both the strategies and to amplify their strengths. Extensive analysis of the
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aromatic CD was done on class A b-lactamases, bovine pancreatic trypsin inhibitor (BPTI) (Sreerama et al., 1999), bovine pancreatic ribonuclease A and S (Koslowski et al., 1996), human tissue factor, dihydrofolate reductase (DHFR) (Grishina and Woody, 1994), and barnase and carbonic anhydrase (Rogers and Hirst, 2004a,b). An important mechanistic analysis of the interactions between aromatic chromophores is done on tryptophan zipper peptide by Keuderling et al. (Roy et al., 2009). The study is performed on the far-UV CD spectra integrating the TD-DFT, as reasonably accurate QM method with the transition dipole coupling method as cheaper computational alternative and included measured CD spectra of the wild peptide and several Trp mutants. TRP zipper peptides are designed b-hairpin peptides, stabilized by hydrophobic interactions which are used for explaining the role of TRP residues and, in particular, the role of TrpTrp coupling in its CD spectra. This coupling is used for studies of the temperature-dependent unfolding of these peptides. The TD-DFT (B3lyp/6-31G** level) calculations including a pair of indoles xed at their positions in the peptide reveal that the CD intensity in 190230 nm result from the coupling of the pp* 1Bb transitions of both the indoles, which are realized from the HOMO, HOMO-1 to LUMO 1, and the LUMO 2 transitions. The calculations reproduce the strong negativepositive couplet in the far-UV and the negative CD intensity in the near-UV region which are experimentally detected as well. This result conrms the nding that the strong far-UV CD in Trp-rich beta hairpins is mainly due to the Trp side chain transitions rather than the backbone ones. The calculations using the more approximate but computationally much cheaper dipole coupling model (DCM) conrm qualitatively the TD-DFT results but do not provide magnitudes well.
IV. Tyrosine Interactions and Contributions in RNase A and RNase S

An interesting example for analysis of aromatic contributions is done by Kurapkat et al. (1997) on the bovine pancreatic ribonuclease A (RNase A) and its subtilisin-modied from (RNase S) using X-ray structures. The authors included all known transitions in the peptide and side-chain groups, most importantly those due to the aromatic (Tyr and Phe) and disulde groups. They performed the computations not only using the
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matrix method but also with the rst-order perturbation theory. The work also included two more methodological improvements: (i) new method for calculations of the transition charge densities and the static charge distributions is implemented; (ii) the effects of local EIs on energies of the electronic excitations are accounted for. The calculations predicted satisfactory the tyrosine contributions as a negative 275 nm band. The model conformed to previous studies and demonstrated that the coupling between Tyr 73 and Tyr 115 is the single most signicant interaction (Fig. 2). The positive band at 240 nm was assigned to the disulde transitions.
V.
Toward Improved Accuracy Using Ab Initio Parameter Sets
Hirst et al. calculated new parameter sets of the aromatic side chain transitions of phenylalanine, tyrosine, and tryptophan in order to be incorporated in the matrix method. They combined these new aromatic parameters with their amide parameters and computed the near- and far-UV CD spectra of 30 proteins (Rogers and Hirst, 2004a,b). They compared the performances of different parameter sets for aromatic amino acids: (i) semiempirical developed by Woody et al. (Sreerama and Woody, 2004); (ii) ab initio developed by Hirst et al. They found that the mean absolute error in the near-UV using the ab initio parameters is twofolds better than the
Y115 Y73
FIG. 2.
Interacting Tyr 73 and Tyr 114 in ribonuclease A.
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same error when semiempirical parameters are used (7500 deg cm2 dmol 1 for semiempirical against 170 deg cm2 dmol 1 for ab initio). For the differential spectra, however, they found that the difference in the semiempirical and ab initio parameters is smaller (98 deg cm2 dmol 1 for semiempirical and 54 deg cm2 dmol 1 for ab initio parameters). Based on calculations on barnase, human carbonic anhydrase II, and BPTI, the authors also suggested that the contributions of side chains to the CD are important when the helicity is calculated at 222 nm. In the case of the phenylalanine chromophore, because its parameters were calculated on benzene, neglect of the vibronic coupling had very small effect on the quality of the calculated rotational strengths. In the case of the tryptophan parameters, the semiempirical parameters have larger number of transitions and the energy of Bb transition is displaced with 21 nm in respect to the ab initio parameter sets and its energy depends on the environment. This sensitivity is proposed to be resulted from the congurational mixing between the 1Bb and 1Ba states (the ratio of the oscillator strengths of both the transitions is 1.1 which could suggest for considerable congurational mixing between them. The direction of the electric dipole moment calculated by semiempirical and ab initio methods differs too ( 15 and 34 , respectively, for semiempirical and ab initio parameters) and the value of the semiempirical one is very close to the experimentally measured. The predicted CD spectra are sensitive to the dipole moment orientation and energy of the 1Bb S0 transition in indole.
VI.
Far-UV Contributions of Tryptophans
Woody et al. (Grishina and Woody, 1994) applied the matrix method to reveal the contribution of Trp Bb transitions to the far-UV CD of several proteins. They calculated strong coupled interactions in exciton type for DHFR, chymotrypsin, and chymotrypsinogen in agreement to the experiment. In particular, these are the interactions between W47W74 in DHFR and W174W215 in chymotrypsinogen. Although the total far-UV CD spectra of these proteins do not predict the experimental data well, the differential spectra between the wild-type and Trp mutants are in reasonable agreement with the measurements which demonstrate the power of the computer methods to be used in combination with the experimental techniques for revealing the CD properties of the systems.
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VII.
Mechanisms of CD Spectra in Class A b-Lactamase A. Strategy of the Studies
Our group performed extensive research which was directed toward systematic investigation of the mechanisms of generation of the near-UV CD spectra of class A b-lactamases. The project was directed toward different complementing aspects of the electronic nature of the CD spectra: A. Analyzing the mechanisms of MGRS coupled oscillators interactions and one-electron mechanism comparative analysis on other b-lactamases from class A total contributions of the aromatic and the disulde transitions B. Individual contributions of each chromophore (from all 14) individual contribution in respect to the total one-electron rotational strength individual contributions in respect to the total coupled oscillator rotational strength individual contributions in respect to the total rotational strength total contributions of all trp, tyr, and disulde chromophores total contribution of each mechanism comparative analysis of other b-lactamases from class A C. Environmental (electrostatic effects) on the CD spectra analysis on the inuence of the MGRS in TEM-1 b-lactamase and total contributions of the aromatic and disulde transitions comparative analysis on three other b-lactamases from class A D. Conformational effects on the CD spectra: inuences on the MGRS inuences on the individual contributions of each chromophore electrostatic perturbation on the conformational sensitivity of the CD spectra E. Effects of deprotonated tyrosine chromophores
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F. Modeling MGRS in other b-lactamases from class A (from Staphylococcus aureus, Streptomyces albus, and Bacillus licheniformis) b-Lactamases are enzymes, responsible for bacterial resistance against antibiotics because they catalyze the hydrolysis of b-lactam antibiotics. Class A enzymes contain catalytic serine in their active site which plays crucial role in the nucleophilic attack on the substrate. TEM-1 b-lactamase is a 29-kDa protein and consists of a polypeptide chain, which has interesting foldingthe central section of chain folds into a globular part which include most of the a-helices, the N- and C-terminals form a vestranded b-sheet and is surrounded by the helical unit and another group of small helices (Vanhove et al., 1998). The chromophoric system of the protein consists of four tryptophans (165, 210 229, and 290), four tyrosines (46, 97, 105, and 264), ve phenylalanines (60, 66, 72, 151, and 230) and one disulde bond (formed between Cys77 and Cys123) (Fig. 3).
B.
Analysis the MGRS of TEM-1 b-Lactamase
We applied the matrix method with the parameters for all chromophores as described in Kurapkat et al. (1997), Christov et al. (2001), and Christov (2002) in order to investigate the mechanisms of the CD phenomena of this enzyme. The theoretical and experimental CD spectra of the free enzyme are presented in Fig. 4. They are characterized with negative band with minima around 280 nm. The predicted CD spectrum without tryptophans is with positive sign; therefore, it concludes that the negative sign is due to the tryptophan chromophores. The strongest contributions exhibit the following transition resulting from the static eld effect on the La and Bb transitions of W210, the transition due mainly to La of W229, those realized by coupling between transitions Lb of Y46 and La of W290 and the excited state which is generated by one-electron mixing of the transitions n4s* and n1s* of the disulde group (C77C123).
C. Electrostatic Effects on the MGRS

The effect of the local EIs on the MGRSs in the free enzyme were analyzed consequently taking into account the energy shift of the excitation energies due to the EIs between the chromophore and its local
99
W290
W229
Y46 F60 Y264 F66 Y05 F72
F230
W210
Y97 C23-C177 W165 F151
FIG. 3. TEM-1 b-lactamase: all near-UV chromophores are shown. Trp-s in blue, Tyr-sin red, and the disulde group in yellow. Copied with permission from Christov and Karabencehva (2010).
environment. The local effects lead to exciton-like couplings generated disbetween W210 and disulde group, which are positioned at 5.83 A and the tance and between W229 and W290 which are spaced 6.66 A coupling interactions between Y97 and Y105 and Y46 and W290 disappear. The EIs inuence the total contributions and this effect is stronger for La transitions of tryptophans and n1s*-transition of the disulde bond. The effective dielectric constant was one, which could be a reason for misestimating of the energy changes; however, the use of higher constant values of the dielectric constant or distance-dependent dielectric constants could be complicated. The tyrosine chromophore is the only one aromatic side chain which could undergo deprotonation at pH not too far from neutrality (pK 9.5; Woody and Dunker, 1996) and might exhibit considerable effect on
100
0 20 40 60 80 100 120 140 160 180 200 260
[q] MRW [deg. cm2. dmol1]
Experimental Theoretical
270
280 Wavelength [nm]
290
300
50
Acyl-enzyme (1 tem) Free enzyme (1 btl) Aclyl-enzyme (1 bt5) Transition state (1 axb) 270 280 290 300
0 260 50
100
150
FIG. 4. Experimental and computed CD spectra of TEM-1 b-lactamase as calculated in Christov and Karabencheva (2004) (left); conformational effects on the computed CD spectra (right): calculations are done using pdb structures with PDB IDs shown on the gure. The gures are copied with permission from Christov and Karabencheva (2004). (See color plate 1).
catalysis, hydrogen bonding, and spectral characteristics (Abraham et al., 2001; Davis et al., 2002; Horsman et al., 2005; Range et al., 2006). Therefore, it is important to predict the inuence of this ionization state on both the CD intensities and MGRS in the near-UV. Applying the matrix method, we demonstrated that deprotonation of the tyrosines inuences the total contributions of other aromatic transitions and, in particular, the
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contributions of tryptophan Lb and La transitions. The strongest effect is predicted for the ionization of Tyr 46. Following the analysis of two types of electrostatic effects: these due to the local EIs around each near-UV chromophore and those due to deprotonation of tyrosines we decided to analyze the accumulative, integrated impact of both the types. Therefore, we performed calculations of the CD spectrum of the TEM-1-free enzyme using ionized tyrosines and incorporating the EIs in the same calculation (Christov et al., 2008a). In the case of Y46, its own Lb and La rotational strengths are with smaller values than those predicted without local environment. In contrast to the calculations without local environment and in analogy of the model including the local electrostatics with all-neutral tyrosines, the exciton-like couplings between W210 and the disulde transitions as between W229 and W290 are generated. But the exciton-like coupling between Y97 and Y105, using classical model, disappears. In the case of Y97 deprotonation, the local electrostatics lead to exciton-like couplings between W210 and the disulde group and between W229 and W290 arise but the interaction between Y46 and W290 (predicted without local effects) disappears. The coupling of Y97 and the disulde chromophore also disappears. The Y105 ionization in contrast to the classical calculations leads to couplings from exciton type that are generated between W210 and disulde transitions and between W229 and W290 in analogy with the calculations of the local effects of all-neutral tyrosines enzyme. In the case of Y246, the interactions between Y46 and W290 as well as between Y97 and Y105 disappear.
D. Conformational Effects on the MGRS

The effects of conformational changes related to interactions with ligands and catalysis on the coupling interactions and one-electron effect were analyzed (Christov and Karabencheva, 2004). The crystal structures of the complex with the inhibitor of deacylation stage6a-(hydroxymethyl) penicilinic acid (PDB code: 1tem_pdb.ent; Maveyraud et al., 1996), of the enzymeinhibitor complex with the acetylation inhibitor imepenem (PDB code: 1bt5_pdb.ent) (Maveyraud et al., 1998a), and of the transition state analog of the acetylation stage (PDB code: 1axb_pdb. ent; Maveyraud et al., 1998b) were used. The rst two represent the structure of the acyl-enzyme intermediate whether the thirdthat of the
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transition state of the acetylation reaction. In the CD spectrum generated by 1tem acyl-enzyme structure, the coupling interactions between Y97 and Y105 and between Y46 and W290 which are characteristic for the free enzyme structure disappear. The acyl-enzyme intermediate 1bt5 is characterized by absence of coupled oscillator types of interactions between Y46 and W290. In the CD generated by the transition state analogous structure 1axb, the coupling from exciton type between Y97 and Y105 and coupling between Lb of Y46 and La of W229 are not presented; however, new exciton type interaction between W220 and W290 is realized. The total rotational strengths of all tryptophan La transitions and all Lb tyrosine transitions exhibit most signicant value in1axb structure. The disulde n1s* and n4s* transitions express strongest alteration in the 1axb structure.
E.
Electrostatic Inuence of the Conformational Sensitivity of MGRS
The separate computational analysis of both the conformational and electrostatic effects on the protein CD mechanisms on class A b-lactamases demonstrates the considerable inuence of both the factors on the MGRS. It is also important to integrate both the factors together and to analyze their integrated inuence on the MGRS and total contributions of the aromatic and disulde transitions. Such an analysis would provide insight into how the conformational sensitivity of the MGRS could be altered (enhanced or suppressed) by the incorporation of the EIs (Christov et al., 2008b). The difference between the simulated MGRS of the acyl-enzyme structure 1tem and that of the free enzyme with EI is more complicated than those that calculated with the classical model. A new coupling interaction arises between transitions of W165, W210, and the disulde group, as well as between W165 and the disulde group. In contrast to classically predicted CD mechanisms, the local effects lead to the following interactions: (i) between W165, W210, and the disulde group; (ii) between W210 and the disulde group; (iii) W165 and the disulde group; and (iv) between Y46, W229, and W290. The difference between the acyl-enzyme structure 1bt5 and the free enzyme structure is characterized with the following specicities when EI are included: (i) a exciton-like type coupling between Y46 and W290 is generated; (ii) phenylalanines F72 and F230 have no negligible
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contributions, which is a rare case in proteins; (iii) two exciton-like couplings between W210 and disulde transitions and a coupling between Y46 and W290 are generated; and (v) the coupling between Y97 and Y105, calculated classically, disappeared. Transition state structure has a key place in the chemical and enzyme mechanisms (Warshel et al., 2006). The simulated MGRS based on the protein component of this structure spectrum including the EI suggests for stronger conformational dependence than the calculations done with the classical model. In contrast to the free enzyme, the interactions between W210 and disulde transitions, as well as those between the transitions of W229 and W290, have a negative sign and could not be discussed as exciton type. The including of the local environment leads to coupling the disulde and W210 transitions. Both crystallographic monomers (A and B) from the b-lactamase crystal structure of B. licheniformis are another nice opportunity for exploration of the dependence of the aromatic rotational strengths to the integrated environmental and conformational effects (Christov et al., 2008b). This enzyme is very similar to TEM-1 and also belongs to class A. Each monomer consists of three tryptophans (210, 229, 251), six tyrosines (60, 97, 105, 129, 241, 274), and seven phenylalanines. In B. lichenifomis enzyme, the catalytically important V loop (residues 163179) is more exible than in TEM-1, where it is restrained by salt bridge interactions (Vijayakumar et al., 1995). The MGRS of conformer A were calculated with the classical model. The main contributions are by W210 and exciton-like couplings between W229 and W251, and Y60-Y274 (Karabencheva and Christov, 2004). Modeling with the same method predicts a deeper minimum for conformer B, displaced slightly to shorter wavelengths compared to conformer A. The accounting of the local effects makes greater contrast between both structures CDs. The spectrum of conformer A is almost the same, whereas the one of conformer B is predicted to exhibit even deeper minimum, shifted to higher. In contrast to structure A, complex coupling with the participation of transitions of W210, W229, and W251 as couplings between W210 and W229 and Y60 and Y241 is realized. The incorporation of local electrostatics decreased the number of the coupled interactions for both conformers, when compared to the classical calculations (Christov et al., 2008b).
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A semiquantitative information of the electrostatics on the conformational sensitivity of the MGRS can be received from relative changes of the calculated total contributions of the aromatic and disulde transitions (these are not individual chromophore contributions which we will discuss later). They are determined as a difference between the particular proteinligand structure and the free enzyme (for TEM-1), and between the conformers B and A (for B. licheniformis). The most signicant inuence of the environment on the rotational strength sensitivity for tryptophan Lb transitions is found in the acyl-enzyme structure 1tem. For tryptophan La transitions, it is found in the structure of transition state analog 1axb, the structure of conformer B of b-lactamase from B. licheniformis (4blmB), and in the structure 1tem. For tyrosine Lb transitions, it is found in the structure 1axb, and for the disulde n1s* and n4s* transitions, in the structure 1tem. The most signicant average electrostatic inuence (over all structures) on the conformational sensitivity of the rotational strengths is found for the tryptophan La transitions. The strongest inuence from the chromophore surroundings is found in the structure 1tem, but a signicant effect is also predicted for the structure 1axb and the conformer B of the enzyme from B. licheniformis.
F. Individual Contributions of Each Aromatic and Disulde Chromophore

CD as experimental technique is characterized with low signal resolution and therefore it is difcult to analyze even contributions of individual chromophores in relatively small organic molecules with moderate size and relatively rigid conformations (Berova et al., 2007). In proteins, which contain large number of chromophores and are characterized with conformational exibility, an analysis of individual chromophore contributions becomes far more difcult. The absence of this insight leads to uncertainty in relating the spectral data to atomistic structure. CD experiments with protein mutants could be used for receiving such information; however, the mutation may lead to subtle structural alterations in the structure which alternate the orientations between the chromophores, consequently their interactions, and lead to misleading conclusions. A fundamental point toward completing this knowledge is to nd an accurate and efcient approach in revealing the role of each individual chromophore, which does not make a perturbation even in smallest
105
details in the structure. Therefore, we developed a strategy, based on the matrix method which cannot be achieved by CD experiment, but successfully complements it (Christov and Karabencehva, 2010). The free enzyme structure, two acyl-enzymes, and the structure of the transition state analog were analyzed thus representing the ne and delicate structural changes which happen during time-resolved experiments. Because numerous experimental techniques alternate the electrostatic environment around chromophores (e.g., varying the ionic strength and pH), a quantitative insight about the sensitivity of the individual chromophore contributions to local changes was done. The individual contribution of each aromatic and the disulde chromophore was analyzed in the following ways (Christov and Karabencehva, 2010): 1. the individual net rotational strength generated by each chromophore over all its transitions 2. the relative contribution of each chromophore with respect to the total CD, generated by the one-electron mechanism 3. the relative contribution of each chromophore with respect to the total CD, generated by coupled oscillator mechanism 4. the relative percentage contribution of each near-UV chromophore, with respect to the total CD intensity 5. analysis of the inuence of the electrostatic changes on the contributions 6. analysis of the total percentage contributions of the aromatic and the disulde chromophores
1.
Contributions of Tryptophans
1.1. W165 This residue is situated in a solvent accessible area in the all-a domain. It generates 4.24% from the total one-electron rotational strength and 3.74% from the total rotational strength in the near-UV of the free enzyme. The structural change leading to 1tem acyl-enzyme structure changes its contributions to the CD spectrum considerably and all types of individual contributions (the contribution to the total one-electron Rotational Strengths (RS) and to the total CD) decreased almost four times in respect to the free enzyme. The structural change related to the formation of the second structure representative of the acyl-enzyme, 1bt5,
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leads to an increase in the individual contribution of W165 with respect to the free enzyme. Its relative participation in the total electron rotational strength is increased from 4.24% in 1btl to 7.13% in 1bt5. Consequently, the relative contribution of that chromophore with respect to the total rotational strength is increased from 3.74% to 6.10%. In the structure 1axb (representative of the transition state), the contribution with respect to the total rotational strength is 11%. The EIs in the free enzyme structure lead to an increase in the individual net contribution and in the individual relative contribution with respect to the total rotational strength. 1.2. W210 It is located in a solvent accessible area, almost opposite W165 within the all-a domain. Its contributions to the one-electron and total rotational strength are almost 10 times higher than those of W165 (i.e., 44% and 39% vs. 4.2% and 3.7%, respectively). In the acyl-enzyme structure, 1tem, the individual net contribution, as well as the contributions to the total one-electron and the overall total rotational strength are even higher than those in the free enzyme. This trend is also preserved in the acyl-enzyme structure, 1bt5, and the transition state structure, 1axb. In the latter structure, W210 exhibits the strongest contributions (1.142 Debye-Bohr magneton (DBM) net contribution, 45% from the total rotational strength, and more than half from the one-electron rotational strength). The accounting for the EIs, however, leads to a lowering of the percentage contribution to the total CD (to 18.25%). 1.3. W229 The residue is located in the ab-domain in a solvent accessible area just at the boundary between a b-sheet and an a-helix. In the free enzyme it provides 38.41% from the one-electron rotational strength and almost 34% from the total rotational strengths in the near-UV. In the acyl-enzyme structures 1tem and 1bt5, the percentage contribution of W229 to the one-electron rotational strength is 32.59% and 29.74%, respectively, and its contribution to the total rotational strengths is 31.10% and 25.45%, respectively. The transition state analog structure, 1axb, is interesting with respect to the CD of this chromophore. It is almost two times lower than in the previous two structures; it generates only 0.63% from the total one-electron rotational strength and the biggest part from the coupled oscillator rotational strength70.95%. Its contribution to the total rotational strength is 13.84%, which is the lowest in all of the structures for this chromophore.
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This chromophore is also sensitive to the local electrostatic environment and the relative contribution to the total one-electron rotational strength is dramatically reduced (from 38.41% to 2.37%). In contrast to the percentage contribution to the coupled oscillators, the rotational strength is dramatically increased from 0% to 29%. The contribution to the total rotational strength is decreased from 34% to 22%. 1.4. W290 W290 is positioned in the ab-domain in a solvent accessible area in front of W229 and at the boundary between a-helix and a b-sheet. In the free enzyme, it has the following contributions: 2.14% with respect to the total one-electron rotational strength, 7.87% with respect to the total coupled oscillators type, and less than 2% from the total near-UV rotational strength of the free enzyme. The conformational change related to the conversion of this structure to the acyl-enzyme structure, 1tem, decreases W290 in the total one-electron rotational strength and dramatically increases its relative contribution to the total couple cluster rotational strength to 98%. The proportion from the total rotational strength due to W290 is increased to 5.25%. In the 1bt5 acyl-enzyme structure, W290 generates 6.24% from the total rotational strength. In the transition state-like structure (1axb), the contribution to the total oneelectron rotational strength is lower than that in the free enzyme and in 1bt5 acyl-enzyme. It takes a considerable part of the total coupled oscillators CD28%. The relative part from the total rotational strength is similar to that in the 1bt56.53%. W290 is very sensitive to electrostatic changes. The relative part from the total one-electron spectrum is increased to 5% and the contribution to the total coupled oscillators CD is increased to almost 21%. It is important to notice that the percentage contribution of W290 to the total CD is increased under electrostatic effects from 1.89% to 16.78%. 2. Contributions of Tyrosines
2.1. Y46 It is located in the ab-domain, at the b-sheet to a-helical part of the domain, in a relatively solvent accessible. Y46 does not take part in the one-electron CD but provides half from the total coupled oscillator spectrum. In the free enzyme, it contributes 6% to the total near-UV CD spectrum. Y46 alters its trend to participate in one-electron and coupled oscillator mechanisms under the electrostatic inuence.
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2.2. Y97 This tyrosine chromophore is located at the top part of the all-a domain. For the free enzyme structure, it has no one-electron CD contribution and provides 24.41% from the coupled oscillator spectrum. The relative participation of Y97 in respect to the total near-UV CD is 2.86%. EIs play a considerable effect on the CD contributions of Y97, decreasing all types of its contribution dramatically in the free enzyme. 2.3. Y105 Y105 is located within the all-a domain at the turn structure that links two 3_10 helices. In the free enzyme, Y105 contributes with 13.78% from the total coupled oscillators RS, does not contribute to the total one-electron CD, and gives 1.61% from the total near-UV CD. The incorporation of EI not only contributes to the decrease in its relative contribution of the near-UV CD by 50% but also alters its mechanism from coupled clusters to one-electron. 2.4. Y264 Y264 is located in the ab-domain on the b-sheet. It exhibits very low contributions to the CD, comparable to those of some of the phenylalanine chromophores. 3. Contributions of the Disulde Chromophore
The disulde group located in the all-a domain connects two a helices. It is closely positioned to W210. The relative part of the total one-electron CD due to the disulde chromophore is 10.41%, and it generates 9.20% from the total near-UV rotational strength. The disulde group keeps the trend to contribute to the total one-electron rotational strength (from 10.41% to 13.59%) in all structures. It is important to mention that in all structures, of the free enzyme and the complexes, the disulde group contributes only to the one-electron spectrum but not to the coupled oscillator type spectrum and it is strongest in 1axb and 1bt5 structures. The disulde part from the total near-UV CD varies between 9.20% and 12.04%. A relationship between the values of the disulde torsion angle (CB-SG-SG-CB) suggests that there is an optimal value of the angle which corresponds to highest individual contribution of the disulde group in respect to the total CD and it is 110.1 (in the 1tem structure). The accounting of EI dramatically alters the qualitative and quantitative picture of the disulde CD. The relative part from the total one-electron
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RS due to the disulde groups is increased three times from 10.41% to 31.88%. Under EI effects, it also contributes to the total coupled oscillator spectrum (23.11%) and the disulde part from the total near-UV CD is increased from 9.20% to 33.56%. It could be concluded that the CD contribution of the disulde group (C23C177) is relatively resistant to structural changes induced by interactions with ligands but is considerably sensitive to a change in the EI. 4. Analysis of the Total Contributions
The total contributions of the aromatic and the disulde chromophores are presented in Table I. All contributions are given with respect to the total rotational strength. In the free enzyme, 1btl, all tryptophan residues provide the main part of the near-UV CD spectrum, 78.24%. The structural change related to the formation of 1bt5 has a most sensitive effect (decreases the contribution to 69%). All tyrosine contributions are more sensitive to the above discussed conformational changes. The effect is highest in the 1bt5 structure (15.42) and lowest in the 1tem structure
Table I Individual Contributions of all Chromophores in the Free Enzyme of TEM-1 b-Lactamase with and without EI
45 40 35 30 25 20 15 10 5 0
6 F6 D 16 5 21 0 22 9 29 0 64 F2 30 0 F1 51 Y4 Y9 05 F7 F6 Y1 W W W W Y2 is ul f 6 7 2
Without EI Including EI
Copied with permission from Christov and Karabencehva (2010).
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(5.81%). The phenylalanine chromophores exhibit very weak total contributionsbetween 0.3% and 1.5%. In total, the aromatic residues provide 91% from the total CD in the free enzyme, which slightly varies under structural changes. The disulde group generates between 9.21% and 12.04% of the total RS. The contributions of all aromatic chromophores decrease from 91% to 68% under EI effects. However, the EI effects on the disulde contribution are opposite to and more sensitive than that of the aromaticstotal disulde contribution is increased three times (from 9.20% to 33.56%). It is also important to estimate the contributions of both the oneelectron and the coupled oscillator mechanisms to the CD. In all structures, with or without EI, it is a common trend that the one-electron mechanism dominates the CD spectrum. However, its relative contribution is inuenced by conformational and electrostatic changes. In the free enzyme, the one-electron rotational strength is 88%; however, in the 1tem structure, the one-electron mechanism is responsible for more than 95% from the spectral intensities. The electrostatic effects decrease the oneelectron character of the spectrum from 88% to 75%. The coupled oscillator mechanism type (m ! m and m ! m mechanisms taken together) provides 11% of the CD intensity in the free enzyme which is increased to 14% in 1bt5 and up to 19% in the 1axb structure, where in the 1tem structure, it decreases to 5%. EIs lead to a considerable increase in the coupled oscillators contributions from 11% to 25%. The changes in the individual contributions of all chromophores under structural changes are presented in Table II. The aromatic chromophores are sensitive structural changes related to catalysis and the disulde group is relatively resistant to them. W210 expresses the strongest contribution to the near-UV spectrum in all structures, which varies between 30 and 45 and the disulde group (small changes between 9% and 11%). The EI (Table I) decreases in the contributions of W210 and W229 but increases the disulde contribution three times more, making the disulde group the most contributive chromophore to the total near-UV CD. The above analysis was extended to other similar b-lactamases from class A (from St. aureus, B. licheniformis, and Str. albus) (Karabencheva et al., 2010) where the individual contributions of the aromatic chromophores were analyzed and in the case for B. licheniformis enzyme demonstrated good agreement with the experiment (Risso et al., 2010).
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Table II Changes in the Individual Contributions Under Structural Changes

50 40 1btl 30 20 10 0
W W W W 0 Y4 6 Y9 7 Y1 05 Y2 64 F2 30 F7 2 F6 0 F1 51 F6 6 D is ul f 5 0 16 21 22 9 29
1tem 1bt5 1axb
Copied with permission from Christov and Karabencehva (2010). (See color plate 1).
VIII. Conclusion
The advances in computational methods for calculation of CD spectra of proteins not only provide increased accuracy in prediction of the spectral features in reference to the experiment but also deepen and strengthen our understanding about the mechanisms of generation of protein rotational strengths and how they are inuenced by structural and environmental changes. The increasing computational power of the clusters in supercomputers together with better performance of the electronic structure codes would make possible applying of accurate methods like TD-DFT for larger systems (e.g., small peptides or clusters of aromatic and sulde chromophores); however, the approximate methods as the matrix method would still be the main computational tool for understanding CD mechanisms of large protein complexes, therefore their proper parameterization makes important role. Understanding the mechanisms of CD spectra will help for better understanding of structure-spectra relationship and will help in providing more accurate details in ligand binding, catalysis, folding and mutational effects. Combination of experimental and theoretical CD of proteins would be important complementary tool in drug design and proteomics.
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Mechanisms of Protein Circular Dichroism Insights From Computational Modeling

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MECHANISMS OF PROTEIN CIRCULAR DICHROISM: INSIGHTS FROM COMPUTATIONAL MODELING

By TATYANA KARABENCHEVA AND CHRISTO CHRISTOV

III. IV. V. VI. VII.

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KARABENCHEVA AND CHRISTOV