Insight into the structural dynamics of light sensitive proteins from time-resolved crystallography and quantum chemical calculations

The structural dynamics underlying molecular mechanisms of light-sensitive proteins can be studied by a variety of experimental and computational biophysical techniques. Here we review recent progress in combining time-resolved crystallography at X-ray free electron lasers and quantum chemical calculations to study structural changes in photoenzymes, photosynthetic proteins, photoreceptors, and photoswitchable fluorescent proteins following photoexcitation.     

Internal motion and electron transfer in proteins: a picosecond fluorescence study of three homologous azurins

We have carried out a picosecond fluorescence study of holo- and apoazurins of Pseudomonas aeruginosa (azurin Pae), Alcaligenes faecilis (azurin Afe), and Alcaligenes denitrificans (azurin Ade). Azurin Pae contains a single, buried tryptophyl residue; azurin Afe, a single surface tryptophyl residue; and azurin Ade, tryptophyl residues in both environments. From anisotropy measurements we conclude that the interiors of azurins Pae and Ade are not mobile enough to enable motion of the indole ring on a nanosecond time scale. The exposed tryptophans in azurins Afe and Ade show considerable mobility on a few hundred picosecond time scale. The quenching of tryptophan fluorescence observed in the holoproteins is interpreted in terms of electron transfer from excited-state tryptophan to Cu(II). The observed rates are near the maximum predicted by Marcus theory for the separation of donor and acceptor. The involvement of protein matrix and donor mobility for electron transfer is discussed. The two single-tryptophan-containing proteins enable the more complex fluorescence behavior of the two tryptophans of azurin Ade to be understood. The single-exponential fluorescence decay observed for azurin Pae and the nonexponential fluorescence decay observed for azurin Afe are discussed in terms of current models for tryptophan photophysics.     

Insight into the conformational dynamics of specific regions of porcine pancreatic phospholipase A2 from a time-resolved fluorescence study of a genetically inserted single tryptophan residue

The effects of Ca2+ and substrate analogue binding on the conformational dynamics of porcine pancreas phospholipase A2 (PLA2) in different regions was explored by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) of the wild-type protein (W3), in the alpha-helix A, was replaced by a phenylalanine residue (W3F), whereafter Trp was substituted either for leucine-31 (W31), located in the calcium binding loop, or for phenylalanine-94 (W94), located at the "back side" of the enzyme. Furthermore, mutants lacking the 62-66 sequence were constructed with the Trp at position 3 (delta W3) or 31 (delta W31). The total fluorescence intensity decays of Trp in each protein, in the protein-calcium and the protein-calcium-substrate analogue complexes, analyzed by the maximum entropy method (MEM) can be interpreted as distributions of separated lifetime classes. In the case of the W94 mutant, a major short-lived excited-state population (tau approximately 50 ps) is observed, probably deactivated by the interaction with two proximate disulfide bridges via a radiationless process. For the four other mutants, the respective barycenters of the four lifetime classes display comparable values, but the amplitude distributions are different for Trp-3 and Trp-31. The rotational mobility of the Trp residue varies along the peptide chain. Trp-3 experiences only a fast hindered motion. Trp-31 is sensitive to an additional local flexibility that is absent in the N-terminal part of the protein. The largest wobbling angle is observed at position 94. No effect of calcium binding occurs on the lifetime distribution of the Trp-3 and Trp-94 residues. Their mobilities are not affected. In contrast, calcium binding displays a strong influence on the excited-state population distribution of Trp-31. A major population decaying with the longest lifetime is selected in the W31 protein and contributes to approximately 50% of the decay. The local flexibility and the amplitude of motion of Trp-31 is wider in the protein-calcium complex than in the unliganded protein. Binding of the monomeric substrate analogue n-dodecylphosphocholine (C12PN) in the presence of calcium slightly affects the Trp-3 excited-state population distribution and its mobility. Trp-31 is more sensitive to this binding. In particular, a more restricted rotation of the Trp-31 residue and a decrease of the peptide local flexibility as protein-calcium complexes are observed in both the W31 and delta W31 mutants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformational forces affecting the folding pathways of dendrotoxins I and K from black mamba venom

The conformations of the major intermediates trapped during the folding of dendrotoxins I and K from venom of black mamba snakes, have been investigated by circular-dichroism spectroscopy. Local alterations to the native, folded conformations are observed in toxins I and K and in a protein of similar sequence, bovine pancreatic trypsin inhibitor. The inability of intermediates (30-51, 14-38) to complete refolding by forming directly the 5-55 disulphide bond is explained. The following observations on the role of secondary structure in the folding of the three proteins are of interest. 1. It is not necessary for the three proteins to acquire elements of secondary structure at the same stage of folding in order to attain similar three-dimensional conformations. 2. The stability of the final folded state is not directly correlated to an early appearance of secondary structure. 3. The degree of secondary structure already present in intermediates (30-51) seems to determine the pathway of refolding preferred by the corresponding protein.     

Protein dynamics. A time-resolved fluorescence, energetic and molecular dynamics study of ribonuclease T1

Studies using time-resolved fluorescence depolarization were performed on the internal motion of Trp 59 of ribonuclease T1 (EC 3.1.27.3) in the free enzyme, 2'-GMP-enzyme complex and 3'-GMP-enzyme complex. The Trp 59 motion was also studied in the free enzyme using molecular dynamics simulations. Energetic analysis of activation barriers to the Trp 59 motion was performed using both the transition state theory and Kramers' theory. The activation parameters showed a dependence on solvent viscosity indicating the transition state approach in aqueous solution to be inadequate. When taking solvent viscosity contributions into account agreement between the transition state and Kramers' theories was obtained. The results indicate the three enzyme forms to have different conformations with the free enzyme and 3'-GMP-enzyme complex being similar. Comparison of the experimental and theoretical results showed a good agreement on the Trp 59 motion in the free enzyme. Trp 59 appears to vibrate rapidly, with a relaxation time of the order of 1 ps, within free space in the protein matrix and to have a slower motion, with a relaxation time of the order of 100 ps, which is related to breathing of the surrounding protein matrix. Molecular dynamics results indicate high mobility in regions of the enzyme involved in the interaction with the guanine base of the inhibitor or substrate while much lower mobility occurred in residues involved in the catalytic mechanism of ribonuclease T1.     

Incorporation of bacterial membrane proteins into liposomes: factors influencing protein reconstitution

Meningococcal and gonococcal outer membrane proteins were reconstituted into liposomes using detergent-mediated dialysis. The detergents octyl glucopyranoside (OGP), sodium cholate and Empigen BB were compared with respect to efficiency of detergent removal and protein incorporation. The rate of OGP removal was greater than for cholate during dialysis. Isopycnic density gradient centrifugation studies showed that liposomes were not formed and hence no protein incorporation occurred during dialysis from an Empigen BB containing reconstitution mixture. Cholate-mediated reconstitution yielded proteoliposomes with only 75% of the protein associated with the vesicles whereas all of the protein was reconstituted into the lipid bilayer during OGP-mediated reconstitution. Essentially complete protein incorporation was achieved with an initial protein-to-lipid ratio of 0.01:1 (w/w) in the reconstitution mixture; however, at higher initial protein-to-lipid ratios (0.02:1) only 75% protein incorporation was achieved. Reconstituted proteoliposomes were observed as large (>300 nm), multilamellar structures using cryo-electron microscopy. Size reduction of these proteoliposomes by extrusion did not result in significant loss of protein or lipid. Extruded proteoliposomes were unilamellar vesicles with mean diameter of about 100 nm.     

Tryptophan dynamics of the FK506 binding protein: time-resolved fluorescence and simulations.

The FK506-binding protein (FKBP12) is important in the immunosuppressant action of FK506 and rapamycin. We have investigated Trp side chain dynamics in FKBP12, with and without a bound immunosuppressant, by measuring the Trp time-resolved fluorescence anisotropy decay r(t). The r(t) for W59 in aqueous uncomplexed FKBP12 at 20 degrees C is well described by a single exponential with a recovered initial anisotropy, r(eff)o, of 0.192 and an overall rotational correlation time for the protein, phi p, of 4.7 ns; r(eff)o = 0.214 and phi p = 4.2 ns for the FKBP12/FK506 complex. Using an expression for the order parameter squared, namely S2 = r(eff)o/rTo, where rTo is the vitrified steady-state excitation anisotropy, we recovered an S2 of 0.75 for W59 fluorescence in uncomplexed FKBP12 and S2 approximately equal to 1 in the FKBP12/FK506 complex. Results obtained for the FKBP12/rapamycin complex are similar to those found for the FKBP12/FK506 complex. Minimum perturbation mapping simulations were performed on the free and complexed forms of FKBP12 and the results were generally in agreement with the experimental data.     

三种温带树种叶片呼吸的时间动态及其影响因子

为认知叶片呼吸(RL)的季节变化格局及其影响因子,以东北东部山区3个主要树种(红松Pinus koraiensis、樟子松P.sylvestris var.mongolica和白桦Betula platyphylla)为对象,采用红外气体分析法在2011年生长季(常绿树4月至10月;落叶树6月至9月)测定了自然条件下叶片气体交换及其相关生理特征的季节变化,探索了RL与空气温度(Tair)和相关叶片特征之间的关系.结果表明:红松和樟子松基于叶面积的RL(RL-area)表现为生长季初期和末期较大,而白桦RL-area则随生长季进程而逐渐减小.在生长季中,RL-area与叶片总光合之比的时间动态明显.红松、樟子松RL-area与Tair关系显著,而白桦RL-area与Tair关系不显著;但3种树种基于叶质量的RL(RL-mass)与Tair均呈显著的指数函数关系.叶片特征(包括可溶性糖、淀粉、氮、比叶面积等参数)也有明显的季节变化.影响RL的叶片特征参数因树种而异,其中可溶性糖浓度对3种树种的RL均有显著影响.可见,RL的季节变化格局受树木的生长节律、温度和叶片特征的联合控制.     

Differences in backbone dynamics of two homologous bacterial albumin-binding modules: implications for binding specificity and bacterial adaptation

Proteins G and PAB are bacterial albumin-binding proteins expressed at the surface of group C and G streptococci and Peptostreptococcus magnus, respectively. Repeated albumin-binding domains, known as GA modules, are found in both proteins. The third GA module of protein G from the group G streptococcal strain G148 (G148-GA3) and the second GA module of protein PAB from P.magnus strain ALB8 (ALB8-GA) exhibit 59% sequence identity and both fold to form three-helix bundle structures that are very stable against thermal denaturation. ALB8-GA binds human serum albumin with higher affinity than G148-GA3, but G148-GA3 shows substantially broader albumin-binding specificity than ALB8-GA. The (15)N nuclear magnetic resonance spin relaxation measurements reported here, show that the two GA modules exhibit mobility on the picosecond-nanosecond time scale in directly corresponding regions (loops and termini). Most residues in G148-GA3 were seen to be involved in conformational exchange processes on the microsecond-millisecond time scale, whereas for ALB8-GA such motions were only identified for the beginning of helix 2 and its preceding loop. Furthermore, and more importantly, hydrogen-deuterium exchange and saturation transfer experiments reveal large differences between the two GA modules with respect to motions on the second-hour time scale. The high degree of similarity between the two GA modules with respect to sequence, structure and stability, and the observed differences in dynamics, binding affinity and binding specificity to different albumins, suggest a distinct correlation between dynamics, binding affinity and binding specificity. Finally, it is noteworthy in this context that the module G148-GA3, which has broad albumin-binding specificity, is expressed by group C and G streptococci known to infect all mammalian species, whereas P.magnus with the ALB8-GA module has been isolated only from humans.     

Insight into the structure, dynamics and the unfolding property of amylosucrases: implications of rational engineering on thermostability

Amylosucrase (AS) is a kind of glucosyltransferases (E.C. 2.4.1.4) belonging to the Glycoside Hydrolase (GH) Family 13. In the presence of an activator polymer, in vitro, AS is able to catalyze the synthesis of an amylose-like polysaccharide composed of only α-1,4-linkages using sucrose as the only energy source. Unlike AS, other enzymes responsible for the synthesis of such amylose-like polymers require the addition of expensive nucleotide-activated sugars. These properties make AS an interesting enzyme for industrial applications. In this work, the structures and topology of the two AS were thoroughly investigated for the sake of explaining the reason why Deinococcus geothermalis amylosucrase (DgAS) is more stable than Neisseria polysaccharea amylosucrase (NpAS). Based on our results, there are two main factors that contribute to the superior thermostability of DgAS. On the one hand, DgAS holds some good structural features that may make positive contributions to the thermostability. On the other hand, the contacts among residues of DgAS are thought to be topologically more compact than those of NpAS. Furthermore, the dynamics and unfolding properties of the two AS were also explored by the gauss network model (GNM) and the anisotropic network model (ANM). According to the results of GNM and ANM, we have found that the two AS could exhibit a shear-like motion, which is probably associated with their functions. What is more, with the discovery of the unfolding pathway of the two AS, we can focus on the weak regions, and hence designing more appropriate mutations for the sake of thermostability engineering. Taking the results on structure, dynamics and unfolding properties of the two AS into consideration, we have predicted some novel mutants whose thermostability is possibly elevated, and hopefully these discoveries can be used as guides for our future work on rational design.     

Vaccinia virus telomeres: interaction with the viral I1, I6, and K4 proteins

The 192-kb linear DNA genome of vaccinia virus has covalently closed hairpin termini that are extremely AT rich and contain 12 extrahelical bases. Vaccinia virus telomeres have previously been implicated in the initiation of viral genome replication; therefore, we sought to determine whether the telomeres form specific protein-DNA complexes. Using an electrophoretic mobility shift assay, we found that extracts prepared from virions and from the cytoplasm of infected cells contain telomere binding activity. Four shifted complexes were detected using hairpin probes representing the viral termini, two of which represent an interaction with the "flip" isoform and two with the "flop" isoform. All of the specificity for protein binding lies within the terminal 65-bp hairpin sequence. Viral hairpins lacking extrahelical bases cannot form the shifted complexes, suggesting that DNA structure is crucial for complex formation. Using an affinity purification protocol, we purified the proteins responsible for hairpin-protein complex formation. The vaccinia virus I1 protein was identified as being necessary and sufficient for the formation of the upper doublet of shifted complexes, and the vaccinia virus I6 protein was shown to form the lower doublet of shifted complexes. Competition and challenge experiments confirmed that the previously uncharacterized I6 protein binds tightly and with great specificity to the hairpin form of the viral telomeric sequence. Incubation of viral hairpins with extracts from infected cells also generates a smaller DNA fragment that is likely to reflect specific nicking at the apex of the hairpin; we show that the vaccinia virus K4 protein is necessary and sufficient for this reaction. We hypothesize that these telomere binding proteins may play a role in the initiation of vaccinia virus genome replication and/or genome encapsidation.     

NMR backbone dynamics of the human type I interferon binding subunit, a representative cytokine receptor

The antiviral and antiproliferative activities of type I interferons (IFNs) are mediated by a common receptor, and its second subunit (IFNAR2) exhibits nanomolar affinity to both IFNalpha and IFNbeta subtypes. We have previously determined the structure of the IFN-binding extracellular domain of IFNAR2 (IFNAR2-EC) using multidimensional NMR [Chill, J. H., Quadt, S. R., Levy, R., Schreiber, G. E., and Anglister, J. (2003) Structure 11, 791-802], showing it to comprise two fibronectin domains linked by a hinge. As the first cytokine receptor structure determined in the unliganded state and in solution, IFNAR2-EC offers an opportunity to characterize the dynamics of the cytokine receptor family and their correlation to biological function. Backbone dynamics of IFNAR2-EC were investigated using 15N relaxation at 11.74 and 18.79 T, and measurements of residual dipolar couplings (RDCs). Dynamics of the binding site distinguish between rigid structural domains, which stabilize the binding site conformation, and a more flexible binding interface which interacts with the ligand. Measurements of diffusional anisotropy and RDCs and model-free analysis all show that the backbone of the hinge interdomain region of IFNAR2-EC is rigid on the picosecond to nanosecond time scale. Signal transduction in cytokines receptors is initiated by ligand-induced juxtaposition of the two receptor subunits, triggering the mutual phosphorylation of kinases associated to their cytoplasmic domains. The rigidity of the hinge ensures correct positioning of the receptor subunits in the ternary signaling complex and modulates the interaction between kinases in the cytoplasm, thereby controlling the rate and efficiency of phosphorylation.     

Insight into the proteome of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis: the major cytosolic and membrane proteins

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic Crenarchaeon, is the host of Nanoarchaeum equitans. Together, they form an intimate association, the first among Archaea. Membranes are of fundamental importance for the interaction of I. hospitalis and N. equitans, as they harbour the proteins necessary for the transport of macromolecules like lipids, amino acids, and cofactors between these organisms. Here, we investigated the protein inventory of I. hospitalis cells, and were able to identify 20 proteins in total. Experimental evidence and predictions let us conclude that 11 are soluble cytosolic proteins, eight membrane or membrane-associated proteins, and a single one extracellular. The quantitatively dominating proteins in the cytoplasm (peroxiredoxin; thermosome) antagonize oxidative and temperature stress which I. hospitalis cells are exposed to at optimal growth conditions. Three abundant membrane protein complexes are found: the major protein of the outer membrane, which might protect the cell against the hostile environment, forms oligomeric complexes with pores of unknown selectivity; two other complexes of the cytoplasmic membrane, the hydrogenase and the ATP synthase, play a key role in energy production and conversion.     

Insight into the stability of the hydrophobic binding proteins of Escherichia coli: assessing the proteins for use as biosensors

Spectroscopic methods were used to monitor the unfolding of the leucine specific (LS) and the leucine-isoleucine-valine (LIV) binding proteins. Our studies indicate that ligand-free protein undergoes a simple two-state unfolding, whereas the protein-ligand complex undergoes a three-state unfolding model. Ligand binding causes significant stabilization of both proteins. There is correlation between ligand hydrophobicity and protein stabilization: the most hydrophobic ligand, isoleucine, causes the most significant stabilization of LIV protein. A disulfide bond present in N-domain of both proteins makes a large contribution to the protein stability of these periplasmic binding receptors.     

Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements

Summary Membrane fragments containing a high density of Na, K-ATPase can be noncovalently labeled with amphiphilic styryl dyes (e.g., RH 421). Phosphorylation of the Na,K-ATPase by ATP in the presence of Na⁺ and in the absence of K⁺ leads to a large increase of the fluorescence of RH 421 (up to 100%). In this paper evidence is presented that the styryl dye mainly responds to changes of the electric field strength in the membrane, resulting from charge movements during the pumping cycle: (i) The spectral characteristic of the ATP-induced dye response essentially agrees with the predictions for an electrochromic shift of the absorption peak. (ii) Adsorption of lipophilic anions to Na, K-ATPase membranes leads to an increase, adsorption of lipophilic cations to the decrease of dye fluorescence. These ions are known to bind to the hydrophobic interior of the membrane and to change the electric field strength in the boundary layer close to the interface. (iii) The fluorescence change that is normally observed upon phosphorylation by ATP is abolished at high concentrations of lipophilic ions. Lipophilic ions are thought to redistribute between the adsorption sites and water and to neutralize in this way the change of field strength caused by ion translocation in the pump protein. (iv) Changes of the fluorescence of RH 421 correlate with known electrogenic transitions in the pumping cycle, whereas transitions that are known to be electrically silent do not lead to fluorescence changes. The information obtained from experiments with amphiphilic styryl dyes is complementary to the results of electrophysiological investigations in which pump currents are measured as a function of transmembrane voltage. In particular, electrochromic dyes can be used for studying electrogenic processes in microsomal membrane preparations which are not amenable to electrophysiological techniques.Deceased (September 13, 1990).     

A comparison of the three isoforms of the light-harvesting complex II using transient absorption and time-resolved fluorescence measurements

In this article we report the characterization of the energy transfer process in the reconstituted isoforms of the plant light-harvesting complex II. Homotrimers of recombinant Lhcb1 and Lhcb2 and monomers of Lhcb3 were compared to native trimeric complexes. We used low-intensity femtosecond transient absorption (TA) and time-resolved fluorescence measurements at 77 K and at room temperature, respectively, to excite the complexes selectively in the chlorophyll b absorption band at 650 nm with 80 fs pulses and on the high-energy side of the chlorophyll a absorption band at 662 nm with 180 fs pulses. The subsequent kinetics was probed at 30–35 different wavelengths in the region from 635 to 700 nm. The rate constants for energy transfer were very similar, indicating that structurally the three isoforms are highly homologous and that probably none of them play a more significant role in light-harvesting and energy transfer. No signature has been found in the transient absorption measurements at 77 K for Lhcb3 which might suggest that this protein acts as a relative energy sink of the excitations in heterotrimers of Lhcb1/Lhcb2/Lhcb3. Minor differences in the amplitudes of some of the rate constants and in the absorption and fluorescence properties of some pigments were observed, which are ascribed to slight variations in the environment surrounding some of the chromophores depending on the isoform. The decay of the fluorescence was also similar for the three isoforms and multi-exponential, characterized by two major components in the ns regime and a minor one in the ps regime. In agreement with previous transient absorption measurements on native LHC II complexes, Chl b → Chl a energy transfer exhibited very fast channels but at the same time a slow component (ps). The Chls absorbing at around 660 nm exhibited both fast energy transfer which we ascribe to transfer from ‘red’ Chl b towards ‘red’ Chl a and slow transfer from ‘blue’ Chl a towards ‘red’ Chl a. The results are discussed in the context of the new available atomic models for LHC II.     

Investigations into sequence and conformational dependence of backbone entropy,inter-basin dynamics and the Flory isolated-pair hypothesis for peptides

The populations and transitions between Ramachandran basins are studied for combinations of the standard 20 amino acids in monomers, dimers and trimers using an implicit solvent Langevin dynamics algorithm and employing seven commonly used force-fields. Both the basin populations and inter-conversion rates are influenced by the nearest neighbor's conformation and identity, contrary to the Flory isolated-pair hypothesis. This conclusion is robust to the choice of force-field, even though the use of different force-fields produces large variations in the populations and inter-conversion rates between the dominant helical, extended beta, and polyproline II basins. The computed variation of conformational and dynamical properties with different force-fields exceeds the difference between explicit and implicit solvent calculations using the same force-field. For all force-fields, the inter-basin transitions exhibit a directional dependence, with most transitions going through extended beta conformation, even when it is the least populated basin. The implications of these results are discussed in the context of estimates for the backbone entropy of single residues, and for the ability of all-atom simulations to reproduce experimental protein folding data.