Inhibition of Receptor Dimerization as a Novel Negative Feedback Mechanism of EGFR Signaling.

Dimerization of the epidermal growth factor receptor (EGFR) is crucial for initiating signal transduction. We employed raster image correlation spectroscopy to continuously monitor the EGFR monomer-dimer equilibrium in living cells. EGFR dimer formation upon addition of EGF showed oscillatory behavior with a periodicity of about 2.5 min, suggesting the presence of a negative feedback loop to monomerize the receptor. We demonstrated that monomerization of EGFR relies on phospholipase Cγ, protein kinase C, and protein kinase D (PKD), while being independent of Ca2+ signaling and endocytosis. Phosphorylation of the juxtamembrane threonine residues of EGFR (T654/T669) by PKD was identified as the factor that shifts the monomer-dimer equilibrium of ligand bound EGFR towards the monomeric state. The dimerization state of the receptor correlated with the activity of an extracellular signal-regulated kinase, downstream of the EGFR. Based on these observations, we propose a novel, negative feedback mechanism that regulates EGFR signaling via receptor monomerization.

In vitro phosphorylation does not influence the aggregation kinetics of WT α-synuclein in contrast to its phosphorylation mutants.

The aggregation of alpha-synuclein (α-SYN) into fibrils is characteristic for several neurodegenerative diseases, including Parkinson's disease (PD). Ninety percent of α-SYN deposited in Lewy Bodies, a pathological hallmark of PD, is phosphorylated on serine129. α-SYN can also be phosphorylated on tyrosine125, which is believed to regulate the membrane binding capacity and thus possibly its normal function. A better understanding of the effect of phosphorylation on the aggregation of α-SYN might shed light on its role in the pathogenesis of PD. In this study we compare the aggregation properties of WT α-SYN with the phospho-dead and phospho-mimic mutants S129A, S129D, Y125F and Y125E and in vitro phosphorylated α-SYN using turbidity, thioflavin T and circular dichroism measurements as well as transmission electron microscopy. We show that the mutants S129A and S129D behave similarly compared to wild type (WT) α-SYN, while the mutants Y125F and Y125E fibrillate significantly slower, although all mutants form fibrillar structures similar to the WT protein. In contrast, in vitro phosphorylation of α-SYN on either S129 or Y125 does not significantly affect the fibrillization kinetics. Moreover, FK506 binding proteins (FKBPs), enzymes with peptidyl-prolyl cis-trans isomerase activity, still accelerate the aggregation of phosphorylated α-SYN in vitro, as was shown previously for WT α-SYN. In conclusion, our results illustrate that phosphorylation mutants can display different aggregation properties compared to the more biologically relevant phosphorylated form of α-SYN.

Mammalian ribosomal and chaperone protein RPS3A counteracts α-synuclein aggregation and toxicity in a yeast model system.

Accumulation of aggregated forms of αSyn (α-synuclein) into Lewy bodies is a known hallmark associated with neuronal cell death in Parkinson's disease. When expressed in the yeast Saccharomyces cerevisiae, αSyn interacts with the plasma membrane, forms inclusions and causes a concentration-dependent growth defect. We have used a yeast mutant, cog6Δ, which is particularly sensitive to moderate αSyn expression, for screening a mouse brain-specific cDNA library in order to identify mammalian proteins that counteract αSyn toxicity. The mouse ribosomal and chaperone protein RPS3A was identified as a suppressor of αSyn [WT (wild-type) and A53T] toxicity in yeast. We demonstrated that the 50 N-terminal amino acids are essential for this function. The yeast homologues of RPS3A were not effective in suppressing the αSyn-induced growth defect, illustrating the potential of our screening system to identify modifiers that would be missed using yeast gene overexpression as the first screening step. Co-expression of mouse RPS3A delayed the formation of αSyn-GFP inclusions in the yeast cells. The results of the present study suggest that the recently identified extraribosomal chaperonin function of RPS3A also acts on the neurodegeneration-related protein αSyn and reveal a new avenue for identifying promising candidate mammalian proteins involved in αSyn functioning.

The characterization of the nuclear dynamics of syntenin-2, a PIP2 binding PDZ protein.

Cellular signaling is largely dependent on the presence, that is, assembly/disassembly, of supramolecular complexes. Postsynaptic density protein, Discs-large, Zona occludens (PDZ) domains play important roles in the assembly of various signaling complexes. Syntenin-2 (S2) is a PDZ protein that interacts with nuclear phosphatidylinositol 4,5-bisphosphate (PIP2 ). Although nuclear lipids emerge as key players in nuclear processes, the global significance of nuclear phosphoinositide-protein interactions is still poorly understood. Those phosphoinositide-protein interactions that have been studied in detail appear to have profound physiological effects. To our knowledge none of these were investigated by dynamic studies such as Fluorescence Correlation Spectroscopy (FCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), or Fluorescence Recovery After Photobleaching (FRAP). Although the exact function of S2 is unknown, siRNA experiments suggest that this PDZ protein plays a role in the organization of nuclear PIP2 , cell division, and cell survival. As a consequence of its PIP2 interaction, its reported self-association in a yeast two-hybrid study and its speculated interaction with many, yet unidentified, proteins one can hypothesize that S2 plays an important role in cell signaling. Therefore, we studied the dynamics of S2 using FCS, FCCS, and FRAP, utilizing an active truncated form deleted for the first 94 amino acids (S2-ΔN). We showed that S2-ΔN self-associates and is distributed in three groups. One immobile group, one slow diffusing group, which interacts with the nuclear environment and one fast diffusing group, which is not incorporated in high molecular weight complexes. In addition, our FCS and FRAP measurements on S2-ΔN mutants affected in their PIP2 binding showed that PIP2 plays an important role in the distribution of S2-ΔN among these groups, and favors the enrichment of S2-ΔN in the slow diffusing and immobile group. This work indicates that S2 relies on nuclear PIP2 to interact with practically immobile structures, possibly chromatin.

Fluorescence lifetime measurements of intrinsically unstructured proteins: application to α-synuclein.

Lifetimes of fluorescent states and their fluorescence intensities are strictly coupled and very sensitive to the environment of the fluorophores. The advantage of measuring lifetimes, next to intensities, comes from the fact that it can reveal heterogeneity and dynamic properties of this environment. In this way lifetime analysis can be used to characterize static and dynamic conformational properties and heterogeneity of fluorescent groups in different areas of a protein and as a function of time for an evolving protein. The phenomena that determine the lifetime of a label are its intrinsic properties, dynamic quenching by neighboring groups, exposure to the solvent, as well as Förster resonance energy transfer (FRET) between different groups. The basic principles of these fluorescence phenomena can be found extensively described in the excellent book of Lakowicz (Principles of fluorescence spectroscopy, 3rd edn. Springer, New York, 2006). The fluorescent groups involved are either natural amino acid side chains like tryptophan (Trp) or tyrosine (Tyr), or fluorescent labels covalently engineered into the protein. Even a single fluorescent group can show indications of heterogeneity in the local environment. If several natural fluorescent groups are present, the properties of the individual groups can be separated using site-directed mutagenesis, and additivity of their contributions can be analyzed (Engelborghs, Spectrochim Acta A Mol Biomol Spectrosc 57(11):2255-2270, 2001). If no fluorescent group is naturally present, site-directed mutagenesis can be used to introduce either a fluorescent amino acid or a cysteine allowing chemical labeling.

Fluorescence correlation spectroscopy to determine the diffusion coefficient of α-synuclein and follow early oligomer formation.

Fluorescence correlation spectroscopy (FCS) can be used to determine the diffusion coefficient of fluorescently labeled α-synuclein. It is a technique based on the use of a confocal microscope. By applying FCS in a combination of short sampling times and repeated measurements, the disappearance of individual α-synuclein molecules (called monomers) and the formation of oligomers can be characterized during the early aggregation process.

Tryptophan side chain conformers monitored by NMR and time-resolved fluorescence spectroscopies.

We have inserted a tryptophan (F77W) in the core of the regulatory domain of cardiac troponin C (cNTnC), and previously determined the structure of this mutant with and without the cosolvent trifluoroethanol (TFE). Interestingly, the orientations of the indole side chain of the Trp are in opposite directions in the two structures (Julien et al., Protein Sci 2009; 18:1165-1174). Fluorescence decay experiments for single Trp-containing proteins often show several lifetimes, which have been interpreted as reflecting conformational heterogeneity of the Trp side chain resulting from different rotamers. To test this interpretation, we monitored the effect of TFE on wild type, F77W and F77W-V82A calcium-saturated cNTnC using 2D (13)C-HSQC NMR and time-correlated single photon counting fluorescence spectroscopies. The time dependence of the Trp fluorescence decay was fit with three lifetimes. Addition of TFE caused a gradual, but limited decrease of the lifetimes due to dynamic quenching. For F77W cNTnC, the amplitude fractions of the lifetimes also changed upon addition of TFE-the long lifetime increased from 13 to 29%, while the middle lifetime decreased from 63 to 50% and the short lifetime remained relatively unchanged. For F77W-V82A cNTnC, comparable NMR changes are observed, confirming the switch in rotamer conformation, but only much smaller changes in fluorescence decay parameters were detected. These data indicate that the balance between the rotamer states can be changed without changing the lifetime amplitude fractions appreciably, and suggest that the environment(s) of the indole ring, responsible for the different lifetimes, can result from factors other than the intrinsic rotamer state of the tryptophan.

Rational design of photoconvertible and biphotochromic fluorescent proteins for advanced microscopy applications.

Advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. Chief among these fluorophores are the photoactivatable fluorescent proteins capable of reversible on/off photoswitching or irreversible green-to-red photoconversion. IrisFP was recently reported as the first fluorescent protein combining these two types of phototransformations. The introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging. However, the spectroscopic properties of IrisFP are far from being optimal and its tetrameric organization complicates its use as a fusion tag. Here, we demonstrate how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins and develop and characterize a new set of such enhanced optical highlighters derived from mEosFP and Dendra2. We present in particular NijiFP, a promising new fluorescent protein with photoconvertible and biphotochromic properties that make it ideal for advanced fluorescence-based imaging applications.

SAP-like domain in nucleolar spindle associated protein mediates mitotic chromosome loading as well as interphase chromatin interaction.

Nucleolar spindle associated protein (NuSAP) is a microtubule-stabilizing protein that localizes to chromosome arms and chromosome-proximal microtubules during mitosis and to the nucleus, with enrichment in the nucleoli, during interphase. The critical function of NuSAP is underscored by the finding that its depletion in HeLa cells results in various mitotic defects. Moreover, NuSAP is found overexpressed in multiple cancers and its expression levels often correlate with the aggressiveness of cancer. Due to its localization on chromosome arms and combination of microtubule-stabilizing and DNA-binding properties, NuSAP takes a special place within the extensive group of spindle assembly factors. In this study, we identify a SAP-like domain that shows DNA binding in vitro with a preference for dsDNA. Deletion of the SAP-like domain abolishes chromosome arm binding of NuSAP during mitosis, but is not sufficient to abrogate its chromosome-proximal localization after anaphase onset. Fluorescence recovery after photobleaching experiments revealed the highly dynamic nature of this NuSAP-chromatin interaction during mitosis. In interphase cells, NuSAP also interacts with chromatin through its SAP-like domain, as evident from its enrichment on dense chromatin regions and intranuclear mobility, measured by fluorescence correlation spectroscopy. The obtained results are in agreement with a model where NuSAP dynamically stabilizes newly formed microtubules on mitotic chromosomes to enhance chromosome positioning without immobilizing these microtubules. Interphase NuSAP-chromatin interaction suggests additional functions for NuSAP, as recently identified for other nuclear spindle assembly factors with a role in gene expression or DNA damage response.

Both substrate hydrolysis and secondary substrate binding determine xylanase mobility as assessed by FRAP.

Xylanases (EC 3.2.1.8) are enzymes that can hydrolyze the xylan backbone internally. Therefore, they are important for biomass breakdown and they are also often added in various biotechnological applications. In this study, the relationship between their substrate binding affinity and hydrolysis, on the one hand, and their movement over natural substrates, on the other hand, was investigated. Fluorescence recovery after photobleaching (FRAP) experiments using different Bacillus subtilis xylanase A (XBS) mutants were conducted on water-unextractable wheat flour arabinoxylan (WU-AX) and insoluble oat spelt xylan (OSX). To assess the importance of substrate hydrolysis, FRAP of a catalytically inactive mutant was compared to that of the wild-type enzyme. For the wild-type enzyme, substrate binding and a complete recovery of fluorescence after photobleaching was observed on both substrates. For the inactive mutant, however, substrate binding but no fluorescence recovery was observed on WU-AX, while very slow recovery was observed on OSX. Furthermore, the importance of substrate binding to a secondary xylan binding site (SBS) for enzyme mobility was studied by testing two mutants with a modified SBS (N54W-N141Q and G56A-T183A-W185A) that showed different behavior on the tested substrates. On OSX, the two modified enzymes both showed higher mobility than the wild-type enzyme. On WU-AX, in contrast, the N54W-N141Q mutant displayed a lower mobility than the wild-type enzyme, while the G56A-T183A-W185A mutant showed higher mobility. The results clearly demonstrate that both substrate hydrolysis and substrate targeting are key factors for XBS mobility.

The transcriptional co-activator LEDGF/p75 displays a dynamic scan-and-lock mechanism for chromatin tethering.

Nearly all cellular and disease related functions of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) involve tethering of interaction partners to chromatin via its conserved integrase binding domain (IBD), but little is known about the mechanism of in vivo chromatin binding and tethering. In this work we studied LEDGF/p75 in real-time in living HeLa cells combining different quantitative fluorescence techniques: spot fluorescence recovery after photobleaching (sFRAP) and half-nucleus fluorescence recovery after photobleaching (hnFRAP), continuous photobleaching, fluorescence correlation spectroscopy (FCS) and an improved FCS method to study diffusion dependence of chromatin binding, tunable focus FCS. LEDGF/p75 moves about in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors. The PWWP domain of LEDGF/p75 is necessary, but not sufficient for in vivo chromatin binding. After interaction with HIV-1 integrase via its IBD, a general protein-protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin. The PWWP is crucial for locking the complex on chromatin. We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting.

The conformation and the aggregation kinetics of α-synuclein depend on the proline residues in its C-terminal region.

The neuronal protein α-synuclein (α-syn) plays a central role in Parkinson's disease (PD). The pathological features of PD are the loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of Lewy bodies. The C-terminal domain of α-syn is characterized by the presence of 15 acidic amino acids and all five proline residues of the protein (P108, P117, P120, P128, and P138). The aggregation of this natively unfolded protein is accelerated in vitro by FK506 binding proteins (FKBPs) showing peptidyl-prolyl cis-trans isomerase activity. These proteins catalyze the cis-trans conformational change of the X-Pro peptide bond, often a rate-limiting step in protein folding. The acceleration of the folding of α-syn by FKBPs may accelerate disease-associated aggregation. To further elucidate the role of the proline residues in the conformation and aggregation of α-syn, we constructed several mutants of α-syn in which one or more proline residues are mutated to alanine via site-directed mutagenesis. For this purpose, we produced and purified His-WT α-syn, a recombinant α-syn with a polyhistidine tag (six His residues) and a linker, and a number of Pro-to-Ala mutants. The aggregation kinetics of these mutants and His-WT α-syn were studied by turbidity, thioflavin T fluorescence, and CD measurements. We can conclude that mutation of the proline residues to alanine accelerates the aggregation kinetics of α-syn while all proline mutants formed fibrils similar to His-WT α-syn, as visualized via transmission electron microscopy. We also demonstrate that the accelerating effect of hFKBP12 is abolished via removal of the proline residues from the C-terminus. Finally, we show that the mutant of His α-syn with all five proline residues mutated to alanine is more structured (more α-helix) than His-WT α-syn, indicating the role of the Pro residues as potential helix breakers in the inhibitory conformation of the C-terminus.

Early aggregation steps in alpha-synuclein as measured by FCS and FRET: evidence for a contagious conformational change.

The kinetics of aggregation of alpha-synuclein are usually studied by turbidity or Thio-T fluorescence. Here we follow the disappearance of monomers and the formation of early oligomers using fluorescence correlation spectroscopy. Alexa488-labeled A140C-synuclein was used as a fluorescent probe in trace amounts in the presence of excess unlabeled alpha-synuclein. Repeated short measurements produce a distribution of diffusion coefficients. Initially, a sharp peak is obtained corresponding to monomers, followed by a distinct transient population and the gradual formation of broader-sized distributions of higher oligomers. The kinetics of aggregation can be followed by the decreasing number of fast-diffusing species. Both the disappearance of fast-diffusing species and the appearance of turbidity can be fitted to the Finke-Watzky equation, but the apparent rate constants obtained are different. This reflects the fact that the disappearance of fast species occurs largely during the lag phase of turbidity development, due to the limited sensitivity of turbidity to the early aggregation process. The nucleation of the early oligomers is concentration-dependent and accompanied by a conformational change that precedes beta-structure formation, and can be visualized using fluorescence resonance energy transfer between the donor-labeled N-terminus and the acceptor-labeled cysteine in the mutant A140C.

Inhibition of FK506 binding proteins reduces alpha-synuclein aggregation and Parkinson's disease-like pathology.

alpha-Synuclein (alpha-SYN) is a key player in the pathogenesis of Parkinson's disease (PD). In pathological conditions, the protein is present in a fibrillar, aggregated form inside cytoplasmic inclusions called Lewy bodies. Members of the FK506 binding protein (FKBP) family are peptidyl-prolyl isomerases that were shown recently to accelerate the aggregation of alpha-SYN in vitro. We now established a neuronal cell culture model for synucleinopathy based on oxidative stress-induced alpha-SYN aggregation and apoptosis. Using high-content analysis, we examined the role of FKBPs in aggregation and apoptotic cell death. FK506, a specific inhibitor of this family of proteins, inhibited alpha-SYN aggregation and neuronal cell death in this synucleinopathy model dose dependently. Knockdown of FKBP12 or FKBP52 reduced the number of alpha-SYN aggregates and protected against cell death, whereas overexpression of FKBP12 or FKBP52 accelerated both aggregation of alpha-SYN and cell death. Thus, FK506 likely targets FKBP members in the cell culture model. Furthermore, oral administration of FK506 after viral vector-mediated overexpression of alpha-SYN in adult mouse brain significantly reduced alpha-SYN aggregate formation and neuronal cell death. Our data explain previously described neuroregenerative and neuroprotective effects of immunophilin ligands and validate FKBPs as a novel drug target for the causative treatment of PD.

Tryptophan conformations associated with partial unfolding in ribonuclease T1.

The origin of the biexponential fluorescence decay of Trp in ribonuclease T1 under mildly destabilizing conditions, such as increased pH or temperature, or the presence of detergent, is still not understood. We have performed two extended replica-exchange molecular dynamics simulations to obtain a detailed representation of the native state at two protonation states corresponding to a high and low pH. At high pH, the appearance of partially unfolded states is evident. We found that this pH-induced destabilization originates from increased global repulsion as well as reduced local favorable electrostatic interactions and reduced H-bonding strength of His(27), His(40), and His(92). At high pH, alternative tryptophan rotamers appear and are linked to a distorted environment of the tryptophan, which also acts as a separate source of ground-state heterogeneity. The total population of these alternative conformations agrees well with the amplitude of the experimentally observed secondary fluorescence lifetime.

Enzymatic investigation of the Staphylococcus aureus type I signal peptidase SpsB - implications for the search for novel antibiotics.

Staphylococcus aureus has one essential type I signal peptidase (SPase), SpsB, which has emerged as a potential target in the search for antibiotics with a new mode of action. In this framework, the biochemical properties of SpsB are described and compared with other previously characterized SPases. Two different substrates have been used to assess the in vitro processing activity of SpsB: (a) a native preprotein substrate immunodominant staphylococcal antigen A and (b) an intramolecularly quenched fluorogenic synthetic peptide based on the sequence of the SceD preprotein of Staphylococcus epidermidis for fluorescence resonance energy transfer-based analysis. Activity testing at different pH showed that the enzyme has an optimum pH of approximately 8. The pH-rate profile revealed apparent pK(a) values of 6.6 and 8.7. Similar to the other SPases, SpsB undergoes self-cleavage and, although the catalytic serine is retained in the self-cleavage product, a very low residual enzymatic activity remained. In contrast, a truncated derivative of SpsB, which was nine amino acids longer at the N-terminus compared to the self-cleavage product, retained activity. The specificity constants (k(cat)/K(m)) of the full-length and the truncated derivative were 1.85 +/- 0.13 x 10(3) m(-1).s(-1) and 59.4 +/- 6.4 m(-1).s(-1), respectively, as determined using the fluorogenic synthetic peptide substrate. These observations highlight the importance of the amino acids in the transmembrane segment and also those preceding the catalytic serine in the sequence of SpsB. Interestingly, we also found that the activity of the truncated SpsB increased in the presence of a non-ionic detergent.

The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144.

The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD(KZ)), originating from Pseudomonas aeruginosa bacteriophage varphiKZ, has been examined using a fusion protein of PBD(KZ) and green fluorescent protein (PBD(KZ)-GFP). A fluorescence recovery after photobleaching analysis of bound PBD(KZ)-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 x 10(7)M(-1) for the PBD(KZ)-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBD(KZ)-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives.

Measuring diffusion of lipid-like probes in artificial and natural membranes by raster image correlation spectroscopy (RICS): use of a commercial laser-scanning microscope with analog detection.

The heterogeneity in composition and interaction within the cellular membrane translates into a wide range of diffusion coefficients of its constituents. Therefore, several complementary microfluorimetric techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) have to be applied to explore the dynamics of membrane components. The recently introduced raster image correlation spectroscopy (RICS) offers a much wider dynamic range than each of these methods separately and allows for spatial mapping of the dynamic properties. RICS is implemented on a confocal laser-scanning microscope (CLSM), and the wide dynamic range is achieved by exploiting the inherent time information carried by the scanning laser beam in the generation of the confocal images. The original introduction of RICS used two-photon excitation and photon counting detection. However, most CLSM systems are based on one-photon excitation with analog detection. Here we report on the performance of such a commercial CLSM (Zeiss LSM 510 META) in the study of the diffusion of the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indodicarbocyanine perchlorate (DiI-C(18)(5)) both in giant unilamellar vesicles and in the plasma membrane of living oligodendrocytes, i.e., the myelin-producing cells of the central nervous system. It is shown that RICS on a commercial CLSM with analog detection allows for reliable results in the study of membrane diffusion by removal of unwanted correlations introduced by the analog detection system. The results obtained compare well with those collected by FRAP and FCS.

How do rotameric conformations influence the time-resolved fluorescence of tryptophan in proteins? A perspective based on molecular modeling and quantum chemistry.

We discuss the dynamics of tryptophan rotamers in the context of the non-exponential fluorescence decay in proteins. The central question is: how does the ground-state conformational heterogeneity influence the time evolution of tryptophan fluorescence? This problem is examined here from the theoretical perspective. Three methods at different levels of theory, and with different scopes and computational requirements are reviewed. The Dead-end elimination method is limited to side-chain dynamics and provides an efficient way to detect the stable tryptophan rotamers in a protein. Its application to the study of heterogeneous emission characteristics is illustrated. Molecular dynamics is aimed at the full phase space of the macromolecule in solution, but must rely on classical force fields and laws of evolution. We examine to what extent the molecular mechanics paradigm yields sufficiently accurate thermodynamic results, and what are the possible kinetic implications. Finally Quantum Chemistry is the only theoretical method that allows a direct assessment of the excited states. It is necessarily restricted to small molecular systems, and thus must be used in a hybrid combination with classical methods and electrostatic models. So far understanding of the emitting state has greatly progressed as a result of these calculations, but the actual treatment of the photophysical decay processes at the quantum level has not yet really started.