Förster (or Fluorescence) Resonance Energy Transfer (FRET) is unique in generating fluorescence signals sensitive to molecular conformation, association, and separation in the 1-10 nm range. We introduce a revised photophysical framework for the phenomenon and provide a systematic catalog of FRET techniques adapted to imaging systems, including new approaches proposed as suitable prospects for implementation. Applications extending from a single molecule to live cells will benefit from multidimensional microscopy techniques, particularly those adapted for optical sectioning and incorporating new algorithms for resolving the component contributions to images of complex molecular systems.

In idiopathic Parkinson's disease, intracytoplasmic neuronal inclusions (Lewy bodies) containing aggregates of the protein ␣-synuclein (␣S) are deposited in the pigmented nuclei of the brainstem. The mechanisms underlying the structural transition of innocuous, presumably natively unfolded, ␣S to neurotoxic forms are largely unknown. Using paramagnetic relaxation enhancement and NMR dipolar couplings, we show that monomeric ␣S assumes conformations that are stabilized by long-range interactions and act to inhibit oligomerization and aggregation. The autoinhibitory conformations fluctuate in the range of nanoseconds to microseconds corresponding to the time scale of secondary structure formation during folding. Polyamine binding and͞or temperature increase, conditions that induce aggregation in vitro, release this inherent tertiary structure, leading to a completely unfolded conformation that associates readily. Stabilization of the native, autoinhibitory structure of ␣S constitutes a potential strategy for reducing or inhibiting oligomerization and aggregation in Parkinson's disease.is the second most common neurodegenerative disease and the most common movement disorder, affecting 1-2% of the population over 65 years of age (1). The cause of PD is as yet unclear due in part to a complex etiology involving a combination of genetic susceptibility and numerous environmental factors (2). Proteinaceous aggregates in motor neurons of the substantia nigra and locus coeruleus are characteristic of idiopathic PD. An abundant component of these so-called Lewy bodies is the presynaptic protein ␣-synuclein (␣S) (3). Three genetic mutations in ␣S (A30P, E46K and A53T) have been identified in autosomal-dominantly inherited early-onset PD (4, 5). In vitro, different conditions such as increased temperature, lower pH, and naturally occurring polyamines accelerate ␣S aggregation (6, 7). Compelling evidence now supports a cytotoxic role in PD for protofibrils, early oligomers of ␣S (8).In other amyloid-related neurological disorders, such as Creutzfeldt-Jakob disease, protein oligomerization͞aggregation requires destabilization of a soluble monomeric protein followed by the formation of highly ordered, ␤-sheet-like fibrillar structures (9). ␣S, however, belongs to the class of natively unfolded proteins with no apparent ordered secondary structure detectable by far-UV CD, Fourier transform IR, or NMR spectroscopy (6, 10, 11), although recent evidence indicates the existence of distinct, functionally relevant intramolecular interactions (refs. 7 and 12 and this work). The challenge is to rationalize in structural terms the inactive state of the soluble, unstructured protein and the potentiation of aggregation by point mutations, ligand binding, or changes in solution conditions. During the past decade numerous NMR techniques have been developed for elucidating the unfolded states of proteins in atomic detail (13). 15 N-relaxation time measurements and paramagnetic relaxation enhancement (PRE) from site-directed spin labeling allo...

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The erbB/HER family of transmembrane receptor tyrosine kinases (RTKs) mediate cellular responses to epidermal growth factor (EGF) and related ligands. We have imaged the early stages of RTK-dependent signaling in living cells using: (i) stable expression of erbB1/2/3 fused with visible fluorescent proteins (VFPs), (ii) fluorescent quantum dots (QDs) bearing epidermal growth factor (EGF-QD) and (iii) continuous confocal laser scanning microscopy and flow cytometry. Here we demonstrate that EGF-QDs are highly specific and potent in the binding and activation of the EGF receptor (erbB1), being rapidly internalized into endosomes that exhibit active trafficking and extensive fusion. EGF-QDs bound to erbB1 expressed on filopodia revealed a previously unreported mechanism of retrograde transport to the cell body. When erbB2-monomeric yellow fluorescent protein (mYFP) or erbB3-monomeric Citrine (mCitrine) were coexpressed with erbB1, the rates and extent of endocytosis of EGF-QD and the RTK-VFP demonstrated that erbB2 but not erbB3 heterodimerizes with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling. QD-ligands will find widespread use in basic research and biotechnological developments.

Many indirect methods have been developed to study the constitution and conformation of macromolecules inside the living cell. Direct analysis by Raman spectroscopy is an ideal complement to techniques using directly labelled fluorescent probes or of indirect labelling with mono- and polyclonal antibodies. The high information content of Raman spectra can characterize biological macromolecules both in solution and in crystals. The positions, intensities and linewidths of the Raman lines (corresponding to vibrational energy levels) in spectra of DNA-protein complexes yield information about the composition, secondary structure and interactions of these molecules, including the chemical microenvironment of molecular subgroups. The main drawback of the method is the low Raman scattering cross-section of biological macromolecules, which until now has prohibited studies at the level of the single cell with the exception of (salmon) sperm heads, in which the DNA is condensed to an exceptionally high degree. Ultraviolet-resonance Raman spectroscopy has been used to obtain single cell spectra (and F. Sureau and P. Y. Turpin, personal communication), but in this method absorption of laser light may impair the integrity of the sample. We have avoided this problem in developing a novel, highly sensitive confocal Raman microspectrometer for nonresonant Raman spectroscopy. Our instrument makes it possible to study single cells and chromosomes with a high spatial resolution (approximately less than 1 micron 3).

Alpha-synuclein is the major component of Lewy bodies and Lewy neurites, which are granular and filamentous protein inclusions that are the defining pathological features of several neurodegenerative conditions such as Parkinson's disease. Fibrillar aggregates formed from alpha-synuclein in vitro resemble brain-derived material, but the role of such aggregates in the etiology of Parkinson's disease and their relation to the toxic molecular species remain unclear. In this study, we investigated the effects of pH and salt concentration on the in vitro assembly of human wild-type alpha-synuclein, particularly with regard to aggregation rate and aggregate morphology. Aggregates formed at pH 7.0 and pH 6.0 in the absence of NaCl and MgCl(2) were fibrillar; the pH 6.0 fibrils displayed a helical twist, as clearly evident by scanning force and electron microscopy. Incubations at pH 7.0 remained transparent during the process of aggregation and exhibited strong thioflavin-T and weak 8-anilino-1-naphthalenesulfonate (ANS) binding; furthermore, they were efficient in seeding fibrillization of fresh solutions. In contrast, incubating alpha-synuclein at low pH (pH 4.0 or pH 5.0) resulted in the rapid formation of turbid suspensions characterized by strong ANS binding, reduced thioflavin-T binding and reduced seeding efficiency. At pH 4.0, fibril formation was abrogated; instead, very large aggregates (dimensions approximately 100 microm) of amorphous appearance were visible by light microscopy. As with acidic conditions, addition of 0.2M NaCl or 10mM MgCl(2) to pH 7.0 incubations led to a shorter aggregation lag time and formation of large, amorphous aggregates. These results demonstrate that the morphology of alpha-synuclein aggregates is highly sensitive to solution conditions, implying that the fibrillar state does not necessarily represent the predominant or most functionally significant aggregated state under physiological conditions.

The aggregation of ␣-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. We demonstrate here that Cu(II) ions are effective in accelerating AS aggregation at physiologically relevant concentrations without altering the resultant fibrillar structures. By using numerous spectroscopic techniques (absorption, CD, EPR, and NMR), we have located the primary binding for Cu(II) to a specific site in the N terminus, involving His-50 as the anchoring residue and other nitrogen͞oxygen donor atoms in a square planar or distorted tetragonal geometry. The carboxylate-rich C terminus, originally thought to drive copper binding, is able to coordinate a second Cu(II) equivalent, albeit with a 300-fold reduced affinity. The NMR analysis of AS-Cu ( amyloid ͉ fibrillation ͉ metallobiology T he protein ␣-synuclein (AS) is the main component of neuronal and glial cytoplasmic inclusions, pathologically described as Lewy bodies, that constitute the hallmark lesions of a group of neurodegenerative diseases collectively referred to as synucleinopathies (1, 2). The identification of point mutations and locus triplication in the AS gene as sole causes of familial inherited Parkinson's disease (PD) (3, 4) has stimulated research on the mechanism of AS neurotoxicity.AS comprises 140 amino acids distributed in three different regions: (i) the amphipathic N terminus (residues 1-60); (ii) the highly hydrophobic self-aggregating sequence known as NAC (non-A␤ component, residues 61-95), which is presumed to initiate fibrillation (5); and (iii) the acidic C-terminal region (residues 96-140). In its native monomeric state, AS adopts an ensemble of conformations with no significant secondary structure (6, 7), although long-range interactions have been shown to stabilize an aggregation-autoinhibited global protein architecture (8, 9). The protein undergoes dramatic conformational transitions from its natively unstructured state to an ␣-helical conformation upon interaction with lipid membranes (10, 11) or to the characteristic crossed ␤-conformation in highly organized amyloid-like fibrils under conditions that trigger aggregation (12, 13). Whereas the mechanism for the ␣-helical transition is well understood, the detailed mechanism of amyloid formation remains to be elucidated.The kinetics of fibrillation of AS are consistent with a nucleation-dependent mechanism (14), being modulated by factors and effectors of different types. Among them, low pH and high temperature (15-17), organic solvents (18), heparin (19), polyamines (14,20), and metal cations (21-23) accelerate AS aggregation. Not only do metal cations exert a physiological influence on protein structure, but transition metals have been also frequently recognized as risk factors in neurodegenerative disorders (24, 25). Brain lesions associated with Alzheimer's disease (AD) are rich in Fe(III), Zn(II), and Cu(II) (26). Recent biophysical and structural studies of the amyloid precursor protein and the amyloid-␤ peptide (A␤) have provided stron...

The aggregation of alpha-synuclein (AS) is characteristic of Parkinson's disease and other neurodegenerative synucleinopathies. Interactions with metal ions affect dramatically the kinetics of fibrillation of AS in vitro and are proposed to play a potential role in vivo. We recently showed that Cu(II) binds at the N-terminus of AS with high affinity (K(d) approximately 0.1 microM) and accelerates its fibrillation. In this work we investigated the binding features of the divalent metal ions Fe(II), Mn(II), Co(II), and Ni(II), and their effects on AS aggregation. By exploiting the different paramagnetic properties of these metal ions, NMR spectroscopy provides detailed information about the protein-metal interactions at the atomic level. The divalent metal ions bind preferentially and with low affinity (millimolar) to the C-terminus of AS, the primary binding site being the (119)DPDNEA(124) motif, in which Asp121 acts as the main anchoring residue. Combined with backbone residual dipolar coupling measurements, these results suggest that metal binding is not driven exclusively by electrostatic interactions but is mostly determined by the residual structure of the C-terminus of AS. A comparative analysis with Cu(II) revealed a hierarchal effect of AS-metal(II) interactions on AS aggregation kinetics, dictated by structural factors corresponding to different protein domains. These findings reveal a strong link between the specificity of AS-metal(II) interactions and the enhancement of aggregation of AS in vitro. The elucidation of the structural basis of AS metal binding specificity is then required to elucidate the mechanism and clarify the role of metal-protein interactions in the etiology of Parkinson's disease.