Dataset Information

Molecular origin of time-dependent fluorescence shifts in proteins.

ABSTRACT: Time-resolved fluorescence spectroscopy is used increasingly to probe molecular motions at the aqueous interfaces of biological macromolecules and membranes. By recording the time variation of the fluorescence frequency, thermal atomic fluctuations in the vicinity of the chromophore can be probed. From such fluorescence Stokes shift (FSS) experiments, it has been inferred that water motions in the hydration layer are slowed down by 1-3 orders of magnitude. To provide a more secure foundation for the interpretation of FSS data, we use molecular dynamics simulations to examine the molecular origin of the FSS from a tryptophan residue in a protein. By using linear response theory to decompose the FSS into its water and protein components, we find that the water component dominates the static FSS but decays rapidly. Thus, after a few picoseconds, the FSS essentially reflects protein dynamics, including the self-motion of the chromophore. Because of its collective nature, the FSS response is insensitive to the motion of individual water molecules. Collective water displacement by slowly fluctuating protein groups introduces a long-time tail in the water autocorrelation function, but this dynamic coupling is hardly manifested in the observed FSS. Our analysis reconciles FSS data with the picture of a highly dynamic hydration layer, derived mainly from magnetic relaxation dispersion and simulation studies, and calls for a revision of previous interpretations of FSS decays in terms of slow hydration dynamics at biomolecular and other interfaces.

SUBMITTER: Nilsson L

PROVIDER: S-EPMC1236546 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Molecular origin of time-dependent fluorescence shifts in proteins.

Nilsson Lennart L Halle Bertil B

Proceedings of the National Academy of Sciences of the United States of America 20050914 39

Time-resolved fluorescence spectroscopy is used increasingly to probe molecular motions at the aqueous interfaces of biological macromolecules and membranes. By recording the time variation of the fluorescence frequency, thermal atomic fluctuations in the vicinity of the chromophore can be probed. From such fluorescence Stokes shift (FSS) experiments, it has been inferred that water motions in the hydration layer are slowed down by 1-3 orders of magnitude. To provide a more secure foundation for ...[more]

PMID: 16162674

Similar Datasets

Project description:ObjectivesThe oral microbiota plays a significant role in oral health. The present study aims to characterize variations in the oral microbiota relative to the collection site, the dynamics of biofilm accumulation, and inherent inter-individual differences.MethodsWhole stimulated saliva and tooth biofilm samples from the 16 defined tooth regions were collected after 1, 2, or 3 days without oral hygiene (accumulation time) in six healthy adults with no signs of active caries or periodontal disease. The routines and conditions before and between sample collections were carefully standardized. Genomic DNA was extracted, and the V3-V4 regions of the 16S rRNA gene were amplified by PCR and sequenced on an Illumina MiSeq platform. Sequences were quality controlled, amplicon sequence variants (ASVs) were clustered, and taxonomic allocation was performed against the expanded Human Oral Microbiome Database (eHOMD). Microbial community profiles were analyzed by multivariate modeling and a linear discriminant analysis (LDA) effect size (LEfSe) method.ResultsThe overall species profile in saliva and tooth biofilm differed between participants, as well as sample type, with a significantly higher diversity in tooth biofilm samples than saliva. On average, 45% of the detected species were shared between the two sample types. The microbiota profile changed from the most anterior to the most posterior tooth regions regardless of whether sampling was done after 1, 2, or 3 days without oral hygiene. Increasing accumulation time led to higher numbers of detected species in both the saliva and region-specific tooth biofilm niches.ConclusionThe present study confirms that the differences between individuals dominate over sample type and the time abstaining from oral hygiene for oral microbiota shaping. Therefore, a standardized accumulation time may be less important for some research questions aiming at separating individuals. Furthermore, the amount of DNA is sufficient if at least two teeth are sampled for microbiota characterization, which allows a site-specific characterization of, for example, caries or periodontitis.

Project description:Autofluorescence from the intracellular metabolite, NAD(P)H, is a biomarker that is widely used and known to reliably screen and report metabolic activity as well as metabolic fluctuations within cells. As a ubiquitous endogenous fluorophore, NAD(P)H has a unique rate of fluorescence decay that is altered when bound to coenzymes. In this work we measure the shift in the fluorescence decay, or average fluorescence lifetime (1-3 ns), of NAD(P)H and correlate this shift to changes in metabolism that cells undergo during apoptosis. Our measurements are made with a flow cytometer designed specifically for fluorescence lifetime acquisition within the ultraviolet to violet spectrum. Our methods involved culture, treatment, and preparation of cells for cytometry and microscopy measurements. The evaluation we performed included observations and quantification of the changes in endogenous emission owing to the induction of apoptosis as well as changes in the decay kinetics of the emission measured by flow cytometry. Shifts in NAD(P)H fluorescence lifetime were observed as early as 15 min post-treatment with an apoptosis inducing agent. Results also include a phasor analysis to evaluate free to bound ratios of NAD(P)H at different time points. We defined the free to bound ratios as the ratio of 'short-to-long' (S/L) fluorescence lifetime, where S/L was found to consistently decrease with an increase in apoptosis. With a quantitative framework such as phasor analysis, the short and long lifetime components of NAD(P)H can be used to map the cycling of free and bound NAD(P)H during the early-to-late stages of apoptosis. The combination of lifetime screening and phasor analyses provides the first step in high throughput metabolic profiling of single cells and can be leveraged for screening and sorting for a range of applications in biomedicine. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Project description:Experiments show that for many two-state folders the free energy of the native state, DeltaG(ND)([C]), changes linearly as the denaturant concentration, [C], is varied. The slope {m = [dDeltaG(ND)([C])]/(d[C])}, is nearly constant. According to the transfer model, the m-value is associated with the difference in the surface area between the native (N) and denatured (D) state, which should be a function of DeltaR(g)(2), the difference in the square of the radius of gyration between the D and N states. Single-molecule experiments show that the R(g) of the structurally heterogeneous denatured state undergoes an equilibrium collapse transition as [C] decreases, which implies m also should be [C]-dependent. We resolve the conundrum between constant m-values and [C]-dependent changes in R(g) using molecular simulations of a coarse-grained representation of protein L, and the molecular transfer model, for which the equilibrium folding can be accurately calculated as a function of denaturant (urea) concentration. In agreement with experiment, we find that over a large range of denaturant concentration (>3 M) the m-value is a constant, whereas under strongly renaturing conditions (<3 M), it depends on [C]. The m-value is a constant above [C] > 3 M because the [C]-dependent changes in the surface area of the backbone groups, which make the largest contribution to m, are relatively narrow in the denatured state. The burial of the backbone and hydrophobic side chains gives rise to substantial surface area changes below [C] < 3 M, leading to collapse in the denatured state of protein L. Dissection of the contribution of various amino acids to the total surface area change with [C] shows that both the sequence context and residual structure are important. There are [C]-dependent variations in the surface area for chemically identical groups such as the backbone or Ala. Consequently, the midpoints of transition of individual residues vary significantly (which we call the Holtzer effect) even though global folding can be described as an all-or-none transition. The collapse is specific in nature, resulting in the formation of compact structures with appreciable populations of nativelike secondary structural elements. The collapse transition is driven by the loss of favorable residue-solvent interactions and a concomitant increase in the strength of intrapeptide interactions with a decreasing [C]. The strength of these interactions is nonuniformly distributed throughout the structure of protein L. Certain secondary structure elements have stronger [C]-dependent interactions than others in the denatured state.

Dataset Information

Molecular origin of time-dependent fluorescence shifts in proteins.

Publications

Molecular origin of time-dependent fluorescence shifts in proteins.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets