Single-molecule fluorescence spectroscopy maps the folding

landscape of a large protein

Menahem Pirchi, Guy Ziv, Inbal Riven, sharona sedghani Cohen, nir Zohar, Yoav Barak & Gilad Haran

Gong, Ping

Department of Chemistry and Biochemistry

University of Delaware

Small

single-domain protein

Two-state folding behavior

Smooth

Minimize the number of

intermediates and kinetic traps

Large protein

with multiple domains

More than 70% of eukaryotic proteome

Metastable intermediates

Complex folding pathway

Single-molecule fluorescence resonance energy transfer

spectroscopy

(smFRET)

3

Reliable "ruler" for measuring structural changes in proteins

Dispelling unknown orientation factor

From PubMed: http://www.ncbi.nlm.nih.gov/pubmed/7577238

4

Adenylate kinase

Labeled at position 73 and 203

Encapsulated within lipid vesicles

Series of GdmCl concentrations

Pirchi, M. et al. Nat. Commun. 2:493 doi:10.1038/ncomms1504 (2011).

In lack of a very long single-molecule temporal trajectory that maps the whole landscape

Multiple short trajectories collected in experimentAvailability of a large number of equilibrium trajectories facilitates reconstruction of the folding landscape

Pirchi, M. et al. Nat. Commun. 2:493 doi:10.1038/ncomms1504 (2011).

(a) Three examples of fluorescence trajectories of individual AK molecules

Single-molecule FRET trajectories of AK

Pirchi, M. et al. Nat. Commun. 2:493 doi:10.1038/ncomms1504 (2011).

(b) Comparison of single-molecule results to the bulk denaturation curve

(c) Comparison of the probability distribution of FRET efficiency values obtained from single-molecule trajectories to a free-diffusion single-molecule experiment

Pirchi, M. et al. Nat. Commun. 2:493 doi:10.1038/ncomms1504 (2011).

GdmCl concentration: 0.65M

Change-point analysis of trajectories

Transition density map constructed from the 0.65M GdmCl data set

Pirchi, M. et al. Nat. Commun. 2:493 doi:10.1038/ncomms1504 (2011).

HMM reveals six states

• HMM: Hidden Markov Model

• State is not directly visible • Output (dependent on the state) is visible • The sequence of tokens generated by an HMM gives some

information about the sequence of states

From wikipedia: http://en.wikipedia.org/wiki/Hidden_Markov_model

• The dynamics obey detailed balance —the flux from i to j equals the flux from j to i.

• Add an extra state presenting the photobleached molecules.

• Baum-Welch algorithm is used to obtain a maximum likelihood estimate of the HMM parameters.

Correlation between the transition density map based on change-point analysis and maps based on the HMM analysis

Focusing on the data set taken at 0.65 M GdmCl

Repeat the HMM analysis for different values of N, from 2 to 14.

Use the HMM parameters to generate a transition map

cross-correlated this map with the one obtained from the change-point analysis.

optimal N is between 5 to 7

Pirchi, M. et al. Nat. Commun. 2:493 doi:10.1038/ncomms1504 (2011).

State probability distribution histograms, as a function of GdmCl concentration

State connectivity changes with denaturant concentration

Pirchi, M. et al. Nat. Commun. 2:493 doi:10.1038/ncomms1504 (2011).

Transition maps at three indicated GdmCl concentrations, constructed from the experimental data using Hmm analysis results.

As the concentration of denaturant increases：

more transitions tend to occur between states of lower FRET efficiency

the fraction of sequential transitions of the type i→i ± 1 increases significantly

Pirchi, M. et al. Nat. Commun. 2:493 doi:10.1038/ncomms1504 (2011).

One-dimensional projections of the folding landscape of AK at the three indicated GdmCl concentrations

The widths of the lines depict the relative productive flux flowing between each pair of states

their colours represent the transition rates.

Pirchi, M. et al. Nat. Commun. 2:493 doi:10.1038/ncomms1504 (2011).

Discussion

• Single-molecule FRET spectroscopy can provide a comprehensive description of the folding landscape of a large, multidomain protein

• The dynamics involve a large set of possible pathways on the landscape

• Provides the experimental means to characterize folding dynamics-- considerably richer than the simple sequential dynamics

• Suggest to combine the results from our smFRET experiment with those obtained from a method like native-state hydrogen exchange, also combine computer simulations and measurements