PROTEIN FOLDING KINETICS

 

Folding of simple proteins is typically a spontaneous and repeatable process. Experiments on small proteins commonly show only two populations of molecules at equilibrium: folded (native) and unfolded (denatured). The specific kinetic equilibrium between the two states changes with temperature and denaturant concentration. In physiological conditions, the unfolded state of most proteins is very low populated. Although, the equilibrium can be reversibly moved toward the denatured state increasing temperature and denaturant concentration.

 

Schematic of the two-state kinetic model.

 

The specific relationship between amino acid sequence and three-dimensional protein structure has been extensively investigated by means of experiments, computer simulations and theoretical modeling, though it still remains essentially unrevealed. Research on protein folding aims to understand which of the many possible amino acid sequences may be able to fold up and how the detailed structure of a folded protein can be predicted from its amino acid sequence. Solving the folding problem may have enormous implications: genetic diseases could be treated more effectively and exact drugs could be designed theoretically without a great deal of experimentation. Simulating protein folding could allow us to go forward with the modeling of the whole cell.

Several mechanism are presumably involved in the complex process of protein folding. One of the simplest and most fundamental events taking place during the folding of a polypeptide is the formation of contacts between two specific chain residues. Measuring the rate of contact formations between two specific amino acids of the same chain provides a tool for the characterization of the elusive unfolded state in native conditions.

Tryptophan (W) is the natural amino acid with the highest absorption cross section in the region of near UV. Form the first excited singlet state, ryptophan can decay in few nanosecond to the ground state (with a certain probability of fluorescence emission) or, differently, can populate the metastable triplet state, from where can decay in tens of microseconds (with a certain probability of phosphorescence emission). The long lifetime of tryptophan triplet state can be used as a kinetic probe for the formation of contacts with a suitable quencher.

 

Electronic energy levels of tryptophan.

 

Cysteine (C) is the most efficient quencher of tryptophan triplet state among natural amino acids. Triplet lifetime for a polypeptide containing a single tryptophan and a single cysteine provides a measure of the rate of contact formation between these two residues, which generally depends on their position in the sequence.

 

Schematic of the tryptophan triplet quenching experiment.

 

Triplet state of tryptophan is characterized by a broad absorbance of blue light in the spectral range ~ 400 – 480 nm. The current instrumental set-up we have implemented for the measure of tryptophan triplet lifetime is based on the detection of the transient absorbance of  triplet state after nanosecond UV excitation.

This technique has been used to investigate contact formation rates in different peptides and proteins. In the absence of cysteine the triplet lifetime is similar to that measured for free tryptophan (~ 40 ms). The presence of a cysteine residue decreases the triplet lifetime accordingly to the rate of contact formation with tryptophan.

 

Decay of the triplet state of tryptophan for N-acetyl tryptophan amide (gray dashed line), for the 66-residue cysteine-free protein CspTm (red points), and for the peptide CAGQW (blue points) in 6 M GdmCl.

 

Quenching of the triplet state of tryptophan by close contact with cysteine has been used to measure the reaction-limited and diffusion-limited rates of loop formation in disordered polypeptides having the sequence cys-(ala-gly-gln)_j-trp (j = 1 - 9). The observed rates have been interpreted simulating the distribution of end-to-end distances for a worm-like chain model with excluded volume and sampling the distribution for short distances. The peptides persistence length and the intra-molecular diffusion coefficient have been extracted for aqueous solutions and strongly denaturing conditions [M. Buscaglia, L. J. Lapidus, W. A. Eaton, J. Hofrichter, “Effects of Denaturants on the Dynamics of Loop Formation in Polypeptides”,  to appear on Biophysical Journal].

Triplet lifetime has been measured for proteins engineered to contain a single tryptophan and a single cysteine. In the denatured state, a shortened triplet lifetime indicates an high rate of contact formation between the two residues. Differently, in the native state, if tryptophan and cysteine occupy distant locations in the three dimensional protein structure, contact formation does not occur. In the case of a two-state slow folding protein, having a kinetics for folding and unfolding slower than the triplet lifetime for free tryptophan in solution, transient absorbance decays show two relaxations attributed to the two populations of native and denatured proteins [M. Buscaglia, B. Schuler, L. J. Lapidus, W. A. Eaton, J. Hofrichter, “Kinetics of Intramolecular Contact Formation in a Denatured Protein”, Journal of Molecular Biology, 332, 9-12 (2003)].

 

Scheme for measuring the rate of contact formation between tryptophan and cysteine of a slow folding proteins (CspTm).

 

Differently, in the case of a two-state fast folding protein, the rates for folding and unfolding can be extracted by fitting the relaxations calculated from a simple kinetic model to the observed triplet decays [M. Buscaglia, J. Kubelka, W. A. Eaton, J. Hofrichter, “Determination of ultrafast protein folding rates from loop formation dynamics”, Journal of Molecular Biology, 347, 657-664 (2005)].

 

Scheme for measuring the rate of contact formation between tryptophan and cysteine of a fast folding proteins (villin headpiece).