Protein Folding

Studies using Stopped-flow Fluorescence Kinetic Spectroscopy with Steady-state and Stopped-flow Circular Dichroism Spectroscopy.

Protein folding is the ultimate process where the information contained in genes is transformed into the final functional unit, an active folded protein from the unfolded primary amino acid sequence.

Misfolded proteins almost always fail to function correctly. Also excess misfolded protein can accumulate and interfere with the functioning of the cell. Consequently misfolded proteins are a feature of a large number of diseases, including Alzheimer's, Creutzfeldt-Jakob disease (CJD), cystic fibrosis, and many cancers. Understanding the difference in the folding process can bring insight into the causes of these diseases at the molecular level.

In many protein production environments, from small scale research applications to large industrial scale bio-pharmaceutical production, it is important to ensure that an expressed and purified protein is correctly folded. Comparison of circular dichroism (CD) spectra, using the Chirascan CD spectrophotometer, a particularly powerful tool for this application.

Understanding the mechanism of protein folding is a very active research areas due to its fundamental importance to biology. The techniques of stopped-flow spectroscopy, particularly using CD and fluorescence, are used widely for this application. Below are two application notes demonstrating CD stopped-flow as a method for investigating protein folding mechanisms and kinetics.

At the bottom there are 5 references utilising APL fluorescence and CD stopped-flow systems to study protein folding. There is also one reference utilising an APL laser flash system to study ultrafast protein folding events.

Multiple Wavelength CD Kinetics — Refolding of the Protein Lysozyme

In protein research applications it is of great interest to study protein refolding as this gives information on the protein characteristics. For example, Hen egg lysozyme refolding occurs through well defined steps, and is fairly typical of a protein refolding reaction.

This application note demonstrates the performance of the Chirascan CD spectrometer equipped with the SF.3 stopped flow accessory for very fast stopped-flow circular dichroism kinetics. In particular, the quality and consistency of stopped-flow CD data sets recorded at single and multiple wavelengths on the Chirascan is demonstrated. Pro-K global analysis software is introduced as a tool for analysing multiple wavelength CD data sets.

 

Steady-state and Kinetic CD — Refolding of Cytochrome-C

Horse heart cytochrome-C is an example of a more complex situation where refolding occurs via several intermediates including a heme misligated side pathway. The refolding process and its individual rates depend upon a number of factors including pH and refolding agent concentration.

Far-UV circular dichroism spectra and very fast stopped-flow CD kinetics are highly demanding spectroscopic techniques. In this application note where an APL CD stopped-flow instrument is used to study horse heart cytochrome-C by acquiring steady-state and kinetic CD data in the far UV. The simultaneous acquisition of CD and fluorescence kinetic data is also demonstrated giving valuable information on the protein.

Relevant References

Listed below are 5 selected reference for studies of protein folding using Applied Photophysics instruments. A complete searchable database with all references can be accessed by logging into the APL members area.

Authors	Title	Year	Keywords	Journal/Proceedings
Naomi Courtemanche and Doug Barrick	Folding thermodynamics and kinetics of the leucine-rich repeat domain of the virulence factor Internalin B [Abstract]   [URL]	2008	repeat protein, leucine-rich repeat, protein folding, kinetics	PROTEIN SCI, 2008, Vol 17, pp 43-53
Abstract: Although the folding of {alpha}-helical repeat proteins has been well characterized, much less is known about the folding of repeat proteins containing β-sheets. Here we investigate the folding thermodynamics and kinetics of the leucine-rich repeat (LRR) domain of Internalin B (InlB), an extracellular virulence factor from the bacterium Lysteria monocytogenes. This domain contains seven tandem leucine-rich repeats, of which each contribute a single β-strand that forms a continuous β-sheet with neighboring repeats, and an N-terminal α-helical capping motif. Despite its modular structure, InlB folds in an equilibrium two-state manner, as reflected by the identical thermodynamic parameters obtained by monitoring its sigmoidal urea-induced unfolding transition by different spectroscopic probes. Although equilibrium two-state folding is common in α-helical repeat proteins, to date, InlB is the only β-sheet-containing repeat protein for which this behavior is observed. Surprisingly, unlike other repeat proteins exhibiting equilibrium two-state folding, InlB also folds by a simple two-state kinetic mechanism lacking intermediates, aside from the effects of prolyl isomerization on the denatured state. However, like other repeat proteins, InlB also folds significantly more slowly than expected from contact order. When plotted against urea, the rate constants for the fast refolding and single unfolding phases constitute a linear chevron that, when fitted with a kinetic two-state model, yields thermodynamic parameters matching those observed for equilibrium folding. Based on these kinetic parameters, the transition state is estimated to comprise 40% of the total surface area buried upon folding, indicating that a large fraction of the native contacts are formed in the rate-limiting step to folding.
Xie, J.-B.; Zhou, J.-M.	Trigger Factor Assisted Folding of Green Fluorescent Protein [Abstract]   [URL]	2008	Protein Folding, FRET	BIOCHEMISTRY-USA, 2008, Vol 47, Issue 1, pp 348-357
Abstract: Guanidine induced equilibrium and kinetic folding of a variant of green fluorescent protein (F99S/M153T/V163A, GFPuv) was studied. Using manual mixing and stopped-flow techniques, we combined different probes, including tryptophan fluorescence, chromophore fluorescence and reactivity with DTNB, to trace the spontaneous and TF-assisted folding of guanidine denatured GFPuv. We found that both unfolding and refolding of GFPuv occurred in a stepwise manner and a stable intermediate was populated under equilibrium conditions. The thermodynamic parameters obtained show that the intermediate state of GFPuv is quite compact compared to the denatured state and most of the green fluorescence is retained in this state. By studying GFPuv folding assisted by TF and a number of TF mutants, we found that wild-type TF catalyzes proline isomerization and accelerates the folding rate at low TF concentrations, but retards GFPuv folding and decelerates the folding rate at high TF concentrations. This reflects the two activities of TF, as an enzyme and as a chaperone. A general mechanism of TF assisted protein folding is discussed.
Beat Fierz, Helmut Satzger, Christopher Root, Peter Gilch, Wolfgang Zinth, and Thomas Kiefhaber	Loop formation in unfolded polypeptide chains on the picoseconds to microseconds time scale [Abstract]   [URL]	2007	conformational substates , femtoseconds spectroscopy , peptide dynamics, protein folding, triplet–triplet energy transfer	PROC NAT ACAD SCI USA, 2007, Vol 104, Iss 7, pp 2163-2168
Abstract: Intrachain loop formation allows unfolded polypeptide chains to search for favorable interactions during protein folding. We applied triplet–triplet energy transfer between a xanthone moiety and naphthylalanine to directly measure loop formation in various unfolded polypeptide chains with loop regions consisting of polyserine, poly(glycine–serine) or polyproline. By combination of femtosecond and nanosecond laserflash experiments loop formation could be studied over many orders of magnitude in time from picoseconds to microseconds. The results reveal processes on different time scales indicating motions on different hierarchical levels of the free energy surface. A minor (<15%) very fast reaction with a time constant of ~3 ps indicates equilibrium conformations with donor and acceptor in contact at the time of the laserflash. Complex kinetics of loop formation were observed on the 50- to 500-ps time scale, which indicate motions within a local well on the energy landscape. Conformations within this well can form loops by undergoing local motions without having to cross major barriers. Exponential kinetics observed on the 10- to 100-ns time scale are caused by diffusional processes involving large-scale motions that allow the polypeptide chain to explore the complete conformational space. These results indicate that the free energy landscape for unfolded polypeptide chains and native proteins have similar properties. The presence of local energy minima reduces the conformational space and accelerates the conformational search for energetically favorable local intrachain contacts.
Sarah Batey and Jane Clarke	Apparent cooperativity in the folding of multidomain proteins depends on the relative rates of folding of the constituent domains [Abstract]   [URL]	2006	alpha-helix, protein folding, spectrin, m value, equilibrium denaturation	PROC NAT ACAD SCI USA, 2006, Vol 103, Iss 48, pp 18113-18118
Abstract: Approximately 75% of eukaryotic proteins contain more than one so-called independently folding domain. However, there have been relatively few systematic studies to investigate the effect of interdomain interactions on protein stability and fewer still on folding kinetics. We present the folding of pairs of three-helix bundle spectrin domains as a paradigm to indicate how complex such an analysis can be. Equilibrium studies show an increase in denaturant concentration required to unfold the domains with only a single unfolding transition; however, in some cases, this is not accompanied by the increase in m value, which would be expected if the protein is a truly cooperative, all-or-none system. We analyze the complex kinetics of spectrin domain pairs, both wild-type and carefully selected mutants. By comparing these pairs, we are able to demonstrate that equilibrium data alone are insufficient to describe the folding of multidomain proteins and to quantify the effects that one domain can have on its neighbor.
Anna Nordlund and Mikael Oliveberg	Folding of Cu/Zn superoxide dismutase suggests structural hotspots for gain of neurotoxic function in ALS: Parallels to precursors in amyloid disease [Abstract]   [URL]	2006	neurodegenerative disease, protein folding, transition state	PROC NAT ACAD SCI USA, 2006, Vol 103, Iss 27, pp 10218-10223
Abstract: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to misfolding of the ubiquitous enzyme Cu/Zn superoxide dismutase (SOD). In contrast to other protein-misfolding disorders with similar neuropathogenesis, ALS is not always associated with the in vivo deposition of protein aggregates. Thus, under the assumption that all protein-misfolding disorders share at primary level a similar disease mechanism, ALS constitutes an interesting disease model for identifying the yet-mysterious precursor states from which the cytotoxic pathway emerges. In this study, we have mapped out the conformational repertoire of the apoSOD monomer through analysis of its folding behavior. The results allow us to target the regions of the SOD structure that are most susceptible to unfolding locally under physiological conditions, leading to the exposure of structurally promiscuous interfaces that are normally hidden in the protein’s interior. The structure of this putative ALS precursor is strikingly similar to those implicated in amyloid disease.