The Protein Folding Problem.

Understanding how a protein’s sequence determines its three-dimensional structure, and consequently, its function is a fundamental problem of modern biology.  Solving this problem, known as the protein folding problem, becomes more urgent as an increasing number of human diseases and conditions are discovered to be caused by misfolded proteins.  Among these diseases are Alzheimer’s Disease (and related prion diseases), Parkinson’s Disease, and cystic fibrosis.  Furthermore, we are beginning to understand that when certain proteins, including the p53 tumor suppressor protein, are incorrectly folded due to genetic alteration they are unable to perform their role in the immune system, leading to an increased susceptibility to a variety of human cancers.  Therefore, solving the protein folding problem holds great promise to improve human health. 

 

          Great progress has been made in the study of protein folding by studying relatively simple proteins and peptides under highly controlled experimental conditions.  The combination of a variety of biophysical and biochemical techniques, including (but not limited to) fluorescence, ultraviolet and NMR spectroscopy, site-directed mutagenesis, and chemical modification, has been very successful at determining the fundamental principles of protein folding when applied to model protein systems.   In fact, the field has developed to the point where it is possible to begin applying the techniques and principles derived for use on these simple proteins to more complicated proteins involved in various metabolic processes.  Two biological processes that I have chosen to study using protein folding techniques are 1) how cells repair oxidative damage and 2) how the intracellular levels of free iron are regulated.

 

Repair of oxidative damage by the enzyme methionine sulfoxide reductase.

          An inevitable consequence of living in an aerobic environment is dealing with damage caused by reactive oxygen species, such as superoxide, hydrogen peroxide, the hydroxyl ion and water.  Modification of proteins, DNA and lipid molecules by reactive oxygen species has been shown to contribute to human disease and the aging process.  Although most of these covalent changes, such as DNA crosslinking and protein fragmentation, are irreversible, two types of protein modifications, cysteine and methionine oxidation, can be reversed enzymatically.  The process by which protein disulfide isomerases reduce cysteine crosslinks (disulfide bridges) is fairly well-understood, but much less is known about methionine sulfoxide reductase (MsrA), the enzyme that reduces methionine sulfoxide back to methionine and restores function to the damaged protein.  Although MsrA is an essential protein – it has been found in the genome of every organism sequenced to date – we are just beginning to understand how it works.

 

          Some of the unanswered questions that I plan to address are:  How does MsrA recognize and repair proteins that contain oxidized methionine residues?  How is MsrA able to interact with such a variety of proteins? What are the biophysical properties of MsrA? What are the thermodynamic parameters of the folding reaction of MsrA and how can we use them to help us understand the physical features that stabilize the protein?  I will use the experimental techniques described below to explore these and other issues.

 

          The figure at left is a three-dimensional representation of bovine peptide methionine sulfoxide reductase (PDB file 1FVG). (Image produced using WebLab ViewerLite.)

The role of iron-responsive element protein 1/aconitase in mammalian iron regulation.

          Although it was thought for many years that each gene produced a single protein with a single function, this simplistic view has been overturned by the discovery of a number of proteins that perform different functions depending on such factors as cell type, changes in oligomeric state, or the cellular concentration of a certain ligand.  One such protein that has two distinct jobs is the mammalian iron-responsive element protein 1 (IRP-1), which plays an important role in the regulation of intracellular iron regulation.  When iron levels are low, IRP-1 bind to regions of mRNA known as iron-responsive elements and by doing so, causes the levels of iron to rise by either up-regulating or down-regulating the production of proteins involved in iron transport and storage.  When the iron concentration is restored to normal levels, IRP-1 assumes its other identity – that of a cytoplasmic aconitase.  (Aconitase, an enzyme in the TCA cycle, is normally found in the mitochondria rather than the cytoplasm.)  As an aconitase, it can no longer bind to RNA and has a structural feature not found in IRP-1: a [4Fe-4S] iron-sulfur cluster. Cytoplasmic aconitase also catalyzes the isomerization of  citrate to isocitrate, just like its mitochondrial counterpart.

 

          I am interested in studying how IRP-1/aconitase converts between its two active forms.  What is the relatively stability (free energy) of these two forms?  Can we learn about the conversion process by determining how these proteins unfold?  What is the effect of mRNA on the stability on IRP-1? Conversely, what is the effect of the iron-sulfur cluster and the natural substrate citrate on the stability of the cytoplasmic aconitase?  Using techniques described below, I will investigate these and other questions.

 

          Figure at right depicts pig heart mitochondrial aconitase (PDB file 7ACN), which is assumed to be homologous in structure to the cytoplasmic form.  (This figure was generated using WebLab ViewerLite.)

Experimental approach

          Over the years, a number of useful experimental methods have been developed to study protein folding.  Many of these are simple but highly informative.  One such approach is to incrementally denature a protein, by chemical or thermal methods, and monitor its spectroscopic properties at each step.  The spectroscopic property can then be plotted as a function of denaturant concentration or temperature.  If the denaturation is performed in a reversible manner, it is possible to extract thermodynamic parameters from the plot such as DG° (free energy), DH° (enthalpy), Tm (the midpoint of thermally-induced unfolding) and m, a parameter that describes exposure of buried surface area as a function of denaturant concentration.  It is also possible to identify stable partially unfolded forms of the protein (folding intermediates) that can provide insight into the function and structural organization of the protein.  We can take advantage of the presence of aromatic amino acid side chains  in the protein to monitor unfolding by a variety of spectroscopic methods including fluorescence emission, ultraviolet, and circular dichroism spectroscopy.  These spectroscopic methods provide complementary information on the tertiary and secondary structure of the protein being studied.  Additional information can be obtained from such techniques as enzyme activity studies, size-exclusion chromatography and site-directed mutagenesis.