RESEARCH REPORT 2001-2003
RESEARCH REPORT 2004-2005
RESEARCH REPORT 2005-2007

Research Report 2008-2009:

How do proteins fold, unfold and misfold?

The polypeptide chain of a protein must coil, turn, bend, loop and twist itself in a very precise manner while folding into the unique structure that enables the protein to function in the cell. The protein folding problem is to understand how structure develops as a protein folds. How proteins fold has been a long-standing, unsolved puzzle in biology, whose solution has obvious biotechnological as well as medical implications. In particular, the improper folding of some proteins, and their consequent aggregation into amyloid fibrils, is a characteristic feature of several neuro-degenerative diseases as well as of the prion diseases. An understanding of the mechanism of protein folding will also lead to a better understanding of the other facet of the protein folding problem, which is how to predict the functional structure of a protein from the amino-acid sequence that specifies it.

My laboratory uses several small proteins, including barstar, monellin, the SH3 domain of the PI3-kinase, α-synuclein, tau, and the mouse prion protein as archetypical model proteins for studying how proteins fold, unfold as well as aggregate. We also study how correct folding is assisted by the chaperone GroEL. We use the tools of protein engineering and physical biochemistry. These include diverse optical spectroscopic methods such as time-resolved fluorescence methods, as well as nuclear magnetic resonance spectroscopy and mass spectrometry methods. Our kinetic measurements span the time domain of 100 microseconds to 10 hours.

Highlights of our recent work on protein folding and unfolding include (1) the demonstration that a dry molten globule forms initially during the unfolding of monellin and that further loss of structure occurs as the polypeptide chain undergoes gradual diffusive swelling; (2) the demonstration that native state fluctuations of a protein play an important role in how the protein unfolds; and (3) the demonstration that GroEL can unfold productive folding intermediates thereby modulating the free energy landscape of folding. Highlights of our recent work on protein misfolding and aggregation include (1) the demonstration that amyloid protofibrils may have different morphologies when formed on different pathways, and that a single mutation in the protein sequence or a change in aggregation conditions can lead to switching between alternative available pathways; and (2) the demonstration that the mouse prion protein forms amyloid protofibrils and fibrils from large soluble oligomers.

Selected publications:

Jha, S.K., Dhar, D., Krishnamoorthy, G. and Udgaonkar, J.B.(2009) Continuous dissolution of structure during the unfolding of a small protein. Proceedings of the National Academy of Science, USA 106, 11113-11118.

Wani, A.H. and Udgaonkar, J.B. (2009). Native state dynamics drive the unfolding of the SH3 domain of PI3 kinase at high denaturant concentration. Proceedings of the National Academy of Science, USA 106, 20711-20716

Kumar, S. and Udgaonkar, J.B. (2009) Structurally distinct amyloid protofibrils form on separate pathways of aggregation of a small protein. Biochemistry, 48, 6441-6449.

Jain, S. and Udgaonkar, J.B. (2008). Evidence for step-wise formation of amyloid fibrils by the mouse prion protein. Journal of Molecular Biology, 382, 1228-1241.

Udgaonkar, J.B. (2008) Multiple routes and structural heterogeneity in protein folding. Annual Reviews of Biophysics, 37, 489-510.

1 Continuous dissolution of structure during protein unfolding

Santosh Kumar Jha

The unfolding kinetics of many small proteins appears to be first order, when measured by ensemble-averaging probes such as fluorescence and circular dichroism. For one such protein, monellin, it has been shown that hidden behind this deceptive simplicity is a complexity that becomes evident with the use of experimental probes that are able to discriminate between different conformations in an ensemble of structures. The unfolding of monellin was probed by measurement of the changes in the distributions of four different intra-molecular distances, using a multi-site, time-resolved fluorescence resonance energy transfer (FRET) methodology. During the course of unfolding, the protein molecules were seen to undergo slow and continuous, diffusive swelling, which could be modeled as the slow diffusive swelling of a Rouse-like chain with some additional non-covalent, intra-molecular interactions. It was shown that specific structure is lost during the swelling process gradually, and not in an all-or-none manner.

Collaborators: Deepak Dhar and G. Krishnamoorthy, Tata Institute of Fundamental Research, Mumbai

2 Direct evidence for a dry molten globule intermediate during protein unfolding Santosh Kumar Jha

Little is known about how proteins begin to unfold. In particular, how and when water molecules penetrate into the protein interior during unfolding, thereby enabling the dissolution of specific structure, is poorly understood. The hypothesis that the native state expands initially into a dry molten globule, in which tight packing interactions are broken, but whose hydrophobic core has not expanded sufficiently to be able to absorb water molecules, has had very little experimental support. An analysis of the earliest events during the unfolding of monellin observed by circular dichroism measurements indicated that a molten globule intermediate is formed initially. Steady-state FRET measurements showed that the C-terminal end of the single helix initially moves rapidly away from the single tryptophan residue which is close to the N-terminal end of the helix. It was shown that at this time, water has yet to penetrate the protein core. The results therefore provided direct evidence for a dry molten globule intermediate at the initial stage of unfolding.

3. Native state dynamics drive the unfolding of the SH3 domain of PI3 kinase at high denaturant concentration

Ajazul Hamid Wani

Little is understood about the role of protein dynamics in directing protein unfolding along a specific pathway, and about the role played by chemical denaturants in modulating the dynamics and the initiation of unfolding. Deuterium-hydrogen exchange (HX) detected by electrospray ionization mass spectrometry (ESI-MS), was used to study the unfolding of a SH3 domain. Unfolding was shown to occur in two steps, both in the absence and in the presence of 1.8 M guanidine hydrochloride (GdnHCl). In both cases, the first step leads to the formation of an intermediate, I_N. ESI-MS analysis of fragments of the protein created by proteolytic digestion, after completion of the HX reaction, showed that I_N has lost protection against HX in the same segments of native structure during unfolding in the absence (at 40 s) and presence (at 5 s) of 1.8 M GdnHCl. Hence, GdnHCl does not appear to play a direct active role in the initiation of unfolding.

4 Chain Collapse precedes structure formation during the folding of a “two-state” folder Amrita Dasgupta

Structure formation is supposed to occur in one concerted, barrier-limited step during the folding of “two-state” folders. Multi-site FRET as well as ANS binding measurements done with 100 microsecond time resolution have indicated that specific structure formation of an archetypal two-state folder, the SH3 domain of PI3 kinase, is preceded by a gradual and synchronized chain collapse reaction.

5 Multi-site FRET measurements of the folding of the SH3 domain of PI3 kinase

Megha Kishore

Structure formation during the folding of the SH3 domain of PI3 kinase is being probed by time-resolved FRET-enabled measurements of multiple intra-molecular distances. Multiple fluorescence lifetimes have been observed to change in a complex manner in equilibrium unfolding studies.

Similar studies of the folding of the mouse prion protein have also been initiated (Roumita Moulick).

6 Association and structure formation during the folding of a hetero-dimeric protein Nilesh Aghera

Chain association and structure formation are being delineated in kinetic studies of the folding of two-chain monellin. Equilibrium folding studies suggest that association occurs by the binding of unstructured chain A to partially structured chain B. Initial kinetic studies suggest that folding pathways may switch when folding conditions become less stabilizing.

7 GroEL can unfold productive folding intermediates

Ashish Kumar Patra

The folding chaperone GroEL was shown to bind very tightly to intermediates on two of the three folding pathways of monellin, in such a manner that the intermediates cannot continue to refold, but remain bound to GroEL. GroEL also binds to an intermediate on the third folding pathway, but in a manner that allows the bound intermediate to continue refolding. This differential binding ability of GroEL was shown to result in the unfolding of late productive folding intermediates through a thermodynamic coupling mechanism.

8 Structurally distinct amyloid protofibrils form on separate pathways of aggregation of a small protein

Santosh Kumar

Understanding the structural as well as mechanistic basis of the conformational polymorphism evident during amyloid protofibril and fibril formation by proteins is an important goal in the study of protein aggregation. A scanning mutagenesis approach was used to determine specific residues important in defining the nature of the aggregation reaction of barstar leading to protofibril and fibril formation from well-defined small soluble oligomers. It was shown that structural polymorphism is not restricted to fibrils, but is also present in protofibrils. The structural heterogeneity seen in fibrils was shown to set in initially during the formation of higher order oligomers and protofibrils. Two alternative pathways for protofibril formation were delineated, and the sequence of structural events on each pathway was defined. Protofibril growth follows conformational conversion on one pathway, while it precedes conformational conversion on the other pathway.

When membrane mimetic conditions were enabled by addition of the alcohol TFE, the amyloid protofibrils formed by barstar appeared to be composed of a β-sheet monolayer. In contrast, protofibrils formed in the absence of TFE appear to possess the pair of bilayer (pair) of β-sheets structural motif, typical of amyloid fibrils. It was shown that the internal structures (measured by FTIR and CD) as well as the stabilities of the two types of protofibrils are distinct from each other, and that their pathways of formation are also distinct.

9 Stepwise formation of amyloid fibrils by the mouse prion protein

Shweta Jain

The mechanism by which the prion protein self-assembles to form amyloid fibrils is poorly understood. In particular, very little is known about the roles played by pre-fibrillar structures such as soluble oligomers and protofibrils, in disease, as well as in the overall aggregation process leading to fibrils. The mechanism of amyloid protofibril formation by the full length mouse prion protein was studied. Using multiple structural probes, the steps involved in protofibril formation were defined. An initially formed soluble β-rich oligomer was shown to transform into a critical higher order oligomer which is competent to form worm-like amyloid fibrils.

The extent to which the amyloidogenic β-rich oligomer is populated can be tuned over a wide range by altering either the sequence of the protein by mutation, or the conditions of aggregation. Hence, the rate of aggregation, which is found to be proportional to the concentration of the β-rich oligomer, is found to be very dependent on mutation and environmental conditions.

10 Amyloid fibril formation by α-synuclein and barstar in different environmental conditions.

Amrita Sekhar

The mechanism by which α-synuclein and barstar form amyloid protofibrils and fibrils in the presence of HFIP is being studied. The structures and sizes of the protofibrils formed by barstar in the presence of HFIP are found to be distinct from those formed in the presence of TFE or in the absence of any solvent additive.

11 Amyloid fibril formation by the human tau protein

Gayathri Ramachandran

The mechanism by which the microtubule binding domain of the human tau protein aggregates to form both amyloid protofibrils and fibrils in the presence of heparin is being studied. Environmental conditions in which fibril formation appears to occur by an isodesmic mechanism, have been established, and the role of protofibrils in the assembly of fibrils is being probed.

12 Aggregation of α-synuclein in cells.

Vishal

Deposits of the protein α-synuclein in Lewy bodies, in specific dopaminergic neurons in the brain are a characteristic feature of Parkinson’s disease. The roles of tau and proteosomal inhibition in facilitating the aggregation of α-synuclein in cells are being studied.

Collaborator: M M Panicker, NCBS