Research Topics

The mechanism of protein folding is one of the most fundamental unanswered questions in modern chemistry and biology. Most prior folding studies have focused on the in vitro folding of fully formed (i.e., full-length) biopolymers starting from non-physiologically relevant unfolded states generated by high concentrations of denaturants, temperature-jumps. But how do proteins "really" fold inside a cell? The goal of our research is to gain a structural and dynamic understanding of the cotranslational and immediately post-translational folding pathways of soluble single-domain proteins in the cell. We work both in the presence and absence of molecular chaperones, under physiologically relevant conditions. This research direction differentiates our group’s efforts from most prior work on both in vivo and in vitro folding.

The specific aims of our group are to:

1. follow the conformational changes relevant to understanding the folding of polypeptides and proteins as they emerge from the ribosome,
2. compare and contrast in vitro and in vivo folding mechanisms, and
3. establish closer links between protein folding theory and experiments.

Our work so far has mainly focused on single-domain, predominantly a-helical proteins.

1. Model studies: the structural aspects of polpeptide chain elongation.

We have prepared and analyzed purified N-terminal polypeptides of increasing length derived from apomyoglobin(apoMb) to follow how protein conformation is modulated by chain elongation. The surprising results that we have obtained show that native-like landscapes only develop after most of the amino acids have been incorporated in the polypeptide chain. Misfolded amyloid-like b-sheet conformations are populated prior to this stage, at short chain lengths. Evolution towards native-like a-helical conformations proceeds linearly with chain elongation, with a steeper slope developing upon incorporation of the last few amino acids (Biochemistry 42, 7090-7099 (2003); Misbehaving Proteins: Protein (Mis)Folding, Aggregation, and Stability, edited by Amos M. Tsai (2005).

2. Cotranslational folding: conformation of ribosome-bound nascent polypeptides

We are developing novel methodologies to follow the cotranslational folding of ribosome-bound nascent polypeptides. This work is based on MALDI mass spectrometry and fluorescence in cell-free systems(Trends in Biotechnology 23(3), 157-162 (2005)). Towards the above goal, we have developed three novel NMR pulse sequences that facilitate high resolution protein analysis in cell-free systems by diffusion-based filtering (J. Biomol. NMR 29, 505-516 (2004)).

3. The role of cotranslationally active chaperones in folding and
misfolding

Crystal structure of the substrate binding domain of DnaK bound to a peptide substrate. The two sub-domains are shown in pink and yellow and the bound substrate is shown in green. Zhu, X., et al. Science 272 pp. 1606 (1996)

Both the research directions outlined in sections 1 and 2 are being pursued in the absence and presence of the cotranslationally-active chaperone DnaK. We have found that the presence of DnaK prevents aggregation of the apomyoglobin fragments and helps them to go into a soluble form.(Biotechnol. Prog., 24 (3), 570-575 (2008)).We are working towards unveiling how the interaction with this chaperone modifies polypeptide secondary structure and folding/misfolding pathways using NMR. (Biotechnol. Prog., 24 (3), 570-575 (2008))

1H,15N-HSQC NMR spectrum of a dynamic complex of an apomyoglobin fragment with DnaK