Identifying Inter-Domain Dynamics of Pin1 With Single Molecule FRET

Department of Biomedical EngineeringPin1, Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1, contains two functional domains, WW domain for the recognition of the peptide substrate and PPIase domain for cis-trans isomerization, linked by a flexible, intrinsically disordered region (IDR). Recent evidences from NMR studies suggest the inter-domain migration in this bivalent protein, the mechanism of which remains puzzling. Therefore, we characterized the inter-domain dynamics of Pin1 from multi-color single molecule fluorescence resonance energy transfer measurements. We measured the dynamics of Pin1 directly tethered to the surface or co-encapsulated in a lipid vesicle with the peptide substrate in order to observe its transient interaction with the substrate. We visualized the structural dynamics between the Pin1 domains as well as their interaction with the peptide substrate in real time to reveal the temporal correlation between the domain recognition and isomerization dynamics. This approach will provide new understanding of the role of the intrinsically disordered regions in coordinating multi-domain protein functions.ope

Cotranslational folding of proteins on the ribosome.

Many proteins in the cell fold cotranslationally within the restricted space of the polypeptide exit tunnel or at the surface of the ribosome. A growing body of evidence suggests that the ribosome can alter the folding trajectory in many different ways. In this review, we summarize the recent examples of how translation affects folding of single-domain, multiple-domain and oligomeric proteins. The vectorial nature of translation, the spatial constraints of the exit tunnel, and the electrostatic properties of the ribosome-nascent peptide complex define the onset of early folding events. The ribosome can facilitate protein compaction, induce the formation of intermediates that are not observed in solution, or delay the onset of folding. Examples of single-domain proteins suggest that early compaction events can define the folding pathway for some types of domain structures. Folding of multi-domain proteins proceeds in a domain-wise fashion, with each domain having its role in stabilizing or destabilizing neighboring domains. Finally, the assembly of protein complexes can also begin cotranslationally. In all these cases, the ribosome helps the nascent protein to attain a native fold and avoid the kinetic traps of misfolding

Accurate Demarcation of Protein Domain Linkers Based on Structural Analysis of Linker Probable Region

In multi-domainproteins, the domainsare connected by a flexible unstructured region called as protein domain linker. The accurate demarcation of these linkers holds a key to understanding of their biochemical and evolutionary attributes. This knowledge helps in designing a suitable linker for engineering stable multi-domain chimeric proteins. Here we propose a novel method for the demarcation of the linker based on a three-dimensional protein structure and a domain definition. The proposed method is based on biological knowledge about structural flexibility of the linkers. We performed structural analysis on a linker probable region (LPR) around domain boundary points of known SCOP domains. The LPR was described using a set of overlapping peptide fragments of fixed size. Each peptide fragment was then described by geometricinvariants (GIs) and subjected to clustering process where the fragments corresponding to actual linker comeupasoutliers.We then discover the actual linkers by finding the longest continuous stretch ofoutlier fragments from LPRs. This method was evaluated on a benchmark dataset of 51 continuous multi-domain proteins, where it achieves F1 score of 0.745 (0.83precision and 0.66recall). When the method was applied on 725 continuous multi-domain proteins, it was able to identify novel linkers that were not reported previously. This method can be used in combination with supervised /sequence based linker prediction methods for accurate linker demarcation.

Expression of Multi-Domain Lytic Peptide Genes in Transgenic Plants for Disease Resistance

Four non-plant multi-domain lytic peptide genes coding for antimicrobial peptides were expressed in Nicotiana benthamiana plants and tested against three fungal pathogens: Sclerotinia sclerotiorum, Rhizoctonia solani, and Pythium sp. Detached-leaf bioassay was performed for the transgenic plants carrying multi-domain lytic peptide constructs and compared with transgenic and wild type control plants. Symptom area of each leaf was measured with high precision using the Compu-Eye software and processed by SAS statistical package. The transgenic lines ORF13 and RSL1 showed substantial resistance to Sclerotinia sclerotiorum infection producing significantly smaller lesion areas compared to control plants. However, these lines were not effective against two other fungal pathogens Rhizoctonia solani and Pythium sp. Advisor: Amitava Mitr

Inferring PDZ Domain Multi-Mutant Binding Preferences from Single-Mutant Data

Many important cellular protein interactions are mediated by peptide recognition domains. The ability to predict a domain's binding specificity directly from its primary sequence is essential to understanding the complexity of protein-protein interaction networks. One such recognition domain is the PDZ domain, functioning in scaffold proteins that facilitate formation of signaling networks. Predicting the PDZ domain's binding specificity was a part of the DREAM4 Peptide Recognition Domain challenge, the goal of which was to describe, as position weight matrices, the specificity profiles of five multi-mutant ERBB2IP-1 domains. We developed a method that derives multi-mutant binding preferences by generalizing the effects of single point mutations on the wild type domain's binding specificities. Our approach, trained on publicly available ERBB2IP-1 single-mutant phage display data, combined linear regression-based prediction for ligand positions whose specificity is determined by few PDZ positions, and single-mutant position weight matrix averaging for all other ligand columns. The success of our method as the winning entry of the DREAM4 competition, as well as its superior performance over a general PDZ-ligand binding model, demonstrates the advantages of training a model on a well-selected domain-specific data set

(1)H, (15)N and (13)C backbone resonance assignments of the Kelch domain of mouse Keap1.

Kelch-like ECH-associated Protein 1 (Keap1) is a multi-domain protein that functions as an inhibitor of the transcription factor nuclear factor E2-related factor 2 (Nrf2) in the cellular response to oxidative stress. Under normal conditions, Keap1 binds to Nrf2 via its C-terminal Kelch domain and the interaction ultimately leads to the ubiquitin-dependent degradation of Nrf2. It has been proposed that designing molecules to selectively disrupt the Keap1-Nrf2 interaction can be a potential therapeutic approach for enhancing the expression of cytoprotective genes. Here, we reported the (1)H, (13)C, and (15)N backbone chemical shift assignments of the Kelch domain of mouse Keap1. Further, unlabeled Nrf2 peptide containing the Kelch-binding motif was added to the (15)N-labeled Kelch sample. (1)H-(15)N HSQC spectra of the protein in the absence and presence of an equimolar concentration of the Nrf2 peptide were presented. A significant number of resonance signals were shifted upon addition of the peptide, confirming the protein-peptide interaction. The results here will not just facilitate the further studies of the binding between Keap1 and Nrf2, it will also be valuable for probing interactions between the Kelch domain and small molecules, as well as a growing list of protein targets that have been identified recently

A minimalist chemical model of matrix metalloproteinases- Can small peptides mimic the more rigid metal binding sites of proteins?

In order to develop a minimalist chemical model of matrix metalloproteinases (MMPs), we synthesized a pentadecapeptide (Ac-KAHEFGHSLGLDHSK-NH2) corresponding to the catalytic zinc(II) binding site of human MMP-13. The multi-domain structural organization of MMPs fundamentally determines their metal binding affinity, catalytic activity and selectivity. Our potentiometric, UV-VIS, CD, EPR, NMR, ESI-MS and kinetic study are aimed to explore the usefulness of flexible peptides to mimic the more rigid metal binding sites of proteins, to examine the intrinsic metal binding properties of this naked sequence, as well as to contribute the development of a minimalist, peptide-based chemical model of MMPs, including the catalytic properties. Since multiimidazole environment is also characteristic for copper(II), and recently copper(II) containing variants of MMPs have been identified, we also studied the copper(II) complexes of the above peptide. Around pH 6-7 the peptide, similarly to MMPs, offers {3Nim} coordinated binding site for both zinc(II) and copper(II). In the case of copper(II), the formation of amide coordinated species at higher pH ceased the analogy with the copper(II) containing MMP variant. On the other hand, the zinc(II)-peptide system mimics some basic features of the MMP active sites: the main species around pH 7 (ZnH2L) possesses {3Nim,H2O} coordination environment, the deprotonation of the zinc-bound water takes place near to the physiological pH, it forms relatively stable ternary complexes with hydroxamic acids, and the species ZnH2L(OH) and ZnH2L(OH)2 have notable hydrolytic activity between pH 7-9

A self-assembling amphiphilic peptide nanoparticle for the efficient entrapment of DNA cargoes up to 100 nucleotides in length

To overcome the low efficiency and cytotoxicity associated with most non-viral DNA delivery systems we developed a purely peptidic self-assembling system that is able to entrap single- and doublestranded DNA of up to 100 nucleotides in length. (HR)3gT peptide design consists of a hydrophilic domain prone to undergo electrostatic interactions with DNA cargo, and a hydrophobic domain at a ratio that promotes the self-assembly into multi-compartment micellar nanoparticles (MCM-NPs). Selfassembled (HR)3gT MCM-NPs range between 100 to 180 nm which is conducive to a rapid and efficient uptake by cells. (HR)3gT MCM-NPs had no adverse effects on HeLa cell viability. In addition, they exhibit long-term structural stability at 4 1C but at 371C, the multi-micellar organization disassembles overtime which demonstrates their thermo-responsiveness. The comparison of (HR)3gT to a shorter, less charged H3gT peptide indicates that the additional arginine residues result in the incorporation of longer DNA segments, an improved DNA entrapment efficiency and an increase cellular uptake. Our unique nonviral system for DNA delivery sets the stage for developing amphiphilic peptide nanoparticles as candidates for future systemic gene delivery

Protein multi-scale organization through graph partitioning and robustness analysis: Application to the myosin-myosin light chain interaction

Despite the recognized importance of the multi-scale spatio-temporal organization of proteins, most computational tools can only access a limited spectrum of time and spatial scales, thereby ignoring the effects on protein behavior of the intricate coupling between the different scales. Starting from a physico-chemical atomistic network of interactions that encodes the structure of the protein, we introduce a methodology based on multi-scale graph partitioning that can uncover partitions and levels of organization of proteins that span the whole range of scales, revealing biological features occurring at different levels of organization and tracking their effect across scales. Additionally, we introduce a measure of robustness to quantify the relevance of the partitions through the generation of biochemically-motivated surrogate random graph models. We apply the method to four distinct conformations of myosin tail interacting protein, a protein from the molecular motor of the malaria parasite, and study properties that have been experimentally addressed such as the closing mechanism, the presence of conserved clusters, and the identification through computational mutational analysis of key residues for binding.Comment: 13 pages, 7 Postscript figure