Publications

Below is a complete list of my publications including a summary of the paper, and a link to a downloadable copy.

Papers in Print

Modulation of frustration in folding by sequence permutation

R Paul Nobrega, Karunesh Aurora, Sagar V. Kathuria, Rita Graceffa, Raul Barrea, Liang Guo, Srinivas Chakravarthy , Osman Bilsel, Thomas C. Irving, Charles L. Brooks, C. Robert Matthews

PNAS, 2014



There are two models to describe the driving forces of folding in CheY. In one, the subdomain model, it is rationalized that the higher contact density of the N-terminal subdomain explains why the N-terminus folds first, nucleating the folding of the C-terminal domain. In another, the ILV cluster model, it is supposed that sequence local contacts of I,L ,and V residues coalesce and drive the folding reaction. To better understand the physicochemical properties that govern the complex folding reaction of CheY, circular permutaions were employed such that the two existing models could be teased apart. We find that Chain entropy plays a crucial role in modulating the energies and structures of partially- folded states on the folding free energy surface of CheY. Further, sequence-local clusters of ILV residues appear to strongly influence the early stages of folding before the global cooperativity engages the entire sequence in the native conformation, while the subdomain model has an apparent role in the productive transition state.

Sub-millisecond time-resolved SAXS using a continuous-flow mixer and X-ray micro-beam

Rita Graceffa, R. Paul Nobrega, Raul A. Barrea, Sagar V. Kathuria, Srinivas Chakravarthy, Osman Bilsel, Thomas C. Irving

JSR, 2013



SAXS is a powerful tool for resolving global dimensional details of biopolymers, however technical limitations put time resolution in contention with the singal-to-noise of the experiemnt. Using turbulent mixers in combination with a Kirkpatrick-Baez mirror-based microbeam system permit submillisecond data to be acquired with high duty cycles via translational scanning of the mixer . This paper details the setup,acquisition control, and analysis of the data that was built at the Biophysics Collaborative Access Team (BioCAT)beamline 18ID at the Advanced Photon Source , Argonne National Laboratory

Microsecond barrier-limited chain collapse observed by time-resolved FRET and SAXS

Sagar V. Kathuria, Can Kayatekin, Raul Barrea, Elena Kondrashkina, Rita Graceffa, Liang Guo, R. Paul Nobrega, Srinivas Chakravarthy, C. Robert Matthews, Thomas C. Irving, Osman Bilsel

JMB, 2014



An independent comprehensive global thermodynamic analysis of Cytochrome C reveals that marginally stable partially folded structures are present in the unfolded GdnHCl denatured state . These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native like conditions.

Advances in turbulent mixing techniques to study microsecond protein folding reactions

Sagar V Kathuria, Alexander Chan, Rita Graceffa, R Paul Nobrega, C Robert Matthews, Thomas C Irving, Blair Perot, Osman Bilsel

Biopolymers, 2013



A brief review of recent activity in micromachining and microfluidics guided by computational simulations. This is intended to discuss the potential for dramatic improvements in time resolution and sample consumption for CF mixers over the next few years.

Minireview: Structural insights into early folding events using continuous-flow time-resolved small-angle X-ray scattering

Sagar V Kathuria, Liang Guo, Rita Graceffa, Raul Barrea, R Paul Nobrega, C Robert Matthews, Thomas C Irving, Osman Bilsel

Biopolymers, 2011



Small-angleX-ray scattering (SAXS) is a versatile tool that ful?lls many of the requirements for quantitatively probing transiently populated protein structures. In this minireview, we focus on the use of time-resolved SAXS to obtain geometric insights into the folding reactions of proteins. Recent developments are highlighted in which continuous-flow mixers have extended the time range of SAXS to gain structural insightsinto early folding events. Experimental constraints involved with the implementation of turbulence and laminar flow-based mixers with SAXS detection are also discussed.

Papers in Preparation

Unique free-energy surfaces in buffer and chemical denaturant for a de novo designed protein

R Paul Nobrega, Laura M Deveau, Sagar V Kathuria, Francessca Massi, C Robert Matthews

2015

De novo designed proteins have successfully been engineered towards stable low energy structures. However, kinetic complexities often arise in folding pathways that demonstrate that these proteins are not yet ideal. One such case is outlined here, where apparent 2-state folding is observed in reference to the GdnHCl unfolded state, however native state HDX experiments show a rugged landscape under native-favoring conditions.

Time-resolved fluorescence of microsecond
kinetic intermediates with micro machined mixers

R Paul Nobrega, Kevin T Halloran, Sagar V Kathuria, C Robert Matthews, Osman Bilsel

2015

Protein folding reactions are observed in the microsecond to millisecond time regime with enhanced resolution by incorporating lifetime measurements into the analysis. This experiment is made possible by combining turbulent flow microfluidic mixing with time correlated single photon counting.

ILV Informatics paper

Sagar V Kathuria, Yvonne Chan, R Paul Nobrega, C Robert Matthews

2015

Surveying the generality of ILV clusters in proteins

Identifying a sub-millisecond kinetic folding intermediate of CheY and subsequent folding pathways using Markov State Model analysis

Jade Shi, TJ Lane , R Paul Nobrega, Sagar V Kathuria, C Robert Matthews, Vijay Pande

2015

Using Folding@Home and SAXS data to gain atomistic details of the folding reaction of CheY.

Other

Innovative nanocalorimeter design for residue specific information of protein unfolding and aggregation reactions with broad biomedical and biopharmaceutical applications

R Paul Nobrega

NIST RAPS
Competition, 2014



This is a proposal that I wrote for a NIST fellowship competition in 2014. Athough it scored well and it was "recommended for funding" by the National Research Counsil, it was ultimately not funded by NIST. The proposal outlines the fabrication and testing of a MEMS based nanocalorimeter interfaced with oxidative labeling capabilities to resolve site specific information on protein folding and aggregation reactions.

Complementary techniques enhance the quality and scope of information obtained from SAXS

David Lambright, Andrew W Malaby, Sagar V Kathuria, R Paul Nobrega, Osman Bilsel, C Robert Matthews, Uma Muthurajan, Karolin Luger, Rajiv Chopra, Thomas C Irving, Srinivas Chakravarthy

ACA Transactions, 2014



The increased technical versatilty of synchrotron beam lines is enabling investigation of increasingly complex biological systems using a variety of biochemical and biophysical tols in combination with Smal Angle X-ray Scatering (SAXS). The high flux delivered by third generation synchrotron sources alow incorporation into SAXS instruments a variety of complementary biochemical or biophysical instrumentation that either directly improves data quality or enhances the depth and resolution of the information obtained. We present here two such instances, 1. Size exclusion chromatography in line with the x-ray beam which faciltates purification of the sample immediately before exposure; 2. Continuous-flow mixer in combination with a micro-focused beam that gives us aces to kinetic information in the sub- milisecond time regime.


Copyright © 2013-2014 Paul Nobrega