TY  - JOUR
A1  - Borgia, Alessandro
A1  - Zheng, Wenwei
A1  - Buholzer, Karin
A1  - Borgia, Madeleine B.
A1  - Schüler, Anja
A1  - Hofmann, Hagen
A1  - Soranno, Andrea
A1  - Nettels, Daniel
A1  - Gast, Klaus
A1  - Grishaev, Alexander
A1  - Best, Robert B.
A1  - Schuler, Benjamin
T1  - Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods
JF  - Journal of the American Chemical Society
N2  - There has been a long-standing controversy regarding the effect of chemical denaturants on the dimensions of unfolded and intrinsically disordered proteins: A wide range of experimental techniques suggest that polypeptide chains expand with increasing denaturant concentration, but several studies using small-angle X-ray scattering (SAXS) have reported no: such increase of the radius of gyration (R-g). This inconsistency challenges our current understanding of the mechanism of chemical denaturants, which are widely employed to investigate protein folding and stability. Here, we use a combination Of single-molecule Forster resonance energy transfer (FRET), SAXS, dynamic light scattering (DLS), and two-focus fluorescence correlation spectroscopy (2f-FCS) to characterize the denaturant dependence of the unfolded state of the spectrin domain R17 and the intrinsically disordered protein ACTR in two different denaturants. Standard analysis of the primary data clearly indicates an expansion of the unfolded state with increasing denaturant concentration irrespective of the protein, denaturant, or experimental method used. This is the first case in which SAXS and FRET have yielded even qualitatively consistent results regarding expansion in denaturant when applied to the same proteins. To more directly illustrate this self-consistency, we used both SAXS and FRET data in a Bayesian procedure to refine structural ensembles representative of the observed unfolded state. This analysis demonstrates that both of these experimental probes are compatible with a common ensemble of protein configurations for each denaturant concentration. Furthermore, the resulting ensembles reproduce the trend of increasing hydrodynamic radius, with denaturant concentration obtained by 2f-FCS,and DLS. We were thus able to reconcile the results from all four experimental techniques quantitatively, to obtain a comprehensive structural picture of denaturant;induced unfolded state expansion, and to identify the Most likely sources of earlier discrepancies.
Y1  - 2016
U6  - https://doi.org/10.1021/jacs.6b05917
SN  - 0002-7863
VL  - 138
SP  - 11714
EP  - 11726
PB  - American Chemical Society
CY  - Washington
ER  - 
TY  - JOUR
A1  - Hofmann, Hagen
A1  - Soranno, Andrea
A1  - Borgia, Alessandro
A1  - Gast, Klaus
A1  - Nettels, Daniel
A1  - Schuler, Benjamin
T1  - Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy
JF  - Proceedings of the National Academy of Sciences of the United States of America
N2  - The dimensions of unfolded and intrinsically disordered proteins are highly dependent on their amino acid composition and solution conditions, especially salt and denaturant concentration. However, the quantitative implications of this behavior have remained unclear, largely because the effective theta-state, the central reference point for the underlying polymer collapse transition, has eluded experimental determination. Here, we used single-molecule fluorescence spectroscopy and two-focus correlation spectroscopy to determine the theta points for six different proteins. While the scaling exponents of all proteins converge to 0.62 +/- 0.03 at high denaturant concentrations, as expected for a polymer in good solvent, the scaling regime in water strongly depends on sequence composition. The resulting average scaling exponent of 0.46 +/- 0.05 for the four foldable protein sequences in our study suggests that the aqueous cellular milieu is close to effective theta conditions for unfolded proteins. In contrast, two intrinsically disordered proteins do not reach the T-point under any of our solvent conditions, which may reflect the optimization of their expanded state for the interactions with cellular partners. Sequence analyses based on our results imply that foldable sequences with more compact unfolded states are a more recent result of protein evolution.
KW  - protein folding
KW  - single-molecule FRET
KW  - coil-globule transition
KW  - polymer theory
Y1  - 2012
U6  - https://doi.org/10.1073/pnas.1207719109
SN  - 0027-8424
VL  - 109
IS  - 40
SP  - 16155
EP  - 16160
PB  - National Acad. of Sciences
CY  - Washington
ER  - 
TY  - JOUR
A1  - Hoffmann, Armin S.
A1  - Kane, Avinash S.
A1  - Nettels, Daniel
A1  - Hertzog, David E.
A1  - Baumgärtel, Peter
A1  - Lengefeld, Jan
A1  - Reichardt, Gerd
A1  - Horsley, David A.
A1  - Seckler, Robert
A1  - Bakajin, Olgica
A1  - Schuler, Benjamin
T1  - Mapping protein collapse with single molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy
Y1  - 2007
UR  - http://www.mendeley.com/research/mapping-protein-collapse-with-singlemolecule-fluorescence-and-kinetic- synchrotron-radiation-circular-dichroism-spectroscopy/#
SN  - 0027-8424
ER  - 
TY  - JOUR
A1  - Nettels, Daniel
A1  - Müller-Späth, Sonja
A1  - Küster, Frank
A1  - Hofmann, Hagen
A1  - Haenni, Domminik
A1  - Rüegger, Stefan
A1  - Reymond, Luc
A1  - Hoffmann, Armin S.
A1  - Kubelka, Jan
A1  - Heinz, Benjamin
A1  - Gast, Klaus
A1  - Best, Robert B.
A1  - Schuler, Benjamin
T1  - Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins
N2  - We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on the dimensions of unfolded proteins. With singlemolecule FRET, this question can be addressed even under nearnative conditions, where most molecules are folded, allowing us to probe a wide range of denaturant concentrations and temperatures. We find a compaction of the unfolded state of a small cold shock protein with increasing temperature in both the presence and the absence of denaturant, with good agreement between the results from single-molecule FRET and dynamic light scattering. Although dissociation of denaturant from the polypeptide chain with increasing temperature accounts for part of the compaction, the results indicate an important role for additional temperaturedependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin suggests that the hydrophobic effect is not the sole source of the underlying interactions. Circular dichroism spectroscopy and replica exchange molecular dynamics simulations in explicit water show changes in secondary structure content with increasing temperature and suggest a contribution of intramolecular hydrogen bonding to unfolded state collapse.
Y1  - 2009
UR  - http://www.pnas.org/content/106/49/20740.full.pdf+html
SN  - 0027-8424
ER  - 
TY  - JOUR
A1  - Kane, Avinash S.
A1  - Hoffmann, Armin S.
A1  - Baumgärtel, Peter
A1  - Seckler, Robert
A1  - Reichardt, Gerd
A1  - Horsley, David A.
A1  - Schuler, Benjamin
A1  - Bakajin, Olgica
T1  - Microfluidic mixers for the investigation of rapid protein folding kinetics using synchrotron radiation circular dichroism spectroscopy
N2  - We have developed a microfluidic mixer optimized for rapid measurements of protein folding kinetics using synchrotron radiation circular dichroism (SRCD) spectroscopy. The combination of fabrication in fused silica and synchrotron radiation allows measurements at wavelengths below 220 nm, the typical limit of commercial instrumentation. At these wavelengths, the discrimination between the different types of protein secondary structure increases sharply. The device was optimized for rapid mixing at moderate sample consumption by employing a serpentine channel design, resulting in a dead time of less than 200 ;s. Here, we discuss the design and fabrication of the mixer and quantify the mixing efficiency using wide-field and confocal epi-fluorescence microscopy. We demonstrate the performance of the device in SRCD measurements of the folding kinetics of cytochrome c, a small, fast-folding protein. Our results show that the combination of SRCD with microfluidic mixing opens new possibilities for investigating rapid conformational changes in biological macromolecules that have previously been inaccessible.
Y1  - 2008
UR  - http://pubs.acs.org/doi/abs/10.1021/ac801764r
SN  - 0003-2700
ER  - 
TY  - JOUR
A1  - Lipman, Everett A.
A1  - Schuler, Benjamin
A1  - Bakajin, Olgica
A1  - Eaton, William A.
T1  - Single-molecule measurement of protein folding kinetics
N2  - In order to investigate the behavior of single molecules under conditions far from equilibrium, we have coupled a microfabricated laminar-flow mixer to a confocal optical system. This combination enables time-resolved measurement of Foerster resonance energy transfer after an abrupt change in solution conditions. Observations of a small protein show the evolution of the intramolecular distance distribution as folding progresses. This technique can expose subpopulations, such as unfolded protein under conditions favoring the native structure, that would be obscured in equilibrium experiments.
Y1  - 2003
UR  - http://www.sciencemag.org/content/301/5637/1233
SN  - 0036-8075
ER  - 
TY  - JOUR
A1  - Tolan, Dean R.
A1  - Schuler, Benjamin
A1  - Beernink, Peter T.
A1  - Jaenicke, Rainer
T1  - Thermodynamic analysis of the dissociation of the aldolase tetramer substituted at one or both of the subunit interfaces
N2  - The fructose-1,6-bis(phosphate) aldolase isologous tetramer tightly associates through two different subunit interfaces defined by its 222 symmetry. Both single- and double-interfacial mutant aldolases have a destabilized quaternary structure, but there is little effect on the catalytic activity. These enzymes are however thermolabile. This study demonstrates the temperature-dependent dissociation of the mutant enzymes and determines the dissociation free energies of both mutant and native aldolase. Subunit dissociation is measured by sedimentation equilibrium in the analytical ultracentrifuge. At 25C the tetramerdimer dissociation constants for each single-mutant enzyme are similar, about 10 -6 M. For the double-mutant enzyme, sedimentation velocity experiments on sucrose density gradients support a tetramermonomer equilibrium. Furthermore, sedimentation equilibrium experiments determined a dissociation constant of 10- 15 M3 for the double-mutant enzyme. By the same methods the upper limit for the dissociation constant of wild-type aldolase A is approximately 10-28 M3, which indicates an extremely stable tetramer. The thermodynamic values describing monomer-tetramer and dimer-tetramer equilibria are analyzed with regard to possible cooperative interaction between the two subunit interfaces.
Y1  - 2003
UR  - http://www.reference-global.com/doi/abs/10.1515/BC.2003.162
ER  - 
TY  - JOUR
A1  - Schuler, Benjamin
A1  - Seckler, Robert
T1  - P22 tailspike folding mutants revisited : effects on thermodynamic stability of the isolated beta-helix domain
Y1  - 1998
ER  - 
TY  - JOUR
A1  - Miller, Stefan
A1  - Schuler, Benjamin
A1  - Seckler, Robert
T1  - A reversibly unfolding fragment of P22 tailspike protein with native structure : the isolated beta-helix domain
Y1  - 1998
ER  - 
TY  - JOUR
A1  - Miller, Stefan
A1  - Schuler, Benjamin
A1  - Seckler, Robert
T1  - Phages P22 tailspike protein: Removal of head-binding domain unmasks efects of folding mutations on native- state thermal stability
Y1  - 1998
ER  - 
TY  - JOUR
A1  - Schuler, Benjamin
A1  - Rachel, Reinhard
A1  - Seckler, Robert
T1  - Formation of fibrous aggregates from a non-native intermediate : the isolated P22 tailspike -helix domain
Y1  - 1999
ER  - 
TY  - JOUR
A1  - Schuler, Benjamin
A1  - Fürst, Frank
A1  - Osterroth, Frank
A1  - Steinbacher, Stefan
A1  - Huber, Robert
A1  - Seckler, Robert
T1  - Plasticity and steric strain in a parallel beta-helix: Rational mutations in P22 tailspike protein
N2  - By means of genetic screens, a great number of mutations that affect the folding and stability of the tailspike protein from Salmonella phage P22 have been identified. Temperature-sensitive folding (tsf) mutations decrease folding yields at high temperature, but hardly affect thermal stability of the native trimeric structure when assembled at low temperature. Global suppressor (su) mutations mitigate this phenotype. Virtually all of these mutations are located in the central domain of tailspike, a large parallel beta-helix. We modified tailspike by rational single amino acid replacements at three sites in order to investigate the influence of mutations of two types: (1) mutations expected to cause a tsf phenotype by increasing the side-chain volume of a core residue, and (2) mutations in a similar structural context as two of the four known su mutations, which have been suggested to stabilize folding intermediates and the native structure by the release of backbone strain, an effect well known for residues that are primarily evolved for function and not for stability or folding of the protein. Analysis of folding yields, refolding kinetics and thermal denaturation kinetics in vitro show that the tsf phenotype can indeed be produced rationally by increasing the volume of side chains in the beta-helix core. The high-resolution crystal structure of mutant T326F proves that structural rearrangements only take place in the remarkably plastic lumen of the beta-helix, leaving the arrangement of the hydrogen-bonded backbone and thus the surface of the protein unaffected. This supports the notion that changes in the stability of an intermediate, in which the beta-helix domain is largely formed, are the essential mechanism by which tsf mutations affect tailspike folding. A rational design of su mutants, on the other hand, appears to be more difficult. The exchange of two residues in the active site expected to lead to a drastic release of steric strain neither enhanced the folding properties nor the stability of tailspike. Apparently, side-chain interactions in these cases overcompensate for backbone strain, illustrating the extreme optimization of the tailspike protein for conformational stability. The result exemplifies the view arising from the statistical analysis of the distribution of backbone dihedral angles in known three-dimensional protein structures that the adoption of straight phi/psi angles other than the most favorable ones is often caused by side-chain interactions.
Y1  - 2000
UR  - http://www3.interscience.wiley.com/cgi-bin/abstract/71001984/START
ER  - 
TY  - JOUR
A1  - Rhoades, E.
A1  - Cohen, M.
A1  - Gussakovsky, E.
A1  - Schuler, Benjamin
A1  - Haran, G.
T1  - Single molecule protein folding
Y1  - 2004
SN  - 0006-3495
ER  - 
TY  - JOUR
A1  - Schuler, Benjamin
A1  - Lipman, Everett A.
A1  - Steinbach, P. J.
A1  - Kumke, Michael Uwe
A1  - Eaton, W. A.
T1  - Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence
N2  - To determine whether Forster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Forster theory, with polyproline treated as a rigid rod. At donor-acceptor distances much less than the Forster radius R-o, the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R-0, they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Forster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor-acceptor distances
Y1  - 2005
SN  - 0027-8424
ER  - 
TY  - JOUR
A1  - Goetz, C.
A1  - Suopanki, J.
A1  - Schuler, Benjamin
A1  - Wanker, E.
A1  - Herrmann, Andreas
T1  - Perturbation of brain lipid membrane by soluble Huntingtin depends on its polyproline tract
Y1  - 2005
SN  - 0006-3495
ER  - 
TY  - JOUR
A1  - Buscaglia, Marco
A1  - Schuler, Benjamin
A1  - Lapidus, Lisa J.
A1  - Eaton, Wiliam A.
A1  - Hofrichter, James
T1  - Kinetics of intramolecular contact formation in a denatured protein
N2  - Quenching of the triplet state of tryptophan by cysteine has provided a new tool for measuring the rate of forming a specific intramolecular contact in disordered polypeptides. Here, we use this technique to investigate contact formation in the denatured state of CspTm, a small cold-shock protein from Thermotoga maritima, engineered to contain a single tryptophan residue (W29) and a single cysteine residue at the C terminus (C67). At all concentrations of denaturant, the decay rate of the W29 triplet of the unfolded protein is more than tenfold faster than the rate observed for the native protein (not, vert, similar104 s;1). Experiments on the unfolded protein without the added C- terminal cysteine residue show that this faster rate results entirely from contact quenching by C67. The quenching rate in the unfolded state by C67 increases at concentrations of denaturant that favor folding, indicating a compaction of the unfolded protein as observed previously in single-molecule Foerster resonance energy transfer (FRET) experiments.
Y1  - 2003
UR  - http://www.sciencedirect.com/science/article/pii/S002228360300891X
SN  - 0022-2836
ER  -