The Gaussian-distributed random coil has been the dominant model for denatured proteins since the 1950s, and it has long been interpreted to mean that proteins are featureless, statistical coils in 6 M guanidinium chloride. Here, we demonstrate that random-coil statistics are not a unique signature of featureless polymers. The random-coil model does predict the experimentally determined coil dimensions of denatured proteins successfully. Yet, other equally convincing experiments have shown that denatured proteins are biased toward specific conformations, in apparent conflict with the random-coil model. We seek to resolve this paradox by introducing a contrived counterexample in which largely native protein ensembles nevertheless exhibit random-coil characteristics. Specifically, proteins of known structure were used to generate disordered conformers by varying backbone torsion angles at random for Ϸ8% of the residues; the remaining Ϸ92% of the residues remained fixed in their native conformation. Ensembles of these disordered structures were generated for 33 proteins by using a torsion-angle Monte Carlo algorithm with hard-sphere sterics; bulk statistics were then calculated for each ensemble. Despite this extreme degree of imposed internal structure, these ensembles have end-to-end distances and mean radii of gyration that agree well with random-coil expectations in all but two cases.T he protein folding reaction, unfolded (U)^native (N), is a reversible disorder^order transition. Typically, proteins are disordered (U) at high temperature, high pressure, extremes of pH, or in the presence of denaturing solvents, but they fold to uniquely ordered, biologically relevant conformers (N) under physiological conditions. With some exceptions (1), the folded state is the biologically relevant form, and it can be characterized to atomic detail by using x-ray crystallography and NMR spectroscopy. In contrast, our understanding of the unfolded state is based primarily on a statistical model, the random-coil model, which was developed largely by Flory (2) and corroborated by Tanford (3) in the 1950s and 1960s.In a random coil, the energy differences among sterically accessible backbone conformers are of order ϷkT (where k is Boltzmann's constant, and T is the absolute temperature). Consequently, there are no strongly preferred conformations, the energy landscape is essentially featureless, and a Boltzmann-weighted ensemble of such polymers would populate this landscape uniformly.Our motivation here is to dispel the belief, which is widespread among protein chemists, that the presence of random-coil statistics for denatured proteins confirms the absence of residual structure in these molecules. Indeed, it is well known to polymer chemists that rods of any stiffness (e.g., steel I-beams) behave as Gaussiandistributed, temperature-dependent random coils if they are long enough. Chains in which the persistence length exceeds one physical link can be treated effectively by rewriting them as polymers of Kuhn segments (ref. 2, pa...

Saturation of Binding ApproximationAccording to De Roe, et al., 1 typical dissociation constants (K d values) for AuNP binding range from 4 to 300 nM. In this work, we assume that AuNPs are covered with N independent, identical binding sites. Assuming a fixed total concentration of AuNPs (M tot ) and a fixed protein (ligand) concentration (L tot ), the degree of binding (ܺ ത ) is given by: 2

Approximately half the structure of folded proteins is either alpha-helix or beta-strand. We have developed a convenient repository of all remaining structure after these two regular secondary structure elements are removed. The Protein Coil Library (http://roselab.jhu.edu/coil/) allows rapid and comprehensive access to non-alpha-helix and non-beta-strand fragments contained in the Protein Data Bank (PDB). The library contains both sequence and structure information together with calculated torsion angles for both the backbone and side chains. Several search options are implemented, including a query function that uses output from popular PDB-culling servers directly. Additionally, several popular searches are stored and updated for immediate access. The library is a useful tool for exploring conformational propensities, turn motifs, and a recent model of the unfolded state.

The role of cysteine residues in the protein binding kinetics and stability on gold nanoparticles (AuNP) was studied using AuNP localized surface plasmon resonance (LSPR) in combination with an organothiol (OT) displacement method. GB3, the third IgG-binding domain of protein G, was used to model protein-AuNP adsorption. While wild-type GB3 (GB30) contains no cysteine residues, bioengineered GB3 variants containing one (GB31) and two (GB32) cysteine residues were also tested. The cysteine content has no significant effect on GB3 binding kinetics with AuNPs, and most protein adsorption occurs within the first few seconds upon protein/AuNP mixing. However, the stability of GB3 on the AuNP surface against OT displacement depends strongly on the cysteine content and the age of the AuNP/GB3 mixture. The GB30 covered AuNPs can be completely destabilized and aggregated by OTs, regardless of the age of the GB30/AuNP mixtures. Long-time incubation of GB31 or GB32 with AuNPs can stabilize AuNPs against the OT adsorption inducted aggregation. This study indicates that multiple forces involved in the GB3/AuNP interaction, and covalent binding between cysteine and AuNP is essential for a stable protein/AuNP complex.

We present a simple method, ARTSY, for extracting 1 J NH couplings and 1 H-15 N RDCs from an interleaved set of two-dimensional 1 H-15 N TROSY-HSQC spectra, based on the principle of quantitative J correlation. The primary advantage of the ARTSY method over other methods is the ability to measure couplings without scaling peak positions or altering the narrow line widths characteristic of TROSY spectra. Accuracy of the method is demonstrated for the model system GB3. Application to the catalytic core domain of HIV integrase, a 36 kDa homodimer with unfavorable spectral characteristics, demonstrates its practical utility. Precision of the RDC measurement is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC spectrum, and is approximately given by 30/(S/N) Hz. KeywordsARTSY; catalytic core domain; HIV integrase; quantitative J correlation; TROSY; RDC Residual dipolar couplings (RDCs) in proteins, as measured by solution state NMR, constitute an important source of both structural and dynamic information (Prestegard et al. 2000;Bax and Grishaev 2005;Tolman and Ruan 2006). Although a wide range of different types of RDCs can be measured, including 1 H-1 H, 1 H-13 C, 1 H-15 N, 15 N-13 C, and 13 C-13 C (Tjandra and Bax 1997;Yang et al. 1999;Permi et al. 2000;Boisbouvier et al. 2003;Vijayan and Zweckstetter 2005), in larger, slowly tumbling proteins measurements are often restricted to the backbone amide 1 H-15 N RDC, 1 D NH . With assignment of the backbone amide signals being a prerequisite for any detailed analysis of protein structure and dynamics, the additional effort needed to collect such couplings for modest size proteins is limited, and they can readily be obtained from the 15 N-{ 1 H} splittings in 2D or 3D NMR spectra, recorded in the absence of 1 H decoupling during 15 N evolution (Tolman et al. 1995). Increased spectral overlap associated with the doubling of the number of resonances in such spectra can be mitigated by separating the two doublet components by the IPAP method (Ottiger et al. 1998;Yao et al. 2009), or by separately recording spectra for each multiplet component using the principle of spin-state selective polarization transfer (Lerche et al. 1999).For larger proteins, a problem associated with the frequency domain measurements is caused by the very different intrinsic line widths of the two 15 N-{ 1 H} doublet components. The downfield component benefits from favorable relaxation interference between the 15 N-1 H dipolar coupling and 15 N chemical shift anisotropy (CSA) mechanisms, resulting in very narrow 15 N line widths, a property utilized in the original TROSY-HSQC method for obtaining spectral resolution enhancement in larger proteins (Pervushin et al. 1997 (Yang et al. 1999;Bhattacharya et al. 2010;Mantylahti et al. 2010). An elegant implementation of this principle distributes the relaxation losses equally over the two doublet components, and appears to offer the highest accuracy at which 1 J NH + 1 D NH splittings can be obtained from frequency domain...