Restriction versus guidance in protein structure prediction.

Conformational restriction by fragment assembly and guidance in molecular dynamics are alternate conformational search strategies in protein structure prediction. We examine both approaches using a version of the associative memory Hamiltonian that incorporates the influence of water-mediated interactions (AMW). For short proteins (<70 residues), fragment assembly, while searching a restricted space, compares well to molecular dynamics and is often sufficient to fold such proteins to near-native conformations (4A) via simulated annealing. Longer proteins encounter kinetic sampling limitations in fragment assembly not seen in molecular dynamics which generally samples more native-like conformations. We also present a fragment enriched version of the standard AMW energy function, AMW-FME, which incorporates the local sequence alignment derived fragment libraries from fragment assembly directly into the energy function. This energy function, in which fragment information acts as a guide not a restriction, is found by molecular dynamics to improve on both previous approaches.

Prediction of native-state hydrogen exchange from perfectly funneled energy landscapes.

Simulations based on perfectly funneled energy landscapes often capture many of the kinetic features of protein folding. We examined whether simulations based on funneled energy functions can also describe fluctuations in native-state protein ensembles. We quantitatively compared the site-specific local stability determined from structure-based folding simulations, with hydrogen exchange protection factors measured experimentally for ubiquitin, chymotrypsin inhibitor 2, and staphylococcal nuclease. Different structural definitions for the open and closed states based on the number of native contacts for each residue, as well as the hydrogen-bonding state, or a combination of both criteria were evaluated. The predicted exchange patterns agree with the experiments under native conditions, indicating that protein topology indeed has a dominant effect on the exchange kinetics. Insights into the simplest mechanistic interpretation of the amide exchange process were thus obtained.

Consequences of localized frustration for the folding mechanism of the IM7 protein.

In the laboratory, IM7 has been found to have an unusual folding mechanism in which an "on-pathway" intermediate with nonnative interactions is formed. We show that this intermediate is a consequence of an unusual cluster of highly frustrated interactions in the native structure. This cluster is involved in the binding of IM7 to its target, Colicin E7. Redesign of residues in this cluster to eliminate frustration is predicted by simulations to lead to faster folding without the population of an intermediate ensemble.

Induced fit, folding, and recognition of the NF-kappaB-nuclear localization signals by IkappaBalpha and IkappaBbeta.

Protein structure prediction codes based on the associative memory Hamiltonian were used to probe the binding modes between the nuclear localization signal (NLS) polypeptide of NF-kappaB and the inhibitors IkappaBalpha and IkappaBbeta. Experimentally, it is known that the NLS polypeptide is unstructured in the NF-kappaB complex with DNA but it forms an extended helical structure with the NLS (residues 301-304) between the two helices in the NF-kappaB/IkappaBalpha complex. The simulations included the NF-kappaB(p65) and (p50) NLS polypeptides and various mutants alone and in the presence of IkappaBalpha and IkappaBbeta. The simulations predict that the NLS polypeptide by itself binds tightly to IkappaBalpha and IkappaBbeta. In the NF-kappaB (p50/p65) heterodimer, the p50 NLS is predicted to remain free to bind to importin alpha. In the interaction with IkappaBalpha, both p65 NLSs are predicted to be bound. In IkappaBbeta, the NLS polypeptide binds to two binding sites, as seen in the crystal structure, with one site heavily favored for stable binding.

CUREs in biochemistry-where we are and where we should go.

Integration of research experience into classroom is an important and vital experience for all undergraduates. These course-based undergraduate research experiences (CUREs) have grown from independent instructor lead projects to large consortium driven experiences. The impact and importance of CUREs on students at all levels in biochemistry was the focus of a National Science Foundation funded think tank. The state of biochemistry CUREs and suggestions for moving biochemistry forward as well as a practical guide (supplementary material) are reported here. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(1):7-12, 2017.

Conformational switching upon phosphorylation: a predictive framework based on energy landscape principles.

We investigate how post-translational phosphorylation modifies the global conformation of a protein by changing its free energy landscape using two test proteins, cystatin and NtrC. We first examine the changes in a free energy landscape caused by phosphorylation using a model containing information about both structural forms. For cystatin the free energy cost is fairly large indicating a low probability of sampling the phosphorylated conformation in a perfectly funneled landscape. The predicted barrier for NtrC conformational transition is several times larger than the barrier for cystatin, indicating that the switch protein NtrC most probably follows a partial unfolding mechanism to move from one basin to the other. Principal component analysis and linear response theory show how the naturally occurring conformational changes in unmodified proteins are captured and stabilized by the change of interaction potential. We also develop a partially guided structure prediction Hamiltonian which is capable of predicting the global structure of a phosphorylated protein using only knowledge of the structure of the unphosphorylated protein or vice versa. This algorithm makes use of a generic transferable long-range residue contact potential along with details of structure short range in sequence. By comparing the results obtained with this guided transferable potential to those from the native-only, perfectly funneled Hamiltonians, we show that the transferable Hamiltonian correctly captures the nature of the global conformational changes induced by phosphorylation and can sample substantially correct structures for the modified protein with high probability.

Simulation studies of the fidelity of biomolecular structure ensemble recreation.

We examine the ability of Bayesian methods to recreate structural ensembles for partially folded molecules from averaged data. Specifically we test the ability of various algorithms to recreate different transition state ensembles for folding proteins using a multiple replica simulation algorithm using input from "gold standard" reference ensembles that were first generated with a Go-like Hamiltonian having nonpairwise additive terms. A set of low resolution data, which function as the "experimental" phi values, were first constructed from this reference ensemble. The resulting phi values were then treated as one would treat laboratory experimental data and were used as input in the replica reconstruction algorithm. The resulting ensembles of structures obtained by the replica algorithm were compared to the gold standard reference ensemble, from which those "data" were, in fact, obtained. It is found that for a unimodal transition state ensemble with a low barrier, the multiple replica algorithm does recreate the reference ensemble fairly successfully when no experimental error is assumed. The Kolmogorov-Smirnov test as well as principal component analysis show that the overlap of the recovered and reference ensembles is significantly enhanced when multiple replicas are used. Reduction of the multiple replica ensembles by clustering successfully yields subensembles with close similarity to the reference ensembles. On the other hand, for a high barrier transition state with two distinct transition state ensembles, the single replica algorithm only samples a few structures of one of the reference ensemble basins. This is due to the fact that the phi values are intrinsically ensemble averaged quantities. The replica algorithm with multiple copies does sample both reference ensemble basins. In contrast to the single replica case, the multiple replicas are constrained to reproduce the average phi values, but allow fluctuations in phi for each individual copy. These fluctuations facilitate a more faithful sampling of the reference ensemble basins. Finally, we test how robustly the reconstruction algorithm can function by introducing errors in phi comparable in magnitude to those suggested by some authors. In this circumstance we observe that the chances of ensemble recovery with the replica algorithm are poor using a single replica, but are improved when multiple copies are used. A multimodal transition state ensemble, however, turns out to be more sensitive to large errors in phi (if appropriately gauged) and attempts at successful recreation of the reference ensemble with simple replica algorithms can fall short.