Bioinformatic Identification of ABC Transporters in Candida auris.

Antifungal resistance mediated by overexpression of ABC transporters is one of the primary roadblocks to effective therapy against Candida infections. Thus, identification and characterization of the ABC transporter repertoire in Candida species are of high relevance. The method described in the chapter is based on our previously developed bioinformatic pipeline for identification of ABC proteins in Candida species. The methodology essentially involves the utilization of a hidden Markov model (HMM) profile of the nucleotide-binding domain (NBD) of ABC proteins to mine these proteins from the proteome of Candida species. Further, a widely used tool to predict membrane protein topology is exploited to identify the true transporter candidates out of the ABC proteins. Even though the chapter specifically focuses on a method to identify ABC transporters in Candida auris , the same can also be applied to any other Candida species.

ABC-finder: A containerized web server for the identification and topology prediction of ABC proteins.

In view of the multiple clinical and physiological implications of ABC transporter proteins, there is a considerable interest among researchers to characterize them functionally. However, such characterizations are based on the premise that ABC proteins are accurately identified in the proteome of an organism, and their topology is correctly predicted. With this objective, we have developed ABC-finder, i.e., a Docker-based package for the identification of ABC proteins in all organisms, and visualization of the topology of ABC proteins using a web browser. ABC-finder is built and deployed in a Linux container, making it scalable for many concurrent users on our servers and enabling users to download and run it locally. Overall, ABC-finder is a convenient, portable, and platform-independent tool for the identification and topology prediction of ABC proteins. ABC-finder is accessible at http://abc-finder.osdd.jnu.ac.in.

Peroneus Longus Tendon Autograft for Anterior Cruciate Ligament Reconstruction: A Safe and Effective Alternative in Nonathletic Patients.

INTRODUCTION: Anterior cruciate ligament (ACL) is a common injury which has been conventionally managed by various graft reconstruction using bone patellar tendon bone, or quadruple hamstring autograft, to name a few. However, all these grafts are associated with many complications. Lately, peroneus longus tendon (PLT) autograft has shown promising results in this field, although there is still a dearth of data on its use. We, therefore, aimed at carrying out a study to evaluate the functional outcome and knee stability results of ACL reconstruction using PLT graft. PATIENTS AND METHODS: Patients with a completely torn ACL were included in the study. The PLT was harvested, and graft length, thickness, and harvesting time were noted intraoperatively. Knee stability and functional scores were evaluated clinically and using Lachman test (primarily) and KT-2000 arthrometer and subjectively with International Knee Documentation Committee (IKDC) score at 6, 12, and 24 months (secondary outcome) postoperatively. Ankle scores were also recorded by making use of American Orthopedic Foot and Ankle Score (AOFAS)-Hindfoot Scale. RESULTS: Forty-eight patients met the inclusion criteria. The graft harvest time was 7.4 min (5-9 min). The mean thickness of the graft on doubling was 7.9 mm (7-9 mm). Ninety-six percent of the patients were satisfied with their results of the knee surgery, and 95% of the patients had no complaints of ankle joint. The mean IKDC score postoperatively was 78.16 ± 6.23, and the mean AOFAS score was 98.4 ± 4.1. None of the patients had any neurovascular deficit. CONCLUSION: ACL reconstruction using PLT graft yields a good functional score (IKDC, KT-2000 arthrometer) even at 2-year follow-up. It is a safe and effective autograft option for ACL reconstruction.

Characterization of heterogeneous intermediate ensembles on the guanidinium chloride-induced unfolding pathway of ß-lactoglobulin.

Folding pathway of ß-LgA (ß-lactoglobulin) evolves through the conformational αâ†’ß transition. The αâ†’ß transition is a molecular hallmark of various neurodegenerative diseases. Thus, ß-LgA may serve as a good model for understanding molecular mechanism of protein aggregation involved in neurodegenerative diseases. Here, we studied the conformational dynamics of ß-LgA in 6 M GdmCl at different temperatures using MD simulations. Structural order parameters such as RMSD, Rg, SASA, native contacts (Q), hydrophobic distal-matrix and free-energy landscape (FEL) were used to investigate the conformational transitions. Our results show that GdmCl destabilizes secondary and tertiary structure of ß-LgA by weakening the hydrophobic interactions and hydrogen bond network. Multidimensional FEL shows the presence of different unfolding intermediates at 400 K. I1 is long-lived intermediate which has mostly intact native secondary structure, but loose tertiary structure. I2 is structurally compact intermediate formed after the partial loss of secondary structure. The transiently and infrequently buried evolution of W19 shows that intermediate conformational ensembles are structurally heterogeneous. We observed that the intermediate conformations are largely stabilized by non-native H-bonds. The outcome of this work provides the molecular details of intermediates trapped due to non-native interactions that may be regarded as pathogenic conformations involved in neurodegenerative diseases.Communicated by Ramaswamy H. Sarma.

Delineating the conformational dynamics of intermediate structures on the unfolding pathway of ß-lactoglobulin in aqueous urea and dimethyl sulfoxide.

The funnel shaped energy landscape model of the protein folding suggests that progression of folding proceeds through multiple pathways, having the multiple intermediates which leads to multidimensional free-energy surface. Herein, we applied all-atom MD simulation to conduct a comparative study on the structure of ß-lactoglobulin (ß-LgA) in aqueous mixture of 8 M urea and 8 M dimethyl sulfoxide (DMSO), at different temperatures. The cumulative results of multiple simulations suggest a common unfolding pathway of ß-LgA, occurred through the stable and meta-stable intermediates (I), in both urea and DMSO. However, the free-energy landscape (FEL) analyses show that the structural transitions of I-states are energetically different. In urea, FEL shows distinct ensemble of intermediates, I1 and I2, separated by the energy barrier of ∼3.0 kcal mol-1. Similarly, we find the population of two distinct I1 and I2 states in DMSO, however, the I1 appeared transiently around ∼30-35 ns and is short-lived. But, the I2 ensemble is observed structurally compact and long-lived (∼50-150 ns) as compared to unfolding in urea. Furthermore, the I1 and I2 are separated through a high energy barrier of ∼6.0 kcal mol-1. Thus, our results provide the structural insights of intermediates which essentially bear the signature of a different unfolding pathway of ß-LgA in urea and DMSO.Abbreviationsß-LgAß-lactoglobulinDMSOdimethyl sulfoxideFELfree-energy landscapeGdmClguanidinium chlorideIintermediate stateMGmolten globule statePMEparticle mesh EwaldQfraction of native contactsRMSDroot mean square deviationRMSFroot mean square fluctuationRgradius of gyrationSASAsolvent Accessible Surface AreascSASAthe side chain SASATrptryptophanCommunicated by Ramaswamy H. Sarma.

Comparative analysis of thermal unfolding simulations of RNA recognition motifs (RRMs) of TAR DNA-binding protein 43 (TDP-43).

TAR DNA-binding protein 43 (TDP-43) inclusions have been found in Amyotrophic lateral sclerosis (ALS) and several other neurodegenerative diseases. Many studies suggest the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy. To elucidate the structural stability and the unfolding dynamics of RRMs, we have carried out atomistic molecular dynamics simulations at two different temperatures (300 and 500 K). The simulations results indicate that there are distinct structural differences in the unfolding pathway between the two domains and RRM1 unfolds faster than RRM2 in accordance with the lower thermal stability found experimentally. The unfolding behaviors of secondary structures showed that the α-helix was more stable than ß-sheet and structural rearrangements of ß-sheets results in formation of additional α-helices. At higher temperature, RRM1 exhibit increased overall flexibility and unfolding than RRM2. The temperature-dependent free energy landscapes consist of multiple metastable states stabilized by non-native contacts and hydrogen bonds in RRM2, thus rendering the RRM2 more prone to misfolding. The structural rearrangements of RRM2 could lead to aberrant protein-protein interactions that may account for enhanced aggregation and toxicity of TDP-43. Our analysis, thus identify the structural and thermodynamic characteristics of the RRMs of TDP-43, which will serve to uncover molecular mechanisms and driving forces in TDP-43 misfolding and aggregation.

Elucidation of stable intermediates in urea-induced unfolding pathway of human carbonic anhydrase IX.

Human carbonic anhydrase IX (CAIX) has evolved as a promising biomarker for cancer prognosis, due to its overexpression in various cancers and restricted expression in normal tissue. However, limited information is available on its biophysical behavior. The unfolding of CAIX in aqueous urea solution was studied using all-atom molecular dynamics simulation approach. The results of this study revealed a stable intermediate state along the unfolding pathway of CAIX. At intermediate concentrations of urea (2.0-4.0 M), the protein displays a native-like structure with a large population of its secondary structure and hydrophobic contacts remaining intact in addition to small confined overall motions. Beyond 4.0 M urea, the unfolding is more gradual and at 8.0 M urea the structure is largely collapsed due to the solvent effect. The hydrophobic contact analysis suggests that the contact in terminal α-helices is separated initially which propagates in the loss of contacts from centrally located ß-sheets. The reduction of 60-65% tertiary contacts in 7.0-8.0 M urea suggested the presence of residual structure in unfolded state and is confirmed with structural snap shot. Free energy landscape analysis suggested that unfolding of CAIX exists through the different intermediate states.

Elucidation of the structural stability and dynamics of heterogeneous intermediate ensembles in unfolding pathway of the N-terminal domain of TDP-43.

The N-terminal domain of the RNA binding protein TDP-43 (NTD) is essential to both physiology and proteinopathy; however, elucidation of its folding/unfolding still remains a major quest. In this study, we have investigated the biophysical behavior of intermediate ensembles employing all-atom molecular dynamics simulations in 8 M urea accelerated with high temperatures to achieve unfolded states in a confined computation time. The cumulative results of the 2.75 µs simulations show that unfolding of the NTD at 350 K evolves through different stable and meta-stable intermediate states. The free-energy landscape reveals two meta-stable intermediates (IN and IU) stabilized by non-native interactions, which are largely hydrophilic and highly energetically frustrated. A single buried tryptophan residue, W80, undergoes solvent exposure to different extents during unfolding; this suggests a structurally heterogeneous population of intermediate ensembles. Furthermore, the structure properties of the IN state show a resemblance to the molten globule (MG) state with most of the secondary structures intact. The unfolding of the NTD is initiated by the loss of ß-strands, and the unfolded (U) states exhibit a population of non-native α-helices. These non-native unfolded intermediate ensembles may mediate protein oligomerization, leading to the formation of pathological, irreversible aggregates, characteristics of disease pathogenesis.