Mohammad Monirul Islam

Crystal structure of an extensively simplified variant of bovine pancreatic trypsin inhibitor in which over one-third of the residues are alanines

We report the high-resolution crystal structures of an extensively simplified variant of bovine pancreatic trypsin inhibitor containing 20 alanines (BPTI-20st) and a reference single-disulfide-bonded variant (BPTI-[5,55]st) at, respectively, 1.39 and 1.09 Å resolutions. The sequence was simplified based on the results of an alanine scanning experiment, as reported previously. The effects of the multiple alanine substitutions on the overall backbone structure were surprisingly small (Cα atom RMSD of 0.53 Å) being limited to small local structural perturbations. Both BPTI variants retained a wild-type level of trypsin inhibitory activity. The side-chain configurations of residues buried in the hydrophobic cores (<30% accessible surface area) were almost perfectly retained in both BPTI-20st and BPTI-[5,55]st, indicating that neither multiple alanine replacements nor the removal of the disulfide bonds affected their precise placements. However, the side chains of three partially buried residues (Q31, R20, and to some extent Y21) and several unburied residues rearranged into alternative dense-packing structures, suggesting some plasticity in their shape complementarity. These results indicate that a protein sequence simplified over its entire length can retain its densely packed, native side-chain structure, and suggest that both the design and fold recognition of natively folded proteins may be easier than previously thought.

Thermodynamic and structural analysis of highly stabilized BPTIs by single and double mutations

Enhancing protein conformational stability is an important aspect of protein engineering and biotechnology. However, protein stabilization is difficult to rationalize as it often results from the small cumulative and intertwined effects of multiple mutations. Here, we analyzed the mechanisms behind a remarkable 13° stabilization produced by a single A14G and a double A14GA38V mutation in BPTI‐[5,55], a natively folded bovine pancreatic trypsin inhibitor variant. Differential scanning calorimetry analysis of three BPTI‐[5,55] variants (A14G, A38V, and A14GA38V) indicated that the A14G mutation stabilized the structure enthalpically, whereas the A38V stabilization was entropy driven. We also determined the structure of the A14GA38V mutant at 1.09 Å resolution, whereas the A38V variant did not crystallize, and we previously reported the A14G variants structure (2ZJX). The overall structures of the A14G and A14GA38V variants were very similar to that of wild‐type BPTI, but small local structure perturbations around residues 14 and 38 strongly suggested potential factors contributing to the enthalpy stabilization. First, the A14G mutation displaced the local backbone structures around residues 14 and 38 by up to 0.7 Å, presumably increasing local van der Waals interactions. Next, this displacement produced steric clashes between neighboring residues side‐chains in all but the variants containing the A14G mutation. Noteworthy, these clashes are not predicted from the wild type BPTI structure. These observations provide one of the first unambiguous analyses of how a subtle interplay between the sidechain and backbone structures can have a major effect on protein stability. Proteins 2009.

Crystal structures of highly simplified BPTIs provide insights into hydration-driven increase of unfolding enthalpy

We report a thermodynamic and structural analysis of six extensively simplified bovine pancreatic trypsin inhibitor (BPTI) variants containing 19–24 alanines out of 58 residues. Differential scanning calorimetry indicated a two-state thermal unfolding, typical of a native protein with densely packed interior. Surprisingly, increasing the number of alanines induced enthalpy stabilization, which was however over-compensated by entropy destabilization. X-ray crystallography indicated that the alanine substitutions caused the recruitment of novel water molecules facilitating the formation of protein–water hydrogen bonds and improving the hydration shells around the alanine’s methyl groups, both of which presumably contributed to enthalpy stabilization. There was a strong correlation between the number of water molecules and the thermodynamic parameters. Overall, our results demonstrate that, in contrast to our initial expectation, a protein sequence in which over 40% of the residues are alanines can retain a densely packed structure and undergo thermal denaturation with a large enthalpy change, mainly contributed by hydration.

A hetero-micro-seeding strategy for readily crystallizing closely related protein variants

Protein crystallization remains difficult to rationalize and screening for optimal crystallization conditions is a tedious and time consuming procedure. Here, we report a hetero-micro-seeding strategy for producing high resolution crystals of closely related protein variants, where micro crystals from a readily crystallized variant are used as seeds to develop crystals of other variants less amenable to crystallization. We applied this strategy to Bovine Pancreatic Trypsin Inhibitor (BPTI) variants, which would not crystallize using standard crystallization practice. Out of six variants in our analysis, only one called BPTI-[5,55]A14G formed well behaving crystals; and the remaining five (A14GA38G, A14GA38V, A14GA38L, A14GA38I, and A14GA38K) could be crystallized only using micro-seeds from the BPTI-[5,55]A14G crystal. All hetero-seeded crystals diffracted at high resolution with minimum mosaicity, retaining the same space group and cell dimension. Moreover, hetero-micro-seeding did not introduce any biases into the mutants structure toward the seed structure, as demonstrated by A14GA38I structures solved using micro-seeds from A14GA38G, A14GA38L and A14GA38I. Though hetero-micro-seeding is a simple and almost naïve strategy, this is the first direct demonstration of its workability. We believe that hetero-micro-seeding, which is contrasting with the popular idea that crystallization requires highly purified proteins, could contribute a new tool for rapidly solving protein structures in mutational analysis studies.

Prevalence and assessment of biochemical parameters of attention-deficit hyperactivity disorder children in Bangladesh.

Background and Objectives: This study aimed to evaluate some new biochemical parameters that help ensuring the early and precise diagnosis of attention-deficit hyperactivity disorder (ADHD) in blood plasma. Design and Settings: A prospective study conducted with patients scheduled for some new biochemical parameters that help ensuring the early and precise diagnosis of ADHD in blood plasma in a Child Development Center of the Chittagong, Bangladesh. Materials and Methods: The study was carried out at two levels. The first level was questionnaire on personal data and disease history while the second was on biochemical examination of the plasma ammonia and lactate status. A total of 100 children (age range 2 years 4 months to 12 years 6 months, mean age 7 years 5 months) were investigated in this study among 75 were male and 25 were female. This study was conducted in Chittagong Maa-O-Shishu General Hospital, Bangladesh. Results: We observed that the level of plasma ammonia and lactate were higher in ADHD children (36-60 μmol/L; P < 0.05 and 22-30 μmol/L; P < 0.05, respectively) compare to a reference value. The prevalence of ADHD is higher in male (75%) than in female (25%) with a ratio of 3:1. Consanguinity increases the risk of having ADHD in the next generation. Conclusion: This study concludes that there might be a correlation between ADHD and increased level of plasma ammonia and lactate level, and those might be an important parameter in the diagnosis of ADHD patients.