CHARMM (Chemistry at HARvard Molecular Mechanics) is a highly versatile and widely used molecu- lar simulation program. It has been developed over the last three decades with a primary focus on molecules of bio- logical interest, including proteins, peptides, lipids, nucleic acids, carbohydrates, and small molecule ligands, as they occur in solution, crystals, and membrane environments. For the study of such systems, the program provides a large suite of computational tools that include numerous conformational and path sampling methods, free energy estima- tors, molecular minimization, dynamics, and analysis techniques, and model-building capabilities. The CHARMM program is applicable to problems involving a much broader class of many-particle systems. Calculations with CHARMM can be performed using a number of different energy functions and models, from mixed quantum mechanical-molecular mechanical force fields, to all-atom classical potential energy functions with explicit solvent and various boundary conditions, to implicit solvent and membrane models. The program has been ported to numer- ous platforms in both serial and parallel architectures. This article provides an overview of the program as it exists today with an emphasis on developments since the publication of the original CHARMM article in 1983.

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CHARMM: the biomolecular simulation program.

BIOE 402. Medical Technology Assessment. 2 or 3 hours. Bioentrepreneur course. Assessment of medical technology in the context of commercialization. Objectives, competition, market share, funding, pricing, manufacturing, growth, and intellectual property; many issues unique to biomedical products. Course Information: 2 undergraduate hours. 3 graduate hours. Prerequisite(s): Junior standing or above and consent of the instructor.

“Bioinformatics” 특집을 내면서

中枢神経系疾患の治療は正常細胞（ニューロン）の機能維持を目的とするが，脳血管障害のように機能障害の原因が細胞の死滅に基づくことは多い．一方，脳腫瘍の治療においては薬物療法や放射線療法といった腫瘍細胞の死滅を目標とするものが大きな位置を占める．いずれの場合にも，細胞死の機序を理解することは各種病態や治療法の理解のうえで重要である．現在のところ最も研究の進んでいる細胞死の型はアポトーシスである．そのなかで重要な位置を占めるミトコンドリアにおける反応および抗アポトーシス因子について概要を紹介する．

Neuroscience 細胞死：最近の知見

Much has been written about theory and practice in the law, and the tension between practitioners and theorists. Judges do not cite theoretical articles often; they rarely "apply" theories to particular cases. These arguments are not revisited. Instead the Essay explores the working and interaction of theory and practice, practitioners and theorists. The Essay starts with a story about solving a legal issue using our intellectual tools - theory, practice, and their progenies: experience and "gut." Next the Essay elaborates on the nature of theory, practice, experience and "gut." The third part of the Essay discusses theories that are helpful to practitioners and those that are less helpful. The Essay concludes that practitioners theorize, and theorists practice. They use these intellectual tools differently because the goals and orientations of theorists and practitioners, and the constraints under which they act, differ. Theory, practice, experience and "gut" help us think, remember, decide and create. They complement each other like the two sides of the same coin: distinct but inseparable.

Of Theory and Practice

Amyloidosis affects millions of people, as a cause of Alzheimer's disease or a complication of dialysis, and also causes rare conditions. The many forms of the disorder have one underlying principle: misfolded proteins. Prompt, correct diagnosis is essential, especially in the inherited forms of amyloidosis. This article reviews the molecular basis of various types of amyloidosis and proposes new ways of treating these disorders.

Molecular mechanisms of amyloidosis.

β2-microglobulin (β2-m) is the nonpolymorphic light chain of the class I major histocompatibility complex (MHC-I). It consists of 99 residues with a single disulfide bridge between the two Cys residues of the sequence at positions 25 and 80 and folds into the classical β-sandwich motif of the immunoglobulin superfamily, as shown by the crystal structure of MHC-I (Bjorkman et al. 1987). The systemic deposition of β2-m fibrils is associated to dialysis-related amyloidosis (DRA; Gejyo et al. 1985), a disease which arises in individuals with chronic renal failure as the inescapable complication of long-term hemodialysis. More than 90% of patients undergoing dialysis for ∼10 y develop symptoms such as destructive arthropathy, bone fractures, and carpal tunnel. Because of the ensuing physical disabilities, against which treatment (including surgery) is only palliative, and the widespread occurrence of renal failure (∼200,000 individuals are steadily under dialysis in Europe alone), this type of amyloidosis can be regarded as a high-cost social disease.

Amyloidoses have been recognized as conformational diseases that arise from the conversion of globular proteins to insoluble fibrillar aggregates. In spite of the diversity of the involved proteins, their amyloid fibrils share a common structure known as cross-β structure, in which partially unfolded conformations aggregate through intermolecular β-strand pairing that are arranged perpendicular to the fiber polymerization axis (Sunde et al. 1997).

Analyses of the material extracted from DRA patients have shown that only full-length wild-type β2-m and proteolysis products thereof occur in fibrils (Linke et al. 1987, 1989; Bellotti et al. 1998), along with some glycation (Miyata et al. 1993) and oxidation derivatives (Capeiller-Blandin et al. 1991) and auxiliary proteins (apoE, serum amyloid P component; Miyata et al. 1993). The most abundant species among the truncation products of β2-m (30% of β2-m in ex vivo fibrils) is the form devoid of the six N-terminal residues (ΔN6β2-m). This species was shown to have a higher tendency to self-aggregate than the full-length protein and did not form a fully folded native state at the end of the refolding procedure (Bellotti et al. 1998). A comparative investigation of full-length β2-m and ΔN6β2-m by 1H NMR, electrospray mass spectrometry, and limited proteolysis enabled us to establish the extent and the location of the structural modifications undergone by β2-m upon removal of the N-terminal hexapeptide (Esposito et al. 2000). By analyzing the changes and the persistences of the NMR pattern of both products, some features of the amyloidogenic conformation of β2-m could be suggested, namely, the identity of the β-strands involved in the intermolecular pairing and a model to explain partial unfolding and aggregation of the truncated and full-length species (Esposito et al. 2000). In a subsequent study on the fibrils of β2-m and ΔN6β2-m, overlapping patterns of limited proteolysis were observed with an additional cleavage for the full-length protein fibrils leading to the truncated species (M. Monti et al. 2001, pers. comm.). These findings, while suggesting a mechanism for the occurrence of ΔN6β2-m in vivo, which implies that the only fibrillogenic species is the full-length protein, strongly support the idea that the truncated species features the amyloidogenic conformation of β2-m, as previously anticipated (Esposito et al. 2000; Chiti et al. 2001). Going further along this rationale, we wondered which could be the structural characteristics that drive the transition of β2-m into an amyloidogenic conformation. The protein occurs naturally in the MHC-I complex and is lost from the cell surface into the plasma to reach the kidneys and eventually to be degraded. When the renal function is compromised and dialysis becomes necessary, only a considerable increase of its plasma concentration is steadily detected (25- to 35-fold; Gejyo et al. 1985) before deposition of amyloid fibrils. Although the isolated protein in solution has been studied by NMR (Okon et al. 1992), only a qualitative analysis of its secondary structure was reported that showed, however, a very close analogy with the crystal structure of the protein in the context of the MHC-I complex (Bjorkman et al. 1987; Saper et al., 1991). Because even minor deviations from the crystal structure may affect significantly the stability of a protein that naturally binds to the polymorphic component of human leukocyte antigen, a determination of the tertiary structure of isolated β2-m in solution is useful, provided the proper level of accuracy is reached to perform a meaningful comparison with the crystal structure. Here we present the 3D solution structure of isolated β2-m obtained from 1H NMR data (Protein Data Bank ID 1JNJ) and show that differences with respect to the crystal structure are indeed observed.

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The solution structure of human β2-microglobulin reveals the prodromes of its amyloid transition

Collagen Plays an Active Role in the Aggregation of β2-Microglobulin under Physiopathological Conditions of Dialysis-related Amyloidosis

Size and shape of sodium deoxycholate micellar aggregates

A /sup 1/H NMR study of the peptide alamethicin, which forms voltage-gated ion channels in membranes, is described. The molecule was studied in methanol as a function of temperature and pH. A complete assignment of the spectra is given, including several stereospecific assignments. Alamethicin was found to have a structure substantially similar to the crystal although, in solution, the C-terminal dipeptide adopts a somewhat extended conformation. The overall conformation was insensitive to the ionization of the side chain of the ionizable group, Glu-18.

High-Resolution 1H NMR Study of the Solution Structure of Alamethicin

Under conditions of cellular stress, small heat shock proteins (sHsps), e.g. Hsp25, stabilize unfolding proteins and prevent their precipitation from solution. 1H NMR spectroscopy has shown that mammalian sHsps possess short, polar and highly flexible C-terminal extensions. A mutant of mouse Hsp25 without this extension has been constructed. CD spectroscopy reveals some differences in secondary and tertiary structure between this mutant and the wild-type protein but analytical ultracentrifugation and electron microscopy show that the proteins have very similar oligomeric masses and quaternary structures. The mutant shows chaperone ability comparable to that of wild-type Hsp25 in a thermal aggregation assay using citrate synthase, but does not stabilize α-lactalbumin against precipitation following reduction with dithiothreitol. The accessible hydrophobic surface of the mutant protein is less than that of the wild-type protein and the mutant is also less stable at elevated temperature. 1H NMR spectroscopy reveals that deletion of the C-terminal extension of Hsp25 leads to induction of extra C-terminal flexibility in the molecule. Monitoring complex formation between Hsp25 and dithiothreitol-reduced α-lactalbumin by 1H NMR spectroscopy indicates that the C-terminal extension of Hsp25 retains its flexibility during this interaction. Overall, these data suggest that a highly flexible C-terminal extension in mammalian sHsps is required for full chaperone activity.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1432-1327.2000.01188.x

Gennaro Esposito

Papers

The solution structure of human β2-microglobulin reveals the prodromes of its amyloid transition

Collagen Plays an Active Role in the Aggregation of β2-Microglobulin under Physiopathological Conditions of Dialysis-related Amyloidosis

Size and shape of sodium deoxycholate micellar aggregates

High-Resolution 1H NMR Study of the Solution Structure of Alamethicin

Mouse Hsp25, a small heat shock protein