Quantitative parameters for amino acid-base interaction: implications for prediction of protein-DNA binding sites.

Inspection of the amino acid-base interactions in protein-DNA complexes is essential to the understanding of specific recognition of DNA target sites by regulatory proteins. The accumulation of information on protein-DNA co-crystals challenges the derivation of quantitative parameters for amino acid-base interaction based on these data. Here we use the coordinates of 53 solved protein-DNA complexes to extract all non-homologous pairs of amino acid-base that are in close contact, including hydrogen bonds and hydrophobic interactions. By comparing the frequency distribution of the different pairs to a theoretical distribution and calculating the log odds, a quantitative measure that expresses the likelihood of interaction for each pair of amino acid-base could be extracted. A score that reflects the compatibility between a protein and its DNA target can be calculated by summing up the individual measures of the pairs of amino acid-base involved in the complex, assuming additivity in their contributions to binding. This score enables ranking of different DNA binding sites given a protein binding site and vice versa and can be used in molecular design protocols. We demonstrate its validity by comparing the predictions using this score with experimental binding results of sequence variants of zif268 zinc fingers and their DNA binding sites.     

Reactions of selenium dioxide free radicals with amino acids and enzymes

Pulse radiolysis of selenium dioxide in aqueous solution has shown the presence of three selenite radicals in acid-base equilibrium within well defined pH ranges: (formula; see text) The selenite radicals react selectively with amino acids, preferentially with the aromatic ones in the order tryptophan greater than tyrosine greater than histidine, independently of the acid-base structure of the radical. Kinetic and spectroscopic data on the reaction of selenite radicals with some proteins and parallel inactivation studies generally reflect knowledge on the amino acid residues mainly involved in the radical attack. The investigations at different pH values on the reactivity of selenite radicals with amino acids and proteins and on the transient spectra of the reaction products exhibit different behaviour for the various acid-base structures of the selenite radicals, reflecting the influence of particular ionizable groups in the reacting molecules and the structure modifications at the level of proteins.     

Isolation of γ-globulin (IgG) from serum: a task that encourages students to consolidate concepts on protein structure and properties

The isolation of γ-globulin from bovine serum by salt precipitation and fractionation through ion-exchange chromatography and gel filtration is proposed as a method to teach protein structure and properties. Students are instructed to evaluate the efficiency of the isolation procedure through SDS-PAGE analysis of protein fractions. Simple techniques to measure antigen-antibody reaction may be used to check preservation of IgG function. Questions are given to get the students to relate protein structure and physical properties and their use in protein purification and quantification. This teaching approach is efficient in identifying misunderstanding of concepts, to follow up students' development and to reinforce ‘learned concepts’.     

The pentose phosphate pathway in the yeasts Saccharomyces cerevisiae and Kloeckera apiculata,an exercise in comparative metabolism for food and wine science students

Comparative cellular metabolism can be a difficult area of biochemistry to teach in the undergraduate laboratory class. Student practicals involving animal tissues generally require approval from animal ethic committees, and the relevance for students whose primary interest in biochemistry is in the area of food and wine sciences, is often questioned. In this report, we present an undergraduate practical exercise in which glucose catabolism via the pentose phosphate pathway is compared in two types of yeast with direct relevance to the wine and food industries, Saccharomyces cerevisiae and Kloeckera apiculata. The exercise is carried out as a demonstration to second year undergraduate students, studying metabolic biochemistry. It is of some value in that it illustrates comparative cellular metabolism in wine yeasts and introduces the students to the safe use of radioisotopes.     

Comparison of secretory protein profiles in developing rat pancreatic rudiments and rat acinar tumor cells

Chemical modification of amino groups in matrix porin solubilized and purified from outer membranes of Escherichia coli in beta- octylglucoside was performed with eosin isothiocyanate and citraconic anhydride. At pH 7 8.5, the former reagent labeled a single amino group in the native protein, while more extensive derivatization was observed with increasing pH or upon denaturation. Citraconic anhydride modified approximately 12-14 residues in native porin and 15-16 of the total of 19 amino groups in the denatured state. Fluorescamine, another amine- specific reagent of intermediate size, derivatized 3 and 16 residues in the native and denatured states, respectively. These results indicate that reactive probes of various sizes may serve as indicators for the surface accessibility of reactive residues in matrix porin. The increased derivatization of lysyl residues at high pH (or in phosphate buffer) suggests the method's sensitivity to different conformational states of the protein. The extent of tyrosine modification (1-2 residues in the native, and approximately 22 in the denatured porin) depended on the state of protein folding, even with reagents of small size. The approach of using various probes with differing properties and specificities thus appears useful for the determination of membrane protein asymmetry, pore topology, and conformational states of transmembrane proteins.     

生物化学教学实用技巧和策略

生物化学是生命科学中最重要的基础专业课程之一。欲使教师讲好它,学生学好并非易事。在学生中经常流传着一句“生理生化,必有一挂”的口头禅,足见学生学习生物化学的难度。本文结合笔者在南京大学讲授生物化学25年多的实践及成功经验,与同行们分享生物化学的教学技巧和策略,如何上好第一次课,如何激发学生学习生物化学的兴趣,如何在教学中培养学生的科学思维,如何把传统课堂教学的手段与其他新型的教学方式结合,如何开展和组织“第二课堂”活动等,希望它们对同行们的生物化学教学有所启发和帮助。     

生物化学教学实用技巧和策略

生物化学是生命科学中最重要的基础专业课程之一。欲使教师讲好它,学生学好并非易事。在学生中经常流传着一句“生理生化,必有一挂”的口头禅,足见学生学习生物化学的难度。本文结合笔者在南京大学讲授生物化学25年多的实践及成功经验,与同行们分享生物化学的教学技巧和策略,如何上好第一次课,如何激发学生学习生物化学的兴趣,如何在教学中培养学生的科学思维,如何把传统课堂教学的手段与其他新型的教学方式结合,如何开展和组织“第二课堂”活动等,希望它们对同行们的生物化学教学有所启发和帮助。     

A single protein research integrated advanced biochemistry laboratory course; spectroscopic determination of tyrosyl side chain pKa

The unique bio-analytical properties of the amino acid tyrosine (Tyr) are the focus of this experiment from the research oriented biochemistry laboratory course at our university. In the present study pK(a(1)), pK(a(2)), and pK(a(3)) values for free Tyr were estimated to be 2.30, 9.40, and 9.97, respectively, when free Tyr was titrated with 1mM NaOH and 1mM HCl using a pH meter. Spectrophotometric analysis of the phenolic side chain pK(a(3)) revealed a value of 10.14, which was consistent with the pK(a)s estimated from the pH meter. The results from this experiment will allow students to compare the free Tyr properties with those present in a protein.     

A suggested module detailing biopharmaceuticals suitable for inclusion in undergraduate applied biochemistry/biochemistry programs

A 26 lecture module is outlined which details the biochemistry and biotechnology of biopharmaceutical substances. It is designed to equip students undertaking programs in applied biochemistry/biochemistry with an understanding of concepts, both academic and applied, directly relevant to working in the biopharmaceutical sector. In addition to the syllabus, a bank of relevant resource material is provided.     

Undergraduate structural biology education: A shift from users to developers of computation and simulation tools

The use of theory and simulation in undergraduate education in biochemistry, molecular biology, and structural biology is now common, but the skills students need and the curriculum instructors have to train their students are evolving. The global pandemic and the immediate switch to remote instruction forced instructors to reconsider how they can use computation to teach concepts previously approached with other instructional methods. In this review, we survey some of the curricula, materials, and resources for instructors who want to include theory, simulation, and computation in the undergraduate curriculum. There has been a notable progression from teaching students to use discipline-specific computational tools to developing interactive computational tools that promote active learning to having students write code themselves, such that they view computation as another tool for solving problems. We are moving toward a future where computational skills, including programming, data analysis, visualization, and simulation, will no longer be considered an optional bonus for students but a required skill for the 21st century STEM (Science, Technology, Engineering, and Mathematics) workforce; therefore, all physical and life science students should learn to program in the undergraduate curriculum.     

Amino acid biosynthesis in plants: Approaching an understanding at the molecular level

Summary The genes encoding GS and EPSP synthase have already been cloned. Clones containing portions of the genes encoding aspartate amino transferase and asparagine synthetase tentatively have been identified by this and other laboratories. It is certain that many other genes encoding other important enzymes involved in amino acid biosynthesis will also be identified in the near future. It is evident that new techniques for the separation of proteins will greatly aid in identifying low abundance genes more rapidly and efficiently. Micro techniques need to be further developed and the necessary equipment made available to laboratories. Hopefully the cost of the required equipment will decrease and commercial enterprises will offer more contract services for work requiring the more expensive equipment. Graduate training in advanced techniques of separating proteins at high resolutions should be encour-aged expanded so that our future scientists are well versed in protein biochemistry as well as in techniques of molecular biology.     

Contribution of proton linkage to the thermodynamic stability of the major cold-shock protein of Escherichia coli CspA

The stability of protein is defined not only by the hydrogen bonding, hydrophobic effect, van der Waals interactions, and salt bridges. Additional, much more subtle contributions to protein stability can arise from surface residues that change their properties upon unfolding. The recombinant major cold shock protein of Escherichia coli CspA an all-beta protein unfolds reversible in a two-state manner, and behaves in all other respects as typical globular protein. However, the enthalpy of CspA unfolding strongly depends on the pH and buffer composition. Detailed analysis of the unfolding enthalpies as a function of pH and buffers with different heats of ionization shows that CspA unfolding in the pH range 5.5-9.0 is linked to protonation of an amino group. This amino group appears to be the N-terminal alpha-amino group of the CspA molecule. It undergoes a 1.6 U shift in pKa values between native and unfolded states. Although this shift in pKa is expected to contribute approximately 5 kJ/mol to CspA stabilization energy the experimentally observed stabilization is only approximately 1 kJ/mol. This discrepancy is related to a strong enthalpy-entropy compensation due, most likely, to the differences in hydration of the protonated and deprotonated forms of the alpha-amino group.     

SAmBA: An interactive software for optimizing the design of biological macromolecules crystallization experiments

SAmBA is a new software for the design of minimal experimental protocols using the notion of orthogonal arrays of strength 2. The main application of SAmBA is the search of protein crystallization conditions. Given a user input defining the relevant effectors/variables (e.g., pH, temperature, salts) and states (e.g., pH: 5, 6, 7 and 8), this software proposes an optimal set of experiments in which all tested variables and the pairwise interactions between them are symmetrically sampled. No a priori restrictions on the number and range of experimental variables is imposed. SAmBA consists of two complementary programs, SAm and BA, using a simulated annealing approach and a backtracking algorithm, respectively. The software is freely available as C code or as an interactive JAVA applet at http://igs-server.cnrs-mrs.fr . Proteins 29:252–257, 1997. © 1997 Wiley-Liss, Inc.     

Analysis on folding of misgurin using two-dimensional HP model

Misgurin is an antimicrobial peptide from the loach, while the hydrophobic-polar (HP) model is a way to study the folding conformations and native states in peptide and protein although several amino acids cannot be classified either hydrophobic or polar. Practically, the HP model requires extremely intensive computations, thus it has yet to be used widely. In this study, we use the two-dimensional HP model to analyze all possible folding conformations and native states of misgurin with conversion of natural amino acids according to the normalized amino acid hydrophobicity index as well as the shortest benchmark HP sequence. The results show that the conversion of misgurin into HP sequence with glycine as hydrophobic amino acid at pH 2 has 1212 folding conformations with the same native state of minimal energy -6; the conversion of glycine as polar amino acid at pH 2 has 13,386 folding conformations with three native states of minimal energy -5; the conversion of glycine as hydrophobic amino acid at pH 7 has 2538 folding conformations with three native states of minimal energy -5; and the conversion of glycine as polar amino acid at pH 7 has 12,852 folding conformations with three native states of minimal energy -4. Those native states can be ranked according to the normalized amino acid hydrophobicity index. The detailed discussions suggest two ways to modify misgurin.     

PBL 教学法在成教班生物化学教学中的应用体会

生物化学是一门"教师难教、学生难学"的医学基础课程,利用成人继续教育学生具备一定的临床经验的优势,将PBL教学法应用于成人继续教育乡医班生物化学教学中,激发了学生的学习兴趣和主动性,提高了学生分析问题和解决问题的能力,同时对教师综合素质提出了更高的要求。与传统教学法相比,PBL教学能够达到学生学与教师教的相互促进,提高了教学效果,符合新形势下教学改革的需求。     

Botanical literacy: What and how should students learn about plants?

Botanists benefit from a scientifically literate society and an interested and botanically literate student population, and we have opportunities to promote literacy in our classes. Unfortunately, scientific illiteracy exists, in part, because students are technologically advanced but lack intellectual curiosity and rigor. Botanical illiteracy results from several interacting factors, including a lack of interest in plants and infrequent exposure to plant science before students reach college. If scientific or botanical literacy is a goal, we must understand what literacy means and how we can help students reach that goal. A model of biological literacy recognizes four levels; students enter courses at the lowest level possessing misconceptions about concepts; however, misconceptions can be used to our advantage, especially by using concept inventories. Inquiry-based instruction is advocated for all science courses, and learning theory supports inquiry. Seven principles of learning inform recommendations about how botanists should teach, including using themes and "thinking botanically" to illustrate all biological concepts. Overall, consideration of the botanical content taught is less critical than the methods used to teach that content. If botanists emphasize thinking and process skills with an understanding of concepts, we will prepare scientifically literate students and citizens and benefit from our efforts.     

pH-induced conformational changes in the soluble manganese-stabilizing protein of photosystem II

In this paper, we analyzed the pH-induced changes in the conformational states of the manganese-stabilizing protein (MSP) of photosystem II. Distinct conformational states of MSP were identified using fluorescence spectra, far-UV circular dichroism, and pressure-induced unfolding at varying suspension pH values, and four different conformational states of MSP were clearly distinguished using the center of fluorescence spectra mass when suspension pH was altered from 2 to 12. MSP was completely unfolded at a suspension pH above 11 and partly unfolded below a pH of 3. Analysis of the center of fluorescence spectral mass showed that the MSP structure appears stably folded around pH 6 and 4. The conformational state of MSP at pH 4 seems more stable than that at pH 6. Studies of peak positions of tryptophan fluorescence and MSP-bound 1-anilinonaphthalene-8-sulfonic acid fluorescence spectra supported this observation. A decrease in the suspension pH to 2 resulted in significant alterations in the MSP structure possibly because of protonation of unprotonated residues at lower pH, suggesting the existence of a large number of unprotonated amino acid residues at neutral pH possibly useful for proton transport in oxygen evolution. The acidic pH-induced conformational changes of MSP were reversible upon increase of pH to neutral pH; however, N-bromosuccinimide modification of tryptophan (Trp241) blocks the recovery of pH-induced conformational changes in MSP, implying that Trp241 is a key residue for the unfolded protein to form a functional structure. Thus, pH-induced structural changes of stable MSP (pH 6-4) may be utilized to analyze its functionality as a cofactor for oxygen evolution.     

Structural dynamics, stability and folding of proteins

The present concepts of protein folding in vitro are reviewed. According to these concepts, amino acid sequence of protein, which has appeared a result of evolutionary selection, determines the native structure of protein, the pathway of protein folding, and the existence of free energy barrier between native and denatured states of protein. The latter means that protein macromolecule can exist in either native or denatured state. And all macromolecules in the native state are identical but for structural fluctuations due to Brownian motion of their atoms. Identity of all molecules in native state is of primary importance for their correct functioning. The dependence of protein stability, which is measured as the difference between free energy of protein in native and denatured states, on temperature and denaturant concentration is discussed. The modern approaches characterizing transition state and nucleation are regarded. The role of intermediate and misfolded states in amorphous aggregate and amyloid fibril formation is discussed.