Stoichiometry and Topology in Protein Folding

Abstract

Crystals of the small ribosomal subunit from Thermus thermophilus diffract to 3Å and exhibit reasonable isomorphism and moderate resistance to irradiation. A 5Å MIR map of this particle shows a similar shape to the part assigned to this particle within the cryo-EM reconstructions of the whole ribosome and contains regions interpretable either as RNA chains or as protein motifs. To assist phasing at higher resolution we introduced recombinant methods aimed at extensive selenation for MAD phasing. We are focusing on several ribosomal proteins that can be quantitatively detached by chemical means. These proteins can be modified and subsequently reconstituted into depleted ribosomal cores. They also can be used for binding heavy atoms, by incorporating chemically reactive binding sites, such as -SH groups, into them. In parallel we are co-crystallizing the ribosomal particles with tailor made ligands, such as antibiotics or cDNA to which heavy-atoms have been attached or diffuse the latter compounds into already formed crystals.     

Cunning Simplicity of a Stoichiometry Driven Protein Folding Thesis

Abstract

Mastoparan B (MP-B) is an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis. NMR spectroscopy was used to study the membrane associated structures of MP-B in various model membrane systems such as 120 mM DPC micelles, 200 mM SDS micelles, and 3%(w/v) DMPC/DHPC (1:2) bicelles. In all systems, MP-B has an amphiphilic α-helical structure from Lys² to Leu¹⁴. NOESY experiments performed on MP-B in nondeuterated SDS micelles show that protons in the indole ring of Trp⁹ are in close contact with methylene protons of SDS micelles. T₁ relaxation data and NOE data revealed that the bound form of MP-B may be dominant in SDS micelles. The interactions between MP-B and zwitterionic DPC micelles were much weaker than those between MP-B and anionic SDS micelles. By substitution of Trp⁹ with Ala⁹, the pore-forming activity of MP-B was decreased dramatically. All of these results imply that strong electrostatic interactions between the positively charged Lys residues in MP-B and the anionic phospholipid head groups must be the primary factor for MP-B binding to the cell membrane. Then, insertion of the indole ring of Trp⁹ into the membrane, as well as the amphiphilic α-helical structures of MP-B may allow MP-B to span the lipid bilayer through the C-terminal portion. These structural features are crucial for the potent antibiotic activities of MP-B.     

蛋白质的折叠

重点介绍了蛋白质折叠的热力学控制学说和动力学控制学说,简单介绍了几种蛋白质折叠模型并分析了多肽链在体内进行快速折叠的原因。     

Cotranslational Protein Folding

The review analyzes the research concerning the folding of proteins in the course of their synthesis on ribosomes. The experimental data obtained for various proteins using various methods give grounds for concluding that a nascent protein largely acquires its spatial structure while still attached to the ribosome, and final folding into the biologically active conformation takes place as soon as the completed protein is released therefrom. Cotranslational folding is characteristic of both bacterial and eukaryotic cells, and appears to be the universal and the most evolutionarily ancient mechanism.     

Chaperonin-mediated Protein Folding

We have been studying chaperonins these past twenty years through an initial discovery of an action in protein folding, analysis of structure, and elucidation of mechanism. Some of the highlights of these studies were presented recently upon sharing the honor of the 2013 Herbert Tabor Award with my early collaborator, Ulrich Hartl, at the annual meeting of the American Society for Biochemistry and Molecular Biology in Boston. Here, some of the major findings are recounted, particularly recognizing my collaborators, describing how I met them and how our great times together propelled our thinking and experiments.     

First-Principles Protein Folding Simulations

Abstract

It is widely believed that the prediction of the three-dimensional structures of proteins from the first principles is impossible. This view is based on the fact that the number of possible structures for each protein is astronomically large. The question is then why a protein folds into its native structure with the proper biological functions in the time scale of milliseconds to minutes, and this is called Levinthal's paradox. In this article I will discuss our strategy for attacking the protein folding problem. Our approach consists of two elements: the inclusion of accurate solvent effects and the development of powerful simulation algorithms that can avoid getting trapped in states of energy local minima. For the former, we discuss several models varying in nature from crude (distance-dependent dielectric function) to rigorous (reference interaction site model). For the latter, we show the effectiveness of Monte Carlo simulated annealing and generalized-ensemble algorithms.     

Ultrafast Protein Folding in Membrane-Mimetic Environments

Proteins fold on timescales from hours to microseconds. In addition to protein size, sequence, and topology, the environment represents an equally important factor in determining folding speed. This is particularly relevant for proteins that require a lipid membrane or a membrane mimic to fold. However, only little is known about how properties of such a hydrophilic/hydrophobic interface modulate the folding landscape of membrane-interacting proteins. Here, we studied the influence of different membrane-mimetic micellar environments on the folding and unfolding kinetics of the helical-bundle protein Mistic. Devising a single-molecule fluorescence spectroscopy approach, we extracted folding and unfolding rates under equilibrium conditions and dissected the contributions from different detergent moieties to the free-energy landscape. While both polar and nonpolar moieties contribute to stability, they exert differential effects on the free-energy barrier: Hydrophobic burial stabilizes the folded state but not the transition state in reference to a purely aqueous environment; by contrast, zwitterionic headgroup moieties stabilize the folded state and, additionally, lower the free-energy barrier to accelerate the folding of Mistic to achieve ultrafast folding times down to 35 μs.     

Programmed Trade-offs in Protein Folding Networks

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蛋白质折叠速率预测研究进展

蛋白质折叠速率预测是当今生物物理学最具挑战性的课题之一。近年来,该领域的研究取得了很大的进展,提出了许多经验参数,例如:接触序、长程序、总接触距离、链拓扑参数、二级结构含量、有效长度、螺旋参数、n-阶接触距离等。这些参数都和蛋白质的折叠速率有很好的相关性,基于这些参数的各种预测方法所得到的预测结果也与实验数据较好地吻合。     

Visualizing Protein Folding and Unfolding

Protein folding/unfolding is a complicated process that defies high-resolution characterization by experimental methods. As an alternative, atomistic molecular dynamics simulations are now routinely employed to elucidate and magnify the accompanying conformational changes and the role of solvent in the folding process. However, the level of detail necessary to map the process at high spatial–temporal resolution provides an overwhelming amount of data. As more and better tools are developed for analysis of these large data sets and validation of the simulations, one is still left with the problem of visualizing the results in ways that provide insight into the folding/unfolding process. While viewing and interrogating static crystal structures has become commonplace, more and different approaches are required for dynamic, interconverting, unfolding, and refolding proteins. Here we review a variety of approaches, ranging from straightforward to complex and unintuitive for multiscale analysis and visualization of protein folding and unfolding.     

Toward Correct Protein Folding Potentials

Empirical protein folding potentialfunctions should have a global minimum nearthe native conformationof globular proteins that fold stably, andthey should give the correct free energy offolding. We demonstrate that otherwise verysuccessful potentials fail to have even alocal minimumanywhere near the native conformation, anda seemingly well validated method ofestimatingthe thermodynamic stability of the nativestate is extremely sensitive to smallperturbations inatomic coordinates. These are bothindicative of fitting a great deal ofirrelevant detail. Here weshow how to devise a robust potentialfunction that succeeds very well at bothtasks, at least for alimited set of proteins, and this involvesdeveloping a novel representation of thedenatured state.Predicted free energies of unfolding for 25mutants of barnase are in close agreementwith theexperimental values, while for 17 mutantsthere are substantial discrepancies.     

蛋白质折叠和分子伴侣

一个有活性的蛋白质分子不但有特定的氨基酸序列,还处于特定的由氨基酸序列决定的三维空间结构。三维结构的完整性受到干扰,生物活性也会发生变化：有时即使只是轻微的破坏,都可能导致其生物活性全部丧失。所以蛋白质的生物功能是与其三维空间结构密切联系在一起的。     

Easily Searched Protein Folding Potentials

In order to calculate the tertiary structure of a protein from its amino acid sequence, the thermodynamic approach requires a potential function of sequence and conformation that has its global minimum at the native conformation for many different proteins. Here we study the behavior of such functions for the simplest model system that still has the essential features of the protein folding problem, namely two-dimensional square lattice chain configurations involving two residue types. First we demonstrate a method for accurately recovering the given contact potential from only a knowledge of which sequences fold to which structures and what the non-native structures are. Second, we show how to derive from the same information more general potential functions having much better positive correlations between potential function value and conformational deviation from the native. These functions consequently permit faster and more reliable searches for the native conformation, given the native sequence. Furthermore, the method for finding such potentials is easily applied to more realistic protein models.     

Experimental and Theoretical Protein Folding

Abstract

Resonance Raman spectra with Q-band excitation are reported for microperoxidase-11, the cytochrome c analog. Spectra were acquired in the mid-frequency range for the oxidized, and reduced forms of the undecapeptide, as well as for the imidazole and carbonyl complexes. Oxidation and spin state marker bands of the undecapeptides are consistent with a six-coordinate, low spin iron in both oxidation states. Porphyrin core size correlations yield a porphyrin-centre to pyrrole-nitrogen distance of 2.00 Å for MP11, suggestive of a six-coordinate species in a distorted heme environment. Molecular dynamics results show that the non-planarity of the heme of the parent cytochrome is conserved in the microperoxidase and its carbonmonoxy analog.     

蛋白质在大肠杆菌间周质中的折叠

随着分子伴侣的发现和外源基因表达技术的发展 ,大肠杆菌间周质蛋白质的折叠成为研究热点。间周质 (ｐｅｒｉｐｌａｓｍ)是革兰氏阴性菌内膜与外膜之间的区域 ,对外界环境变化的适当能力很脆弱 ,例如ｐＨ、温度、渗透压[1] 。重组技术表达的重要蛋白质大多含有二硫键 ,二硫键的形成在真核生物是在内质网中完成的 ,而大肠杆菌是在间周质中进行的。催化大肠杆菌间周质蛋白质折叠会遇到两个限速步骤 ,被两类酶催化 :二硫键氧化还原酶 (Ｄｓｂ)和肽酰 脯氨酰顺反异构酶 (ｐｅｐｔｉｄｙｌ ｐｒｏｌｙｌｃｉｓ ｔｒａｎｓｉｓｏ ｍｅｒａｓｅ ,…     

Protein Vivisection Reveals Elusive Intermediates in Folding

Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here, we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu → Glu⁻) to destabilize and unfold a specific region of the protein. We applied this strategy to ubiquitin, reversibly trapping a folding intermediate in which the β5-strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high-energy states.