Biochemistry Biochemistry最新文献_第10页

Recurrent Neurodevelopmentally Associated Variants of the Pre-mRNA Splicing Factor U2AF2 Alter RNA Binding Affinities and Interactions 前核糖核酸剪接因子 U2AF2 的神经发育相关变异改变了核糖核酸的结合亲和力和相互作用

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-10 DOI: 10.1021/acs.biochem.4c0034410.1021/acs.biochem.4c00344

Debanjana Maji, Jermaine L. Jenkins, Paul L. Boutz and Clara L. Kielkopf*,

{"title":"Recurrent Neurodevelopmentally Associated Variants of the Pre-mRNA Splicing Factor U2AF2 Alter RNA Binding Affinities and Interactions","authors":"Debanjana Maji, Jermaine L. Jenkins, Paul L. Boutz and Clara L. Kielkopf*, ","doi":"10.1021/acs.biochem.4c0034410.1021/acs.biochem.4c00344","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00344https://doi.org/10.1021/acs.biochem.4c00344","url":null,"abstract":"De novo mutations affecting the pre-mRNA splicing factor U2AF2 are associated with developmental delays and intellectual disabilities, yet the molecular basis is unknown. Here, we demonstrated by fluorescence anisotropy RNA binding assays that recurrent missense mutants (Arg149Trp, Arg150His, or Arg150Cys) decreased the binding affinity of U2AF2 for a consensus splice site RNA. Crystal structures at 1.4 Å resolutions showed that Arg149Trp or Arg150His disrupted hydrogen bonds between U2AF2 and the terminal nucleotides of the RNA site. Reanalysis of publicly available RNaseq data confirmed that U2AF2 depletion altered splicing of transcripts encoding RNA binding proteins (RBPs). These results confirmed that the impaired RNA interactions of Arg149Trp and Arg150His U2AF2 variants could contribute to dysregulating an RBP-governed neurodevelopmental program of alternative splicing.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"63 21","pages":"2718–2722 2718–2722"},"PeriodicalIF":2.9,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00344","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577533","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Delivery Aspects for Implementing siRNA Therapeutics for Blood Diseases. 实施血液病 siRNA 治疗的传输方面。

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-10 DOI: 10.1021/acs.biochem.4c00327

Saba Abbasi Dezfouli, Marcel E Michailides, Hasan Uludag

{"title":"Delivery Aspects for Implementing siRNA Therapeutics for Blood Diseases.","authors":"Saba Abbasi Dezfouli, Marcel E Michailides, Hasan Uludag","doi":"10.1021/acs.biochem.4c00327","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00327","url":null,"abstract":"Hematological disorders result in significant health consequences, and traditional therapies frequently entail adverse reactions without addressing the root cause. A potential solution for hematological disorders characterized by gain-of-function mutations lies in the emergence of small interfering RNA (siRNA) molecules as a therapeutic option. siRNAs are a class of RNA molecules composed of double-stranded RNAs that can degrade specific mRNAs, thereby inhibiting the synthesis of underlying disease proteins. Therapeutic interventions utilizing siRNA can be tailored to selectively target genes implicated in diverse hematological disorders, including sickle cell anemia, β-thalassemia, and malignancies such as lymphoma, myeloma, and leukemia. The development of efficient siRNA silencers necessitates meticulous contemplation of variables such as the RNA backbone, stability, and specificity. Transportation of siRNA to specific cells poses a significant hurdle, prompting investigations of diverse delivery approaches, including chemically modified forms of siRNA and nanoparticle formulations with various biocompatible carriers. This review delves into the crucial role of siRNA technology in targeting and treating hematological malignancies and disorders. It sheds light on the latest research, development, and clinical trials, detailing how various pharmaceutical approaches leverage siRNA against blood disorders, mainly concentrating on cancers. It outlines the preferred molecular targets and physiological barriers to delivery while emphasizing the growing potential of various therapeutic delivery methods. The need for further research is articulated in the context of overcoming the shortcomings of siRNA in order to enrich discussions around siRNA's role in managing blood disorders and aiding the scientific community in advancing more targeted and effective treatments.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142398687","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

KRas4b–Calmodulin Interaction with Membrane Surfaces: Role of Headgroup, Acyl Chain, and Electrostatics KRas4b-钙调蛋白与膜表面的相互作用：头基、酰基链和静电的作用

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-09 DOI: 10.1021/acs.biochem.4c0011610.1021/acs.biochem.4c00116

Shweta Shree, Mark A. McLean, Andrew G. Stephen and Stephen G. Sligar*,

{"title":"KRas4b–Calmodulin Interaction with Membrane Surfaces: Role of Headgroup, Acyl Chain, and Electrostatics","authors":"Shweta Shree, Mark A. McLean, Andrew G. Stephen and Stephen G. Sligar*, ","doi":"10.1021/acs.biochem.4c0011610.1021/acs.biochem.4c00116","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00116https://doi.org/10.1021/acs.biochem.4c00116","url":null,"abstract":"KRas4b is a small plasma membrane-bound G-protein that regulates signal transduction pathways. The interaction of KRas4b with the plasma membrane is governed by both its basic C-terminus, which is farnesylated and methylated, and the lipid composition of the membrane itself. The signaling activity of KRas4b is intricately related to its interaction with various binding partners at the plasma membrane, underlining the critical role played by the lipid environment. The calcium-binding protein calmodulin binds farnesylated KRas4b and plays an important role in the dynamic spatial cycle of KRas4b trafficking in the cell. We utilize Biolayer Interferometry to assay the role of lipid headgroup, chain length, and electrostatics in the dissociation kinetics of fully post-translationally modified KRas4b from Nanodisc bilayers with defined lipid compositions. Our results suggest that calmodulin promotes the dissociation of KRas4b from an anionic membrane, with a comparatively slower displacement of KRas4b from PIP2 relative to PS containing bilayers. In addition to this headgroup dependence, KRas4b dissociation appears to be slower from Nanodiscs wherein the lipid composition contains mismatched, unsaturated acyl chains as compared to lipids with a matched acyl chain length. These findings contribute to understanding the role of the lipid composition in the binding of KRas4b and release from lipid bilayers, showing that the overall charge of the bilayer, the identity of the headgroups present, and the length and saturation of the acyl chains play key roles in KRas4b release from the membrane, potentially providing insights in targeting Ras-membrane interactions for therapeutic interventions.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"63 21","pages":"2740–2749 2740–2749"},"PeriodicalIF":2.9,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577703","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Flavones Suppress Aggregation and Amyloid Fibril Formation of Human Lysozyme under Macromolecular Crowding Conditions. 黄酮类化合物抑制大分子拥挤条件下人溶菌酶的聚集和淀粉样纤维的形成

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-09 DOI: 10.1021/acs.biochem.4c00362

Shabnam, Rajiv Bhat

{"title":"Flavones Suppress Aggregation and Amyloid Fibril Formation of Human Lysozyme under Macromolecular Crowding Conditions.","authors":"Shabnam, Rajiv Bhat","doi":"10.1021/acs.biochem.4c00362","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00362","url":null,"abstract":"The crowded milieu of a biological cell significantly impacts protein aggregation and interactions. Understanding the effects of macromolecular crowding on the aggregation and fibrillation of amyloidogenic proteins is crucial for the treatment of many amyloid-related disorders. Most in vitro studies of protein amyloid formation and its inhibition by small molecules are conducted in dilute buffers, which do not mimic the complexity of the cellular environment. In this study, we used PEGs to simulate macromolecular crowding and examined the inhibitory effects of flavones DHF, baicalein, and luteolin on human lysozyme (HuL) aggregation at pH 2. Naturally occurring flavones have been effective inhibitors of amyloid formation in some proteins. Our findings indicate that while flavones inhibit HuL aggregation and fibrillation in dilute buffer solutions, complete inhibition is observed with a combination of flavones and PEGs, as shown by ThT fluorescence, light scattering, TEM, and AFM studies. The species formed in the presence of PEG 8000 and flavones were less hydrophobic, less toxic, and α-helix-rich compared to control samples, which were hydrophobic and β-sheet-rich, as demonstrated by ANS hydrophobicity, MTT assay, and CD spectroscopy. Fluorescence titration studies of flavones with HuL showed a significant increase in binding constant values under crowding conditions. These findings highlight the importance of macromolecular crowding in modulating protein aggregation and amyloid inhibition. Further studies using disease-causing mutants of HuL and other amyloidogenic proteins are needed to explore the role of macromolecular crowding in small-molecule-mediated modulation and inhibition of protein aggregation and amyloid formation.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":""},"PeriodicalIF":2.9,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386373","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Atomistic Insights into Sequence-Mediated Spontaneous Association of Short RNA Chains 从原子论角度洞察序列介导的短 RNA 链自发关联

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-08 DOI: 10.1021/acs.biochem.4c0029310.1021/acs.biochem.4c00293

Manas Mondal, and , Yi Qin Gao*,

{"title":"Atomistic Insights into Sequence-Mediated Spontaneous Association of Short RNA Chains","authors":"Manas Mondal,  and , Yi Qin Gao*, ","doi":"10.1021/acs.biochem.4c0029310.1021/acs.biochem.4c00293","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00293https://doi.org/10.1021/acs.biochem.4c00293","url":null,"abstract":"RNA–RNA association and phase separation appear to be essential for the assembly of stress granules and underlie RNA foci formation in repeat expansion disorders. RNA molecules are found to play a significant role in gene-regulatory functions via condensate formation among themselves or with RNA-binding proteins. The interplay between driven versus spontaneous processes is likely to be an important factor for controlling the formation of RNA-mediated biomolecular condensate. However, the sequence-specific interactions and molecular mechanisms that drive the spontaneous RNA–RNA association and help to form RNA-mediated phase-separated condensate remain unclear. With microseconds-long atomistic molecular simulations here, we report how essential aspects of RNA chains, namely, base composition, metal ion binding, and hydration properties, contribute to the association of the series of simplest biologically relevant homopolymeric and heteropolymeric short RNA chains. We show that spontaneous processes make the key contributions governed by the sequence-intrinsic properties of RNA chains, where the definite roles of base-specific hydrogen bonding and stacking interactions are prominent in the association of the RNA chains. Purine versus pyrimidine contents of RNA chains can directly influence the association properties of RNA chains by modulating hydrogen bonding and base stacking interactions. This study determines the impact of ionic environment in sequence-specific spontaneous association of short RNA chains, hydration features, and base-specific interactions of Na+, K+, and Mg2+ ions with RNA chains.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"63 21","pages":"2916–2936 2916–2936"},"PeriodicalIF":2.9,"publicationDate":"2024-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577654","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Pin1 WW Domain Ligand Library Synthesized with an Easy Solid-Phase Phosphorylating Reagent 用简易固相磷酸化试剂合成 Pin1 WW 结构域配体库

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-08 DOI: 10.1021/acs.biochem.4c0023110.1021/acs.biochem.4c00231

Xingguo R. Chen, Ana Y. Mercedes-Camacho, Kimberly A. Wilson, Jill J. Bouchard, Jeffrey W. Peng* and Felicia A. Etzkorn*,

{"title":"Pin1 WW Domain Ligand Library Synthesized with an Easy Solid-Phase Phosphorylating Reagent","authors":"Xingguo R. Chen, Ana Y. Mercedes-Camacho, Kimberly A. Wilson, Jill J. Bouchard, Jeffrey W. Peng* and Felicia A. Etzkorn*, ","doi":"10.1021/acs.biochem.4c0023110.1021/acs.biochem.4c00231","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00231https://doi.org/10.1021/acs.biochem.4c00231","url":null,"abstract":"Cell cycle regulatory enzyme Pin1 both catalyzes pSer/Thr-cis/trans-Pro isomerization and binds the same motif separately in its WW domain. To better understand the function of Pin1, a way to separate these activities is needed. An unnatural peptide library, R1CO–pSer–Pro–NHR2, was designed to identify ligands specific for the Pin1 WW domain. A new solid-phase phosphorylating reagent (SPPR) containing a phosphoramidite functional group was synthesized in one step from Wang resin. The SPPR was used in the preparation of the library by parallel synthesis. The final 315-member library was screened with our WW-domain-specific, enzyme-linked enzyme-binding assay (ELEBA). Four of the best hits were resynthesized, and the competitive dissociation constants were measured by ELEBA. NMR chemical-shift perturbations (CSP) of ligands with 15N-labeled Pin1 were used to measure Kd for the best four ligands directly, demonstrating that they were specific Pin1 WW domain ligands. Models of the ligands bound to the Pin1 WW domain were used to visualize the mode of binding in the WW domain.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"63 21","pages":"2803–2815 2803–2815"},"PeriodicalIF":2.9,"publicationDate":"2024-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.biochem.4c00231","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142577655","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Retinal Chromophore Configuration in the O Intermediate of Sensory Rhodopsin II from Natronomonas pharaonis Natronomonas pharaonis 的感官罗多普勒蛋白 II O 中间体的视网膜色团构型

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-08 DOI: 10.1021/acs.biochem.4c0042010.1021/acs.biochem.4c00420

Tomotsumi Fujisawa*, Nozomi Tanaka, Jun Tamogami and Masashi Unno,

引用次数: 0

Mammalian Esterase Activity: Implications for Peptide Prodrugs 哺乳动物的酯酶活性：对多肽原药的影响

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-03 DOI: 10.1021/acs.biochem.4c0044610.1021/acs.biochem.4c00446

Yana D. Petri, Ruben Verresen, Clair S. Gutierrez, Volga Kojasoy, Erika Zhang, Nile S. Abularrage, Evans C. Wralstad, Kaya R. Weiser and Ronald T. Raines*,

{"title":"Mammalian Esterase Activity: Implications for Peptide Prodrugs","authors":"Yana D. Petri, Ruben Verresen, Clair S. Gutierrez, Volga Kojasoy, Erika Zhang, Nile S. Abularrage, Evans C. Wralstad, Kaya R. Weiser and Ronald T. Raines*, ","doi":"10.1021/acs.biochem.4c0044610.1021/acs.biochem.4c00446","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00446https://doi.org/10.1021/acs.biochem.4c00446","url":null,"abstract":"As a traceless, bioreversible modification, the esterification of carboxyl groups in peptides and proteins has the potential to increase their clinical utility. An impediment is the lack of strategies to quantify esterase-catalyzed hydrolysis rates for esters in esterified biologics. We have developed a continuous Förster resonance energy transfer (FRET) assay for esterase activity based on a peptidic substrate and a protease, Glu-C, that cleaves a glutamyl peptide bond only if the glutamyl side chain is a free acid. Using pig liver esterase (PLE) and human carboxylesterases, we validated the assay with substrates containing simple esters (e.g., ethyl) and esters designed to be released by self-immolation upon quinone methide elimination. We found that simple esters were not cleaved by esterases, likely for steric reasons. To account for the relatively low rate of quinone methide elimination, we extended the mathematics of the traditional Michaelis–Menten model to conclude with a first-order intermediate decay step. By exploring two regimes of our substrate → intermediate → product (SIP) model, we evaluated the rate constants for the PLE-catalyzed cleavage of an ester on a glutamyl side chain (kcat/KM = 1.63 × 103 M–1 s–1) and subsequent spontaneous quinone methide elimination to regenerate the unmodified peptide (kI = 0.00325 s–1; t1/2 = 3.55 min). The detection of esterase activity was also feasible in the human intestinal S9 fraction. Our assay and SIP model increase the understanding of the release kinetics of esterified biologics and facilitate the rational design of efficacious peptide prodrugs.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"63 20","pages":"2580–2593 2580–2593"},"PeriodicalIF":2.9,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142436621","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Fingerprinting Tertiary Structure in Complex RNAs Using Single-Molecule Correlated Chemical Probing 利用单分子相关化学探针对复杂 RNA 的三级结构进行指纹识别

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-03 DOI: 10.1021/acs.biochem.4c0034310.1021/acs.biochem.4c00343

Ana C. Tan, Patrick S. Irving, Jordan T. Koehn, Shouhong Jin, David Y. Qiu and Kevin M. Weeks*,

{"title":"Fingerprinting Tertiary Structure in Complex RNAs Using Single-Molecule Correlated Chemical Probing","authors":"Ana C. Tan, Patrick S. Irving, Jordan T. Koehn, Shouhong Jin, David Y. Qiu and Kevin M. Weeks*, ","doi":"10.1021/acs.biochem.4c0034310.1021/acs.biochem.4c00343","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00343https://doi.org/10.1021/acs.biochem.4c00343","url":null,"abstract":"Single-molecule correlated chemical probing (smCCP) is an experimentally concise strategy for characterizing higher-order structural interactions in RNA. smCCP data yield rich, but complex, information about base pairing, conformational ensembles, and tertiary interactions. To date, through-space communication specifically measuring RNA tertiary structure has been difficult to isolate from structural communication reflective of other interactions. Here, we introduce mutual information as a filtering metric to isolate tertiary structure communication contained within smCCP data and use this strategy to characterize the structural ensemble of the SAM-III riboswitch. We identified an smCCP fingerprint that is selective for states containing a tertiary structure that forms concurrently with cognate ligand binding. We then successfully applied mutual information filters to independent RNAs and isolated through-space tertiary interactions in riboswitches and large RNAs with complex structures. smCCP, coupled with mutual information criteria, can now be used as a tertiary structure discovery tool, including to identify specific states in an ensemble that have a higher-order structure. These studies pave the way for the use of the straightforward smCCP experiment for discovery and characterization of tertiary structure motifs in complex RNAs.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"63 20","pages":"2648–2657 2648–2657"},"PeriodicalIF":2.9,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142448983","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Slow Conformational Exchange between Partially Folded and Near-Native States of Ubiquitin: Evidence for a Multistate Folding Model 泛素部分折叠态与近原态之间的缓慢构象交换：多态折叠模型的证据

IF 2.9 3区生物学

Biochemistry Biochemistry Pub Date : 2024-10-01 DOI: 10.1021/acs.biochem.4c0032110.1021/acs.biochem.4c00321

Sri Teja Adhada, and , Siddhartha P. Sarma*,

{"title":"Slow Conformational Exchange between Partially Folded and Near-Native States of Ubiquitin: Evidence for a Multistate Folding Model","authors":"Sri Teja Adhada,  and , Siddhartha P. Sarma*, ","doi":"10.1021/acs.biochem.4c0032110.1021/acs.biochem.4c00321","DOIUrl":"https://doi.org/10.1021/acs.biochem.4c00321https://doi.org/10.1021/acs.biochem.4c00321","url":null,"abstract":"The mechanism by which small proteins fold, i.e., via intermediates or via a two-state mechanism, is a subject of intense investigation. Intermediate states in the folding pathways of these proteins are sparsely populated due to transient lifetimes under normal conditions rendering them transparent to a majority of the biophysical methods employed for structural, thermodynamic, and kinetic characterization, which attributes are essential for understanding the cooperative folding/unfolding of such proteins. Dynamic NMR spectroscopy has enabled the characterization of folding intermediates of ubiquitin that exist in equilibrium under conditions of low pH and denaturants. At low pH, an unlocked state defined as N′ is in fast exchange with an invisible state, U″, as observed by CEST NMR. Addition of urea to ubiquitin at pH 2 creates two new states F′ and U′, which are in slow exchange (kF′→U′ = 0.14 and kU′→F′ = 0.28 s–1) as indicated by longitudinal ZZ-magnetization exchange spectroscopy. High-resolution solution NMR structures of F′ show it to be in an “unlocked” conformation with measurable changes in rotational diffusion, translational diffusion, and rotational correlational times. U′ is characterized by the presence of just the highly conserved N-terminal β1−β2 hairpin. The folding of ubiquitin is cooperative and is nucleated by the formation of an N-terminal β-hairpin followed by significant hydrophobic collapse of the protein core resulting in the formation of bulk of the secondary structural elements stabilized by extensive tertiary contacts. U′ and F′ may thus be described as early and late folding intermediates in the ubiquitin folding pathway.","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":"63 20","pages":"2565–2579 2565–2579"},"PeriodicalIF":2.9,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142450601","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0